CN1373811A

CN1373811A - Herbicide-tolerant protoporphyrinogen oxidase

Info

Publication number: CN1373811A
Application number: CN 00812881
Authority: CN
Inventors: M·A·约翰逊; S·L·沃尔拉斯; P·B·海菲茨; M·D·洛尔
Original assignee: Syngenta Participations AG
Current assignee: Syngenta Participations AG
Priority date: 1999-08-13
Filing date: 2000-06-30
Publication date: 2002-10-09
Also published as: AU5536000A; EP1200611A1; CA2381927A1; AR024642A1; JP2003507019A; WO2001012825A1

Abstract

The present invention provides novel DNA sequences coding for protoporphyrinogen oxidase (protox) enzymes from soybean, wheat, cotton, sugar beet, oilseed rape, rice, sorghum, and sugar cane. In addition, the present invention teaches modified forms of protox enzymes that are herbicide tolerant. Plants expressing herbicide tolerant protox enzymes taught herein are also provided. These plants may be engineered for resistance to protox inhibitors via mutation of the native protox gene to a resistant form or they may be transformed with a gene encoding a herbicide tolerant form of a plant protox enzyme.

Description

The proporphyrinogen oxidase of herbicide-resistant

The enzyme of herbicide-resistant form of the present invention relates to encode is the dna molecular of proporphyrinogen oxidase (" protox ").The invention further relates to the plant of herbicide-resistant and be used for tissue culture screening and based on the method for the herbicide application of the protox of these herbicide-resistant forms.

The invention provides the modified forms of plant proporphyrinogen oxidase (protox), their tolerances suppress the compound of the naturally occurring plant protox enzyme of unmodified; And the present invention also provides the dna molecular of the plant protox enzyme of anti-this class inhibitor of encoding.

For clarity sake, with some term definition of using in this specification sheets be described below:

Be correlated with/be operably connected: two kinds of dna sequence dnas that refer on the structure or on function, be correlated with.For example, think that promotor or regulate dna sequence dna and coding RNA or protein DNA sequence " relevant ", condition are that these two kinds of sequences are operably connected or locate, make that regulating dna sequence dna influences and encode or the expression level of structural DNA sequence.

Mosaic gene: a kind of recombinant DNA sequence, wherein promotor or regulate dna sequence dna and encode mRNA or be expressed as the protein DNA sequence and be operably connected or combine makes described adjusting dna sequence dna can regulate transcribing or expressing of associated dna sequence.The adjusting dna sequence dna of this mosaic gene is not operably connected with the associated dna sequence that occurring in nature is found usually.

The dna encoding sequence: a kind of being translated in vivo produces the protein DNA sequence.

Be equivalent to: in the context of the present invention, " be equivalent to " to refer to when the aminoacid sequence with various protox enzymes carries out such as the sequence contrast among the table 1A each other, the amino acid of some position of enumerating is consistent with the arrangement of these position sequences among the table 1A but determines on positions of numeral and those nonessential amino acid at these with regard to the aminoacid sequence of specific protox enzyme among " being equivalent to " table 1A.Equally, when the aminoacid sequence with the aminoacid sequence of specific protox enzyme (for example soybean protox enzyme) and reference protox enzyme (for example the mouse ear mustard of representing with SEQ ID NO:2 belongs to the protox-1 sequence) carried out the sequence contrast, it was that series arrangement with these positions of SEQ ID NO:2 is consistent but determine on positions of numeral at these of soybean protox enzyme amino acid sequence and those nonessential amino acid that some of " being equivalent to " SEQ ID NO:2 enumerated amino acid in the soybean protox sequence of position.

Weedicide: a kind of chemical substance that is used to kill and wound or suppress plant, vegetable cell, plant seed or plant tissue growth.

Allogeneic dna sequence: a kind of not with the natural relevant dna sequence dna of the host cell that is imported, comprise the multiple copied that the non-natural of naturally occurring dna sequence dna exists.

Homologous DNA sequence: a kind of and the natural relevant dna sequence dna of the host cell that is imported.

Homogeneity: refer to a kind of all plastids and be upward identical plant, plant tissue or vegetable cell of heredity.At different tissues or in the etap, described plastid can be with multi-form appearance, for example chloroplast(id), proplastid, etioplast, amyloplast, chromoplast etc.

Inhibitor: a kind of chemical substance that makes such as biosynthetic enzyme, acceptor, signal transducer, structure gene product or the such plant-growth of translocator or the requisite proteinic enzymic activity inactivation of surviving.In the context of the present invention, inhibitor is the chemical substance of the enzymic activity inactivation of a kind of protox of making.Term used herein " weedicide " has defined a kind of inhibitor that is applied to plant, vegetable cell, plant seed or plant tissue.

Isolating: in the context of the present invention, isolated nucleic acid molecule or isolating enzyme are to break away from that its natural surroundings exists and be not the nucleic acid molecule or the enzyme of natural product thus with manual type.Isolated nucleic acid molecule or enzyme can exist or may reside in such as in the such non-natural environment of for example genetically modified host cell with purified form.

Minimal promoter: make the promoter activity inactivation or do not having remarkable promoter element, particularly the TATA element that reduces promoter activity under the activatory situation of upstream.Under the situation that has suitable transcription factor to exist, minimal promoter plays a part to allow to transcribe.

Modification enzyme activity: be different from the enzymic activity of naturally occurring enzymic activity in plant (i.e. naturally occurring enzymic activity under not having by the active situation of artificial directly or indirectly this class of manipulation), its tolerance can suppress the inhibitor of naturally occurring enzymic activity.

Nucleic acid molecule: can separate from strand of originating arbitrarily or the linear fragment of double-stranded DNA or RNA.In the context of the present invention, preferably a kind of dna fragmentation of described nucleic acid molecule.

Plant: any plant or the plant parts that refer to any etap.Wherein also comprise the culture and the seed that downcut part, cell or tissue.Include but not limited to any one group of vegetable cell of complete plant, vegetable cell, plant organ, plant seed, protoplastis, callus, cell culture and formation structure and/or functional unit as the term that uses with the present invention " plant tissue ".

Plastom(e): the genome of plastid.

Protox-1: chloroplast(id) protox.

Protox-2: plastosome protox.

Significantly increase: the increase of enzymic activity is greater than inherent margin of error in the determination techniques, preferably under the situation that has inhibitor to exist than the enzymic activity of wild-type increase about more than 2 times or 2 times, more preferably increase about more than 5 times or 5 times and most preferably increase about more than 10 times or 10 times.

Basically similar: with regard to nucleic acid, nucleic acid molecule and reference nucleic acid molecule have at least 60% identity.In a preferred embodiment, similar basically dna sequence dna has at least 80% identical with the reference dna sequence dna; In a preferred embodiment, it is 90% identical with the reference dna sequence dna that similar basically dna sequence dna has at least; And in a most preferred embodiment, it is 95% identical with the reference dna sequence dna that similar basically dna sequence dna has at least.Basically similar nucleotide sequence is generally being hybridized with reference nucleic acid molecule or its fragment under the following condition: hybridize among 7% sodium lauryl sulphate (SDS) under 50 ℃, 0.5M NaPO4 pH7.0, the 1mM EDTA; Wash with 2X SSC, 1%SDS down at 50 ℃.With regard to protein or peptide, similar basically aminoacid sequence be have at least 90% identical with the aminoacid sequence of reference protein matter or peptide and have substantially the same active aminoacid sequence with reference protein matter or peptide.

Tolerance/resistance: the ability that when contacting, keeps normal growth or function with inhibitor or weedicide.

Transform: a kind of process allogeneic dna sequence DNA transfered cell, tissue or plant.Be understandable that cell transformed, tissue or plant not only comprise the end product of conversion process but also comprise its transgenosis filial generation.

Transit peptides: a kind of signal polypeptide that is translated with protein by dna molecule encode, thus form a kind of polypeptide precursor.To the process of the position of selecting, for example chloroplast(id), produce activity or maturation protein in intracellular transport thereby can fall described transit peptides from the remainder cracking of described polypeptide precursor.

Transform: refer to such as the such organism of plant that has imported the allogeneic dna sequence DNA molecule.This dna molecular stably can be integrated into the genome of described plant, wherein the genome of this plant comprises nuclear gene group, plastom and Mitochondrial Genome Overview.In by plant transformed, described dna molecular can also exist as extrachromosomal molecule.The outer molecule of this chromosomoid can carry out self-replacation." unconverted " plant refers to wild-type organisms, promptly a kind of plant that does not contain the allogeneic dna sequence DNA molecule.

Change plastom(e): a kind of plastom of conversion.

Nucleotide is represented by its base of following standardized abbreviations representative: VITAMIN B4 (A); Cytosine(Cyt) (C); Thymus pyrimidine (T); And guanine (G).Amino acid is represented by following standardized abbreviations equally: L-Ala (ala; A); Arginine (Arg; R); L-asparagine (Asn; N); Aspartic acid (Asp; D); Halfcystine (Cys; C); Glutamine (Gln; Q); L-glutamic acid (Glu; E); Glycine (Gly; G); Histidine (His; H); Isoleucine (Ile; I); Leucine (Leu; L); Methionin (lys; K); Methionine(Met) (Met; M); Phenylalanine (Phe; F); Proline(Pro) (Pro; P); Serine (Ser; S); Threonine (Thr; T); Tryptophane (Trp; W); Tyrosine (Tyr; Y); And Xie Ansuan (Val; V).In addition, (Xaa; X) represent amino acid arbitrarily.

The invention discloses the plant proporphyrinogen oxidase (protox) of modified forms, their tolerances suppress the compound of the naturally occurring plant protox enzyme of unmodified; And the invention also discloses the dna molecular of the plant protox enzyme of anti-this class inhibitor of coding.

Therefore, a kind of dna molecular of coded plant protox enzyme is disclosed in one aspect of the invention, it can be introduced into the DNA construct that is used to transform the plant that contains wild-type herbicide sensitive protox, wherein said dna molecular has a point mutation at least for the wild-type dna molecular of coded plant protox, make that described plant contains this dna molecular when transforming with described DNA construct, this dna molecular can make described plant that the weedicide of the naturally occurring plant protox of the inhibition of using is produced tolerance.

The present invention be more particularly directed to a kind of nucleic acid molecule, it comprises that coding has the nucleotide sequence of the active modifying enzyme of proporphyrinogen oxidase (protox), wherein said modifying enzyme can tolerate the inhibitor of the natural existence form of described enzyme, and wherein said modifying enzyme comprises at least a in the following amino acid subsequence:

(a) KA Δ ₁₈F, wherein Δ ₁₈Be the amino acid of a non-L-Ala;

(b) Q Δ ₁₉H, wherein Δ ₁₉Be a non-leucic amino acid;

(c) AP Δ ₁F, wherein Δ ₁Be a non-arginic amino acid, and especially leucine or halfcystine;

(d) F Δ ₂S, wherein Δ ₂It is leucine;

(e) Y Δ ₃G, wherein Δ ₃It is Isoleucine;

(f) Δ ₇IG, wherein Δ ₇Be Histidine or L-Ala;

(g) T Δ ₁₆G, wherein Δ ₁₆Be a non-leucic amino acid; With the YV Δ ₁₇G, wherein Δ ₁₇Be the amino acid of a non-L-Ala;

The nucleotide sequence of natural existence form of described enzyme of wherein encoding derives from a kind of plant.

In a preferred embodiment, the present invention relates to a kind of nucleic acid molecule, it comprises that coding has the nucleotide sequence of the active modifying enzyme of proporphyrinogen oxidase (protox), wherein said modifying enzyme can tolerate the inhibitor of the natural existence form of described enzyme, and wherein said modifying enzyme comprises at least a amino acid subsequence that is selected from the following amino acid subsequence:

(a) KA Δ ₁₈F, wherein Δ ₁₈Be the amino acid of a non-L-Ala;

(b) Q Δ ₁₉H, wherein Δ ₁₉Be a non-leucic amino acid;

(c) AP Δ ₁F, wherein Δ ₁Be a non-arginic amino acid, and leucine especially;

(d) F Δ ₂S, wherein Δ ₂It is leucine;

(e) Y Δ ₃G, wherein Δ ₃It is Isoleucine;

(f) Δ ₇IG, wherein Δ ₇Be Histidine or L-Ala;

The described nucleotides sequence of described modifying enzyme of wherein encoding be listed under the following condition with the nucleotide sequence of representing with SEQ IDNO:1, SEQ ID NO:5, SEQ ID NO:9, EQ ID NO:11, SEQ ID NO:15, SEQID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23 or SEQ ID NO:36 in any one hybridization:

(i) hybridize among 7% sodium lauryl sulphate (SDS) under 50 ℃, 0.5M NaPO4 pH7.0, the 1mM EDTA; With

(ii) with 2X SSC, 1%SDS under 50 ℃ washing.

Preferably a kind of in addition nucleic acid molecule, it comprises that coding has the nucleotide sequence of the active modifying enzyme of proporphyrinogen oxidase (protox), wherein said modifying enzyme can tolerate the inhibitor of the natural existence form of described enzyme, and wherein said modifying enzyme comprises at least a in the following amino acid subsequence:

(a) KA Δ ₁₈F, wherein Δ ₁₈Be the amino acid of a non-L-Ala;

(b) Q Δ ₁₉H, wherein Δ ₁₉Be a non-leucic amino acid;

(c) AP Δ ₁F, wherein Δ ₁It is leucine;

(d) Δ ₇IG, wherein Δ ₇It is Histidine;

Preferably a kind of nucleic acid molecule, wherein said modifying enzyme comprise amino acid subsequence KA Δ ₁₈F, wherein Δ ₁₈Be the amino acid of a non-L-Ala.

Preferably a kind of in addition nucleic acid molecule, wherein Δ ₁₈Be Threonine or Xie Ansuan.

Also preferred a kind of nucleic acid molecule, wherein said modifying enzyme comprise amino acid subsequence Q Δ ₁₉H, wherein Δ ₁₉Be a non-leucic amino acid.

Also preferred a kind of nucleic acid molecule, wherein Δ ₁₉It is Serine.

Also preferred a kind of nucleic acid molecule, wherein said modifying enzyme comprise amino acid subsequence AP Δ ₁F, wherein Δ ₁Be leucine or halfcystine.

Also preferred a kind of nucleic acid molecule, wherein said modifying enzyme comprise amino acid subsequence Δ ₇IG, wherein Δ ₇Be Histidine or L-Ala and Histidine especially.

Also preferred a kind of nucleic acid molecule, wherein said modifying enzyme comprises: amino acid subsequence T Δ ₁₆G, wherein Δ ₁₆It is Serine; With amino acid subsequence YV Δ ₁₇G, wherein Δ ₁₇It is Threonine.

The present invention further provides a kind of nucleic acid molecule, modifying enzyme wherein of the present invention further comprises at least a additional amino acid subsequence that is selected from the following amino acid subsequence:

(e) Q Δ ₁₁S, wherein Δ ₁₁Be the amino acid of a non-proline(Pro);

(f) IGG Δ ₁₂, Δ wherein ₁₂Be the amino acid of a non-Threonine;

(g) SWXL Δ ₁₃, Δ wherein ₁₃Be the amino acid of a non-Serine;

(h) L Δ ₁₄Y, wherein Δ ₁₄Be the amino acid of a non-l-asparagine;

(i) G Δ ₁₅XGL, wherein Δ ₁₅Be the amino acid of a non-tyrosine.

Preferably a kind of nucleic acid molecule, wherein said additional subsequence is the Q Δ ₁₁S, wherein Δ ₁₁Be the amino acid of a non-proline(Pro).

Preferably a kind of in addition nucleic acid molecule, wherein Δ ₁₁It is leucine.

Preferably a kind of in addition nucleic acid molecule, wherein said additional subsequence is the IGG Δ ₁₂, Δ wherein ₁₂Be the amino acid of a non-Threonine.

Also preferred a kind of nucleic acid molecule, wherein Δ ₁₂Be Isoleucine or L-Ala.

Also preferred a kind of nucleic acid molecule, wherein said additional subsequence is the SWXL Δ ₁₃, Δ wherein ₁₃Be the amino acid of a non-Serine.

Also preferred a kind of nucleic acid molecule, wherein Δ ₁₃It is leucine.

Also preferred a kind of nucleic acid molecule, wherein said additional subsequence is the L Δ ₁₄Y, wherein Δ ₁₄Be the amino acid of a non-l-asparagine.

Also preferred a kind of nucleic acid molecule, wherein Δ ₁₄It is Serine.

Also preferred a kind of nucleic acid molecule, wherein said additional subsequence is the G Δ ₁₅XGL, wherein Δ ₁₅Be the amino acid of a non-tyrosine.

Also preferred a kind of nucleic acid molecule, wherein Δ ₁₅It is halfcystine.

Particularly preferably be a kind of nucleic acid molecule of coded plant protox enzyme, described plant protox endonuclease capable is introduced into the DNA construct that is used to transform the plant that contains wild-type herbicide sensitive protox, at least has a point mutation for the wild-type dna molecular of the plant protox that wherein said nucleic acid molecule is as indicated above with respect to coding, make when transforming with described DNA construct described plant contain can to make described plant to the tolerific dna molecular of weedicide of the naturally occurring plant protox of the inhibition of using, wherein said nucleic acid molecule can be available from an a kind of kind of plant that is selected from the following plants: mouse ear mustard belongs to, corn, wheat, soybean, cotton, beet, rape, rice, Chinese sorghum and sugarcane.

The sequence of this class dna molecular is with SEQ ID NOs:9 (wheat), 11 (soybean), and 15 (cottons), 17 (beets), 19 (rapes), 21 (rice), 23 (Chinese sorghums) and 36 (sugarcanes) are represented.Preferably a kind of can be available from the nucleic acid molecule of corn or cotton.

The present invention also comprises a kind of active modifying enzyme of proporphyrinogen oxidase (protox) that has, by mentioned earlier of the present invention nucleotide sequence coded arbitrarily of wherein said modifying enzyme.

The present invention further comprises the modified forms of mosaic gene and naturally occurring protox gene, and they can express the plant protox enzyme of anti-inhibitor in plant.

The present invention be more particularly directed to a kind of mosaic gene, it is included in has the active promotor that operationally is connected with nucleic acid molecule of the present invention in the plant.

The present invention further comprises recombinant carrier molecule, and it comprises mosaic gene of the present invention.

The gene of plant protox enzyme of the anti-inhibitor of coding can be used for the weedicide that suppresses protox being produced resistance and as the selective marker in the vegetable cell method for transformation complete plant.Therefore, the present invention also comprise contain the coding these modified the expression of plants gene of protox enzyme plant, comprise its offspring, plant tissue and plant seed.These plants, plant tissue and plant seed can tolerate usually the protox inhibitor under the active inhibited concentration of naturally occurring protox in the plant.

The present invention be more particularly directed to a kind of vegetable cell that comprises nucleic acid molecule of the present invention.

The invention further relates to the plant, vegetable cell or the plant seed that comprise nucleic acid molecule of the present invention, comprise its filial generation, wherein said nucleic acid molecule is expressed in described plant, plant tissue, vegetable cell or plant seed and thus the active inhibitor of naturally occurring protox is produced tolerance.

The plant that the present invention includes comprises that especially those may be the plants of target thing that suppresses the weedicide of protox, particularly such as corn and the important like this farm crop of other cereal farm crop, such as barley, wheat, Chinese sorghum, rye, oat, mud grass and forage grass, millet and rice.Also comprise other crop plants, such as sugarcane, soybean, cotton, beet, rape and tobacco.

Preferably a kind of plant, plant tissue, vegetable cell or plant seed that comprises nucleic acid molecule of the present invention, comprise that its filial generation, wherein said plant, plant tissue, vegetable cell or plant seed are selected from the group that mouse ear mustard genus, soybean, cotton, beet, rape, corn, wheat, Chinese sorghum, rice and sugarcane are formed.

The invention still further relates to the conversion of plastid and relate to the expression of nucleic acid molecule of the present invention in plant plastid.In a preferred embodiment, in plant plastid, express adorned plant protox enzyme as indicated above so that obtain herbicide resistant plants.

The present invention be more particularly directed to a kind of mosaic gene that is included in the promotor that operationally is connected with nucleic acid molecule of the present invention that works in the plant plastid, preferred described promotor is the clpP gene promoter.

In another embodiment, the present invention relates to a kind of mosaic gene, it comprises: (a) separate the dna molecular from a kind of plant, polypeptide and a kind of maturing enzyme that this dna molecular is encoded under its native state and comprised plastid transit peptides, described maturing enzyme is by the natural plastid that is directed to described plant of described plastid transit peptides, and wherein said dna molecular is modified to not can encoding function character body transit peptides; (b) can express the promotor of the dna molecular in the plastid, wherein this promotor operationally is connected with dna molecular of the present invention.Can modify this dna molecular, promptly from described dna molecular, lack to the natural plastid transit peptide-coding sequence of small part.On the other hand, can modify described dna molecular,, make coded plastid transit peptides become non-functional thus even one or more Nucleotide in the natural plastid transit peptide-coding sequence are undergone mutation.The invention still further relates to the chloroplast gene group that contains this class mosaic gene is the plant of homogeneity.In a preferred embodiment, described dna molecule encode is by the enzyme of the natural inhibition of herbicidal compounds.In this case, the tolerance of this class plant can natural inhibition by the weedicide of the enzyme of dna molecule encode of the present invention.

Another embodiment of the invention relates to a kind of plastid conversion carrier, and it comprises some or all element mentioned above, comprises nucleic acid molecule of the present invention.

Further comprise a kind of plant plastid, it comprises plastid conversion carrier of the present invention.

The invention still further relates to dna molecular of the present invention and transform plastom and the plant of the herbicide-resistant for preparing and relate to the preparation method of this class plant.The present invention further relates to the plant, plant tissue, vegetable cell or the plant seed that comprise plant plastid of the present invention, comprises its filial generation that the plant protox enzyme of wherein said modification is expressed and made described plant, plant tissue, vegetable cell or plant seed that the active inhibitor of naturally occurring protox is produced tolerance thus thus in described plant, plant tissue, vegetable cell or plant seed.

The invention further relates to and be used to the method for producing plant, comprising plant material, the described plant that comprises plant material such as plant tissue for example; Protoplastis; Cell; Callus; Organ; Plant seed; Embryo; Pollen; Ovum; Zygote; And other breeding thing and plant parts arbitrarily, such as the flower, stem, fruit, leaf, the root that for example derive from the filial generation that transgenic plant or its transform with the inventive method in advance, they can produce the plant protox enzyme that this paper provides anti-inhibitor form.Can the be encoded structure gene of described resistance protox of this class plant stably transforms or prepares by the orthoselection technology, thus the strain of herbicide-resistant is separated, is carried out characterization and make its growth.

The present invention further provides a kind of method that is used to screen the dna molecular plant transformed cell of the present invention of the plant protox that uses coding herbicide-resistant form.This method comprises the following steps: described dna molecular is imported vegetable cell, the restraining effect sensitivity that the growth of this vegetable cell produces weedicide, and described weedicide is produced tolerance by the protox of described dna molecule encode, form a kind of plant transformed cell thus.The plant transformed cell of the weedicide of the anti-selection of this growth is identified by screening under the weedicide concentration that suppresses unconverted plant cell growth.

The present invention also provides a kind of novel method of selecting to change the vegetable cell of plastom(e), and this method comprises the following steps: above-mentioned mosaic gene is imported the plastom(e) of vegetable cell; In the plastid of this vegetable cell, express the enzyme of coding; With the tolerific cell of selecting the described enzymic activity of natural inhibition of herbicidal compounds, this tolerance cell comprises the plastid of conversion thus.In a preferred embodiment, come the described enzyme of natural inhibition and transgenic plant under the consumption of the herbicidal compounds of the described enzymic activity of natural inhibition, to grow by herbicidal compounds.

Another aspect, the present invention relates to a kind of method that unwanted vegetation grows that is used to control on certain position, wherein cultivated the useful crop plants of herbicide-resistant, the dna molecular of the present invention of the plant protox of its herbicide-resistant form that is encoded transforms.This method comprises the step to the active weedicide of the naturally occurring protox of inhibition of using significant quantity on the position of being protected.The present invention be more particularly directed to a kind of method that does not need vegetation growth that is used to control, this method comprises the step of inhibitor of using the described enzyme of significant quantity to above-mentioned plant population.

In a preferred embodiment, the present invention relates to a kind of method that is used to control unwanted vegetation growth, the weedicide of wherein said inhibition protox is selected from following one group of material: aryi-uracile (aryluracil); Phenyl ether; Oxidation pyrazoles (oxidiazole); Imide preferably has the imide of general formula V, VI, VII, VIIa, VIII, IX, IXa or IXb; Phenylpyrazole preferably has the phenylpyrazole of general formula X XIV; Pyridyl pyrazoles preferably has the pyridyl pyrazoles of general formula X XIIIa or XXIIIb; Pyridine derivate; 3-replaces-2-aryl-4,5,6, the 7-tetrahydrochysene indazole; Phenopyrate; With the O-Phenylpyrrolidine of described phenopyrate also-and the similar thing of piperidino-(1-position only) carbamate.

The invention further relates to a kind of method of weed growth that selectivity suppresses to contain the field of the crop seeds of many plantations or plant that is used for, this method comprises the following steps:

(a) farm crop or the crop seeds of plantation herbicide-resistant, they are plant of the present invention or plant seed; With

(b) weedicide of using the inhibition protox that suppresses the active consumption of naturally occurring protox for the farm crop in field or crop seeds and weeds, wherein this weedicide suppresses weed growth and can significantly not suppress the growths of described farm crop.

In one aspect, the present invention relates to the isolated DNA molecule of a kind of coding from the proporphyrinogen oxidase (this paper is called " protox ") of sugarcane.The part dna encoding sequence and the amino acid sequence corresponding of sugarcane protox enzyme are represented with SEQ ID NOs:36 and 37 respectively.

Therefore, what the present invention comprised in addition is the isolated DNA molecule of coding from proporphyrinogen oxidase (protox) zymoprotein of sugarcane plants, and wherein said protein comprises the aminoacid sequence of representing with SEQ IDNO:37.

In one aspect of the method, the present invention relates to a kind of sugarcane protox enzyme and comprise and the DNA isolation molecule of the nucleotide sequence of the encoding sequence hybridization represented with SEQ ID NO:36 that described crossover process is carried out under following hybridization and wash conditions of encoding:

(a) 7% sodium lauryl sulphate (SDS), the 0.5M NaPO under 50 ℃ ₄, hybridize among pH7.0, the 1mM EDTA; With

(b) wash among 2X SSC, the 1%SDS under 50 ℃.

The invention further relates to the plant protox enzyme that is used for detecting the anti-inhibitor form of coding gene existence and the protox transcript concentration of the anti-inhibitor of plant tissue carried out quantitative probe and method.These methods can be used to identify or screen the plant or the plant tissue of gene that contains and/or express the plant protox enzyme of the anti-as disclosed herein inhibitor form of coding.

Protox sequence that can the present invention is complete or its part as can with the probe of protox encoding sequence and messenger RNA(mRNA) specific hybrid.In order to finish specific hybrid under various conditions, this class probe is included in the protox encoding sequence be unique and preferred length be at least 10 Nucleotide and most preferably length be at least the sequence of 20 Nucleotide.Can this class probe is used to increase and analyze protox encoding sequence by well-known step in the polymerase chain reaction (PCR) from the organism of selecting.This technology can be used for separating the diagnostic assay method of adding the protox encoding sequence or existing as protox encoding sequence the mensuration organism from required organism.

The factor that influences hybrid stability has determined the severity of hybridization.A kind of this class factor is melting temperature(Tm) T _m, can calculate T easily according to the formula that provides in the following document _m: dna probe (DNAPROBES), George H.Keller and Mark M.Manak, Macmillan PublishersLtd, 1993, chapter 1: molecular hybridization (Molecular HybridizationTechnology); Page 8 ff.

Preferred hybridization temperature is being less than about the melting temperature(Tm) T of calculating _mAbout 25 ℃ scope and preferably be less than about the melting temperature(Tm) T of calculating _mAbout 12-15 ℃ scope, and with regard to the situation of oligonucleotide, hybridization temperature is being less than about melting temperature(Tm) T _mAbout 5-10 ℃ scope.

Present invention resides under the medium stringent condition with as hereinbefore defined dna molecule hybridize of the present invention and the dna molecular of preferred and oligonucleotide probe hybridization, described oligonucleotide probe is available from comprising that length is at least the dna molecular of the contiguous sections of the proporphyrinogen oxidase of 10 Nucleotide (protox) sequence.

The present invention further comprises the application that can be at least the nucleotide probe of the plant protox gene of 10 Nucleotide or mRNA specific hybrid with length in the polymerase chain reaction (PCR).

In another embodiment, the invention provides can be with the coding active eukaryotic DNA sequence of proporphyrinogen oxidase or with the probe of corresponding mRNA specific hybrid with use the method for the dna sequence dna in the probe in detecting most eukaryotes of the present invention.

Can also use based on the standard technique of probe and genome protox sequence selective hybridization Protox specific hybrid probe of the present invention is used for being mapped in the position of the natural eucaryon protox gene of the organism genome selected.These technology include but not limited in the protox probe sequence to identify or the evaluation of the dna polymorphism that comprises with this class polymorphism is used for subsequently separate the protox gene relevant with other mark of the known figure spectral position of mapping population, described mapping population derive from two kinds of polymorphic parental lines hybrid selfing (for example, referring to Helentjaris etc., molecular biology of plants (Plant Mol.Biol.) 5:109 (1985); Biotechnologys such as Sommer (Biotechniques) 12:82 (1992); Molecular biology of plants (Plant Mol.Biol.) 15:169 (1990) such as D ' Ovidio).Although what pay close attention to is the probe that the protox gene mapping that any eucaryon protox sequence comes from any most eukaryotes with opposing is used, preferred probes is those from protox sequence and the most preferred probe of the organism that is closely related more with the organism of selecting is those protox sequences from selected organism.What paid close attention to is the plant that by this way the protox gene mapping is specially adapted to breeding objective.For example, flanking DNA mark (for example, referring to Helentjaris, Trends Genet.3:217 (1987)) can be identified in the genetic map position of the mutant protox gene by known generation Herbicid resistant from the reference genetic map.The Herbicid resistant feature is gradually being infiltrated in the process of new breeding strain, these marks can be used for monitoring subsequently the degree that the chromosomal DNA flank that still is present in recurrent parent after each circulation of backcrossing connects protox that is positioned at.

Can also use such as the such standard technique of the careful engram analysis of promise Protox specific hybrid probe of the present invention is used for the level of organism protox mRNA is carried out quantitatively.Can be with this technology as detecting the diagnostic assay method that the protox expression level changes, the change of described expression level may with human body in relevant such as the so specific unfavorable condition of the autosomal dominant pathology that with neuropsychopathy symptom and skin lesion is feature, these unfavorable conditions and the active level decline of protox relevant (Brenner and Bloomer, New Engl.J.Med.302:765 (1980)).

Another embodiment of the invention is a kind of production method of dna molecular, and this dna molecular comprises the DNA part, the protein that this part coding has the enzymic activity of proporphyrinogen oxidase (protox), and described method comprises the following steps:

(a) preparation can with the nucleotide probe of plant protox gene or mRNA specific hybrid, wherein this probe comprises that length is at least the contiguous sections of the protox albumen coded sequence of the present invention of 10 Nucleotide;

(b) use the nucleotide probe for preparing according to step (a) to survey from the cloned genes group dna fragmentation of the organism of selecting or other protox encoding sequence in the segmental colony of cDNA; With

(c) separate and amplification comprises partly the encode protein of enzymic activity with proporphyrinogen oxidase (protox) of the dna molecular of DNA part, described DNA.

Another embodiment of the invention be a kind of from any plant that comprises DAN part the method for DNA isolation molecule, partly the encode protein of enzymic activity of described DNA with proporphyrinogen oxidase (protox).

(c) separate the dna molecular comprise the DNA part, partly the encode protein of enzymic activity of described DNA with proporphyrinogen oxidase (protox).

The present invention comprises that further a kind of coding demonstrates the protein of enzymic activity of proporphyrinogen oxidase (protox) and the production method of pure basically dna sequence dna, and this method comprises the following steps:

(a) use suitable cloning vector to prepare genome or cDNA library by the organism in suitable source;

(b) make the hybridization of described library and probe molecule of the present invention; With

(c) identify described probe and forward hybridization from the dna clone in described library, described dna clone is the clone that possible contain the nucleotide sequence of the aminoacid sequence that is equivalent to proporphyrinogen oxidase (protox).

The present invention comprises that further a kind of coding demonstrates the production method of proteinic pure basically dna sequence dna of the enzymic activity of proporphyrinogen oxidase (protox), and this method comprises the following steps:

(a) prepare total DNA by genome or cDNA library;

(b) DNA with step (a) is used as the template that PCR reacts, and the primer of the low degeneracy part of the aminoacid sequence of representing proporphyrinogen oxidase of the present invention (protox) is used in this PCR reaction.

Another object of the present invention is the measuring method of inhibitor of the enzymic activity of a kind of evaluation proporphyrinogen oxidase (protox), and this method comprises the following steps:

(a) first kind of sample with proporphyrinogen oxidase of the present invention (protox) and substrate thereof is incubated;

(b) mensuration is from the reactivity that does not suppress of the proporphyrinogen oxidase (protox) of step (a);

(c) under the situation that has second kind of sample that comprises inhibitor compound to exist, first kind of sample of proporphyrinogen oxidase (protox) and substrate thereof is incubated;

(d) mensuration is from the reactivity that is suppressed of the proporphyrinogen oxidase (protox) of step (c); With

(e) reactivity that is suppressed of proporphyrinogen oxidase (protox) and the reactivity that does not suppress are compared.

Another object of the present invention is a kind of analytical procedure of identifying proporphyrinogen oxidase (protox) mutant of anti-inhibitor, and this method comprises the following steps:

(a) first kind of sample with proporphyrinogen oxidase of the present invention (protox) and substrate thereof is incubated under the situation that the second kind of sample existence that comprises proporphyrinogen oxidase (protox) inhibitor arranged;

(b) mensuration is from the not reactivity of sudden change of the proporphyrinogen oxidase (protox) of step (a);

(c) under the situation that has second kind of sample that comprises proporphyrinogen oxidase (protox) inhibitor to exist, the proporphyrinogen oxidase (protox) of sudden change and first kind of sample of substrate thereof are incubated;

(d) mensuration is from the reactivity of the sudden change of the proporphyrinogen oxidase (protox) of the sudden change of step (c); With

(e) reactivity of the reactivity of the sudden change of proporphyrinogen oxidase (protox) with not sudden change compared.

Another object of the present invention is a kind of protox enzyme inhibitors that obtains by the inventive method.

For the recombinant production of described enzyme in three kinds of organisms, protox encoding sequence of the present invention can be inserted as the expression cassette of selected host's design and import this host, wherein it is carried out recombinant production.The horizontal extent that the selection of regulating sequence such as the such specificity of promotor, signal sequence, 5 ' and 3 ' the untranslated sequence and enhanser is belonged to those of ordinary skills.The gained molecule that contains each element that connects in the suitable frame can be inserted the carrier that can be transformed into host cell.Just such as intestinal bacteria (for example, referring to Studier and Moffatt, molecular biology magazine (J.Mol.Biol.) 189:113 (1986); Brosius, DNA 8:759 (1989)), yeast (for example, referring to Schneider and Guarente, Enzymology method (Meth.Enzymol.) 194:373 (1991)) and insect cell (for example, referring to Luckow and Summers, Bio/Technol.6:47 (1988)) such host organisms, the suitable expression vector and the method that are used for the protein recombinant production are well-known.Concrete example comprises: plasmid, such as pBluescript (Stratagene, La Jolla, CA); PFLAG (InternationalBiotechnologies, Inc., New Haven, CT); PTrcHis (Invitrogen, LaJolla, CA); And rhabdovirus expression vector, for example those derive from the genomic rhabdovirus expression vector of Autographicacalifornica nuclear polyhedrosis virus (AcMNPV).Preferred baculovirus/insect system be the pVl11392/Sf21 cell (Invitrogen, La Jolla, CA).

The eucaryon protox enzyme of recombinant production is used for various purposes.For example, it can be used in the external protox of providing enzymic activity.It can also be used to screen the in vitro tests of the still undetermined known weedicide chemical of target thing and whether can suppress protox so that determine them.Can also be with this class in vitro tests as screening method more generally so that identify that can to suppress protox active and be the chemical of weedicide material standed for thus.The eucaryon protox enzyme of recombinant production can also be used to identify the test (referring to being disclosed as WO95/34659 on International Application PCT/IB95/00452, the December 21 nineteen ninety-five of submitting to June 8 nineteen ninety-five, the full content of the document being incorporated herein by reference) of the protox mutant of anti-inhibitor.On the other hand, the protox enzyme of recombinant production can be used for further making the relationship characteristicization of itself and known inhibitor, thereby can reasonably design the enzyme of novel inhibition weedicide and described herbicide-resistant form.

In one aspect of the method, the present invention has instructed and can modify so that produce the anti-inhibitor form of this kind of enzyme the aminoacid sequence of any eucaryon proporphyrinogen oxidase (this paper is called " protox ").Preferred described eucaryon protox enzyme is a kind of plant protox enzyme.The present invention relates to have this paper instruction this modification anti-inhibitor the protox enzyme, relate to the dna molecular of these modifying enzymes of encoding and relate to the mosaic gene that can in plant, express these modifying enzymes.

The invention discloses a kind of isolated DNA molecule, its coding has at least a amino acid modified modification eucaryon proporphyrinogen oxidase (protox), the wherein said amino acid modified characteristic that has the tolerance of protox inhibitor, the protox that promptly wherein modifies tolerance can suppress the inhibitor of the consumption of naturally occurring eucaryon protox." inhibition " used herein refers to is having test-compound to exist under the situation observed activity ratio not having test-compound to exist under the situation observed activity level low, and wherein the per-cent that reduces of level preferably is at least 10%, more preferably is at least 50% and most preferably be at least 90%.

The present invention discloses a kind of dna molecular especially, its coding belongs to the eucaryon proporphyrinogen oxidase (protox) of the modification of a kind of plant protox, the protox tolerance of wherein being modified can suppress the weedicide of the active consumption of naturally occurring protox, particularly a kind of at least a amino acid modified protox of having of following one group of enzyme that is selected from: mouse ear mustard belongs to the protox enzyme; Corn protox enzyme; Wheat protox enzyme; Soybean protox enzyme; Cotton protox enzyme; Beet protox enzyme; Rape protox enzyme; Rice protox enzyme; Chinese sorghum protox enzyme; With sugarcane protox enzyme, the protox that is wherein modified tolerance can suppress the weedicide of the active consumption of naturally occurring protox.

Term used herein " Bao Shou aminoacid sequence basically " refers to amino acid whose homologous region between the polypeptide of the protox enzyme that comprises different sources.In the present invention, 17 of difference called after 1-19 conservative basically amino acid subsequences are as shown in table 1B.Those skilled in the art can carry out the sequence contrast so that identify the subsequence of conservative aminoacid sequence basically that wherein constitutes this paper definition by the aminoacid sequence to the protox enzyme of different sources as shown in table 1A.Described another kind of mode is the homology subsequence that a kind of described subsequence of source " is equivalent to " different sources.Those skilled in the art can determine subsequently whether the subsequence of being identified has open and feature required for protection among the application.

Therefore, the invention discloses a kind of nucleic acid molecule, it comprises that separation has the nucleotide sequence of the active enzyme of proporphyrinogen oxidase (protox) from the coding of a kind of plant, wherein said nucleic acid molecule can be imported into the nucleic acid construct that is used to transform the plant that contains wild-type herbicide sensitive protox, wherein said nucleotide sequence is compared with the wild-type nucleotide sequence of coded plant protox has a point mutation at least, make that described plant has been endowed herbicide tolerant when transforming with described nucleic acid construct.

An embodiment preferred of the present invention relates to a kind of nucleic acid molecule, it comprises that separation has the nucleotide sequence of the active modifying enzyme of proporphyrinogen oxidase (protox) from the coding of a kind of plant, wherein said modifying enzyme tolerates the inhibitor of naturally occurring protox enzyme, and wherein said modifying enzyme comprises at least a in the following amino acid subsequence:

(a) AP Δ ₁F, wherein Δ ₁The arginic amino acid of right and wrong, and especially leucine or halfcystine;

(b) F Δ ₂S, wherein Δ ₂It is leucine;

(c) Y Δ ₃G, wherein Δ ₃It is Isoleucine;

(d) Δ ₇IG, wherein Δ ₇Be Histidine or L-Ala;

(e) KA Δ ₁₈F, wherein Δ ₁₈Be the amino acid of non-L-Ala;

(f) Q Δ ₁₉H, wherein Δ ₁₉The leucic amino acid of right and wrong;

(g) T Δ ₁₆G, wherein Δ ₁₆The leucic amino acid of right and wrong; With the YV Δ ₁₇G, wherein Δ ₁₇Be the amino acid of non-L-Ala.

(table 1B; Subsequence 1-10 and 18-19 and 16-17).

Preferably a kind of nucleic acid molecule, it comprises that separation has the nucleotide sequence of the active modifying enzyme of proporphyrinogen oxidase (protox) from the coding of a kind of plant, wherein said modifying enzyme tolerates the inhibitor of naturally occurring protox enzyme, and wherein said modifying enzyme comprises amino acid subsequence AP Δ ₁F, wherein Δ ₁Be leucine or halfcystine.

Preferably a kind of nucleic acid molecule, it comprises that separation has the nucleotide sequence of the active modifying enzyme of proporphyrinogen oxidase (protox) from the coding of a kind of plant, wherein said modifying enzyme tolerates the inhibitor of naturally occurring protox enzyme, and wherein said modifying enzyme comprises amino acid subsequence F Δ ₂S, wherein Δ ₂It is leucine.

Preferably a kind of nucleic acid molecule, it comprises that separation has the nucleotide sequence of the active modifying enzyme of proporphyrinogen oxidase (protox) from the coding of a kind of plant, wherein said modifying enzyme tolerates the inhibitor of naturally occurring protox enzyme, and wherein said modifying enzyme comprises amino acid subsequence Y Δ ₃G, wherein Δ ₃It is Isoleucine.

Preferably a kind of nucleic acid molecule, it comprises that separation has the nucleotide sequence of the active modifying enzyme of proporphyrinogen oxidase (protox) from the coding of a kind of plant, wherein said modifying enzyme tolerates the inhibitor of naturally occurring protox enzyme, and wherein said modifying enzyme comprises amino acid subsequence Δ ₇IG, wherein Δ ₇Be Histidine or L-Ala, and Histidine especially.

Further preferably a kind of nucleic acid molecule, it comprises that separation has the nucleotide sequence of the active modifying enzyme of proporphyrinogen oxidase (protox) from the coding of a kind of plant, wherein said modifying enzyme tolerates the inhibitor of naturally occurring protox enzyme, and wherein said modifying enzyme comprises amino acid subsequence KA Δ ₁₈F, wherein Δ ₁₈Be the amino acid of non-L-Ala.Δ most preferably ₁₈Be Threonine or Xie Ansuan.

Preferably a kind of nucleic acid molecule, it comprises that separation has the nucleotide sequence of the active modifying enzyme of proporphyrinogen oxidase (protox) from the coding of a kind of plant, wherein said modifying enzyme tolerates the inhibitor of naturally occurring protox enzyme, and wherein said modifying enzyme comprises amino acid subsequence Q Δ ₁₉H, wherein Δ ₁₉The leucic amino acid of right and wrong.Δ most preferably ₁₉It is Serine.

Another embodiment preferred of the present invention relates to a kind of nucleic acid molecule, it comprises that separation has the nucleotide sequence of the active modifying enzyme of proporphyrinogen oxidase (protox) from the coding of a kind of plant, wherein said modifying enzyme tolerates the inhibitor of naturally occurring protox enzyme, and wherein said modifying enzyme comprises at least a in the following amino acid subsequence:

(a) KA Δ ₁₈F, wherein Δ ₁₈Be the amino acid of non-L-Ala;

(b) Q Δ ₁₉H, wherein Δ ₁₉The leucic amino acid of right and wrong;

(c) AP Δ ₁F, wherein Δ ₁The arginic amino acid of right and wrong, and especially leucine or halfcystine;

(d) F Δ ₂S, wherein Δ ₂It is leucine;

(e) Y Δ ₃G, wherein Δ ₃It is Isoleucine;

(f) Δ ₇IG, wherein Δ ₇Be Histidine or L-Ala;

(g) T Δ ₁₆G, wherein Δ ₁₆The leucic amino acid of right and wrong; With the YV Δ ₁₇G, wherein Δ ₁₇Be the amino acid of non-L-Ala;

(table 1B; Subsequence 1-10 and 18-19 and 16-17), and wherein said modifying enzyme further comprises at least a additional amino acid subsequence that is selected from following one group of amino acid subsequence:

(c) (d) Q Δ ₁₁S, wherein Δ ₁₁Be the amino acid of non-proline(Pro);

(d) (e) IGG Δ ₁₂, Δ wherein ₁₂Be the amino acid of non-Threonine;

(e) SWXL Δ ₁₃, Δ wherein ₁₃Be the amino acid of non-Serine;

(f) L Δ ₁₄Y, wherein Δ ₁₄Be the amino acid of non-l-asparagine; With

(g) G Δ ₁₅XGL, wherein Δ ₁₅Be the amino acid of non-tyrosine.

Another embodiment preferred of the present invention relates to a kind of nucleic acid molecule, it comprises that separation has the nucleotide sequence of the active modifying enzyme of proporphyrinogen oxidase (protox) from the coding of a kind of plant, wherein said modifying enzyme tolerates the inhibitor of naturally occurring protox enzyme, and wherein said modifying enzyme comprises: amino acid subsequence Δ ₇IG, wherein Δ ₇Be the amino acid of non-tyrosine; Amino acid subsequence IGG Δ ₁₂, Δ wherein ₁₂Be the amino acid of non-Threonine; With amino acid subsequence SWXL Δ ₁₃, Δ wherein ₁₃Be the amino acid of non-Serine.Δ most preferably ₇Be Isoleucine, Δ ₁₂Be Isoleucine and Δ ₁₃It is leucine.

Another embodiment preferred of the present invention relates to a kind of nucleic acid molecule, it comprises that separation has the nucleotide sequence of the active modifying enzyme of proporphyrinogen oxidase (protox) from the coding of a kind of plant, wherein said modifying enzyme tolerates the inhibitor of naturally occurring protox enzyme, and the feature of wherein said nucleotide sequence further is to satisfy at least a in the following condition:

(a) described nucleotide sequence has coded amino acid subsequence AP Δ ₁The sequence of F, wherein Δ ₁It is leucine;

(b) described nucleotide sequence has coded amino acid subsequence F Δ ₂The sequence of S, wherein Δ ₂It is leucine;

(c) described nucleotide sequence has coded amino acid subsequence Y Δ ₃The sequence of G, wherein Δ ₃It is Isoleucine;

(d) described nucleotide sequence has coded amino acid subsequence Δ ₇The sequence of IG, wherein Δ ₇Be L-Ala or Histidine;

(e) described nucleotide sequence has coded amino acid subsequence Y Δ ₃The sequence of G, wherein Δ ₃Be the amino acid of non-L-Ala, and preferred halfcystine or Isoleucine, more preferably Isoleucine; And described nucleotide sequence also has the sequence that coding one of is selected from following one group of subsequence:

(1) subsequence Q Δ ₁₁S, wherein Δ ₁₁Be the amino acid of non-proline(Pro);

(2) subsequence IGG Δ ₁₂, Δ wherein ₁₂Be the amino acid of non-Threonine;

(3) subsequence SWXL Δ ₁₃, Δ wherein ₁₃Be the amino acid of non-Serine;

(4) subsequence L Δ ₁₄Y, wherein Δ ₁₄Be the amino acid of non-l-asparagine; With

(5) subsequence G Δ ₁₅XGL, wherein Δ ₁₅Be the amino acid of non-tyrosine;

(f) described nucleic acid molecule has coded amino acid subsequence Δ ₇The sequence of IG, wherein Δ ₇Be the amino acid of non-junket, and preferred Threonine, L-Ala or Histidine, more preferably L-Ala or Histidine and most preferred group propylhomoserin; And described nucleic acid molecule also has the sequence of coding one of in following one group of subsequence:

(5) subsequence G Δ ₁₅XGL, wherein Δ ₁₅Be the amino acid of non-tyrosine; With

(g) described nucleic acid molecule has coded amino acid subsequence T Δ ₁₆The sequence of G, wherein Δ ₁₆The leucic amino acid of right and wrong; And described nucleotide sequence also has coded amino acid subsequence YV Δ ₁₇The sequence of G, wherein Δ ₁₇Be the amino acid of non-L-Ala;

(h) KA Δ ₁₈F, wherein Δ ₁₈Be the amino acid of non-L-Ala;

(i) Q Δ ₁₉H, wherein Δ ₁₉The leucic amino acid of right and wrong.

Preferred described nucleotide sequence has coded amino acid subsequence T Δ ₁₆The sequence of G, wherein Δ ₁₆The leucic amino acid of right and wrong; And described nucleotide sequence also has coded amino acid subsequence YV Δ ₁₇The sequence of G, wherein Δ ₁₇Be the amino acid of non-L-Ala.Preferred described nucleotide sequence has coded amino acid subsequence T Δ ₁₆The sequence of G, wherein Δ ₁₆It is Serine; And described nucleotide sequence also has coded amino acid subsequence YV Δ ₁₇The sequence of G, wherein Δ ₁₇It is Threonine.

Also preferred a kind of dna molecular, its coding comprises the proporphyrinogen oxidase (protox) of the modification of plant protox, wherein the arginine that occurs on the position that is equivalent to SEQ ID NO:6 the 88th amino acids is by another kind of aminoacid replacement, and the tolerance of wherein said modification can suppress the weedicide of the active consumption of naturally occurring protox.Particularly preferably be arginine by the dna molecular of leucine or halfcystine replacement.

Also preferred a kind of dna molecular, its coding comprises the proporphyrinogen oxidase (protox) of the modification of plant protox, the halfcystine that wherein is equivalent to occur on the position of SEQ ID NO:6 the 159th amino acids is replaced by leucine.

Also preferred a kind of dna molecular, its coding comprises the proporphyrinogen oxidase (protox) of the modification of plant protox, the L-Ala that wherein is equivalent to occur on the position of SEQ ID NO:6 the 175th amino acids is by another kind of aminoacid replacement, and the tolerance of wherein said modification can suppress the weedicide of the active consumption of naturally occurring protox.Particularly preferably be L-Ala by the dna molecular of Xie Ansuan or Threonine replacement.

Also preferred a kind of dna molecular, its coding comprises the proporphyrinogen oxidase (protox) of the modification of plant protox, the leucine that wherein is equivalent to occur on the position of SEQ ID NO:6 the 337th amino acids is by another kind of aminoacid replacement, and the tolerance of wherein said modification can suppress the weedicide of the active consumption of naturally occurring protox.Particularly preferably be the dna molecular that leucine is replaced by Serine.

Also preferred a kind of dna molecular, its coding comprises the proporphyrinogen oxidase (protox) of the modification of plant protox, the tyrosine that wherein is equivalent to occur on the position of SEQ ID NO:16 the 428th amino acids is replaced the preferred group propylhomoserin by Histidine or L-Ala.

Also preferred a kind of dna molecular, its coding comprises the proporphyrinogen oxidase (protox) of the modification of plant protox, and the L-Ala that wherein is equivalent to occur on the position of SEQ ID NO:2 the 220th amino acids is replaced, preferably replaced by Isoleucine by Isoleucine or tyrosine.

Also preferred a kind of dna molecular, its coding comprises the proporphyrinogen oxidase (protox) of the modification of plant protox, the tyrosine that wherein is equivalent to occur on the position of SEQ ID NO:2 the 426th amino acids is replaced by L-Ala.

Also preferred a kind of dna molecular, its coding comprises the proporphyrinogen oxidase (protox) of modification of plant protox, and the leucine that wherein is equivalent to occur on the position of SEQ ID NO:6 the 347th amino acids is replaced by Serine and the L-Ala that is equivalent to occur on the position of SEQ ID NO:6 the 453rd amino acids is replaced by Threonine.

The present invention further discloses a kind of dna molecular, its coding comprises the proporphyrinogen oxidase (protox) of the modification of plant protox, and described plant protox has the replacement of first seed amino acid and second seed amino acid replaces; First seed amino acid replaces the characteristic with generation inhibitor of anti-protox; And second seed amino acid replace have that raising is replaced by first seed amino acid and especially coding comprise the characteristic of the tolerance that the dna molecular of proporphyrinogen oxidase (protox) of the modification of plant protox produces; Wherein said plant is selected from corn, wheat, soybean, cotton, beet, rape, rice, Chinese sorghum, sugarcane and mouse ear mustard and belongs to the group of forming.Preferably a kind of dna molecular, wherein the replacement of first seed amino acid occurs on the position that is selected from following one group of position:

(a) be equivalent to the arginic position of SEQ ID NO:6 the 88th amino acids;

(b) be equivalent to the position of the L-Ala of SEQ ID NO:6 the 175th amino acids; With

(c) be equivalent to the leucic position of SEQ ID NO:6 the 337th amino acids.

Particularly preferably be a kind of dna molecular, wherein the replacement of first seed amino acid occurs on the position that is selected from following one group of position:

(a) be equivalent to the arginic position of SEQ ID NO:6 the 88th amino acids;

(b) be equivalent to the position of the L-Ala of SEQ ID NO:6 the 164th amino acids;

(c) be equivalent to the position of the glycine of SEQ ID NO:6 the 165th amino acids;

(d) be equivalent to the position of the tyrosine of SEQ ID NO:6 the 370th amino acids;

(e) be equivalent to the position of the halfcystine of SEQ ID NO:6 the 159th amino acids;

(f) be equivalent to the position of the Isoleucine of SEQ ID NO:6 the 419th amino acids;

(g) be equivalent to the position of the Xie Ansuan of SEQ ID NO:10 the 356th amino acids;

(h) be equivalent to the position of the Serine of SEQ ID NO:10 the 421st amino acids;

(i) be equivalent to the position of the Xie Ansuan of SEQ ID NO:10 the 502nd amino acids;

(j) be equivalent to the position of the L-Ala of SEQ ID NO:10 the 211st amino acids;

(k) be equivalent to the position of the glycine of SEQ ID NO:10 the 212nd amino acids;

(l) be equivalent to the position of the Isoleucine of SEQ ID NO:10 the 466th amino acids;

(m) be equivalent to the position of the proline(Pro) of SEQ ID NO:12 the 369th amino acids;

(n) be equivalent to the position of the L-Ala of SEQ ID NO:12 the 226th amino acids;

(o) be equivalent to the position of the tyrosine of SEQ ID NO:12 the 432nd amino acids;

(p) be equivalent to the position of the Xie Ansuan of SEQ ID NO:12 the 517th amino acids;

(q) be equivalent to the position of the tyrosine of SEQ ID NO:16 the 428th amino acids;

(r) be equivalent to the position of the proline(Pro) of SEQ ID NO:16 the 365th amino acids;

(s) be equivalent to the position of the tyrosine of SEQ ID NO:18 the 449th amino acids;

(t) be equivalent to the position of the L-Ala of SEQ ID NO:6 the 175th amino acids; With

(u) be equivalent to the leucic position of SEQ ID NO:6 the 337th amino acids; And

Wherein the replacement of second seed amino acid occurs on the position that is selected from following one group of position:

(a) be equivalent to the position of the Serine of SEQ ID NO:2 the 305th amino acids;

(b) be equivalent to the position of the Threonine of SEQ ID NO:2 the 249th amino acids;

(c) be equivalent to the position of the proline(Pro) of SEQ ID NO:2 the 118th amino acids;

(d) be equivalent to the position of the l-asparagine of SEQ ID NO:2 the 425th amino acids; With

(e) be equivalent to the position of the tyrosine of SEQ ID NO:2 the 498th amino acids.

Preferred especially a kind of nucleic acid molecule, wherein the replacement of first seed amino acid occurs on the position that is selected from following one group of position:

(a) be equivalent to the arginic position of SEQ ID NO:6 the 88th amino acids;

(c) be equivalent to the leucic position of SEQ ID NO:6 the 337th amino acids; And

Preferred especially a kind of dna molecular, wherein the arginine that occurs on the position that is equivalent to the 88th residue of SEQ ID NO:6 is replaced by halfcystine or leucine.

More preferably a kind of dna molecular, the halfcystine that wherein is equivalent to occur on the position of the 159th residue of SEQ ID NO:6 is replaced by leucine.

Preferred especially a kind of dna molecular, the L-Ala that wherein is equivalent to occur on the position of the 175th residue of SEQ ID NO:6 is replaced by being selected from Threonine or Xie Ansuan.

Preferred especially a kind of dna molecular, the leucine that wherein is equivalent to occur on the position of the 337th residue of SEQ ID NO:6 is replaced by Serine.

More preferably a kind of dna molecular, the tyrosine that wherein is equivalent to occur on the position of the 428th residue of SEQ ID NO:16 is replaced by Histidine or L-Ala.

The invention further relates to a kind of dna molecular, its coding comprises the proporphyrinogen oxidase (protox) of the modification of the plant protox with dual aminoacid replacement, and wherein the replacement of two seed amino acids is needed to protox inhibitor generation tolerance.Preferably a kind of coding comprises the dna molecular of the proporphyrinogen oxidase (protox) of the modification of plant protox, and wherein said plant is selected from corn, wheat, soybean, cotton, beet, rape, rice, Chinese sorghum, sugarcane and mouse ear mustard and belongs to the group of forming.More preferably a kind of coding comprises the dna molecular of the proporphyrinogen oxidase (protox) of the modification of plant protox, and wherein said plant is a corn.

Preferably a kind of dna molecular with dual aminoacid replacement, a kind of aminoacid replacement wherein takes place on the leucine position that is equivalent to SEQ IDNO:6 the 347th amino acids, and the replacement of second seed amino acid wherein takes place on the L-Ala position that is equivalent to SEQ ID NO:6 the 453rd amino acids.

Particularly preferably be a kind of dna molecular with dual aminoacid replacement, wherein replaced by Serine, and replaced by Threonine at the L-Ala that is equivalent to occur on SEQ ID NO:6 the 453rd amino acids position at the leucine that is equivalent to occur on SEQID NO:6 the 347th amino acids position.

The invention further relates to a kind of coding and comprise that the dna molecular of the proporphyrinogen oxidase (protox) of the modification of plant protox, wherein said plant protox comprise the aminoacid sequence that is selected from the group that SEQ ID NO:37 forms.

The invention further relates to expression cassette and the recombinant vectors that comprises this expression cassette, described expression cassette mainly comprises a kind of promotor, and especially a kind ofly in plant, having an active promotor that operationally is connected with a kind of dna molecular, described dna molecule encode the present invention is from the proporphyrinogen oxidase (protox) of most eukaryotes.Expression cassette of the present invention also further comprises the signal sequence that operationally is connected with described dna molecular, and wherein this signal sequence can be with protein directed importing chloroplast(id) or the plastosome by described dna molecule encode.

The present invention relates to a kind of mosaic gene, it comprises a kind of expression cassette, this expression cassette mainly comprises a kind of promotor, and especially a kind ofly in plant, having an active promotor that operationally is connected with a kind of allogeneic dna sequence DNA molecule, the present invention of described allogeneic dna sequence DNA molecule encoding is from the proporphyrinogen oxidase (protox) of most eukaryotes.Preferred a kind of mosaic gene, wherein said dna molecule encode is from the proporphyrinogen oxidase (protox) of sugarcane.

More preferably a kind of mosaic gene that has the active promotor that operationally is connected in the plant that is included in a kind of allogeneic dna sequence DNA molecule, wherein said allogeneic dna sequence DNA molecule encoding is from the proporphyrinogen oxidase (protox) of sugarcane, and it comprises the sequence of representing with SEQ ID NO:37.

Particularly preferably be a kind of mosaic gene, wherein said dna molecule encode has the active protein of protox-1 from sugarcane, and preferred wherein said protein comprises the sequence of representing with SEQ ID NO:37.

The present invention also comprises a kind of mosaic gene, it comprises a kind of expression cassette, this expression cassette mainly comprises a kind of promotor, and especially a kind ofly in plant, has an active promotor that operationally is connected with a kind of dna molecular, described dna molecule encode the present invention is from the proporphyrinogen oxidase (protox) of most eukaryotes, and this kind of enzyme tolerance can suppress the weedicide under the concentration of enzyme of corresponding unmodified form.Preferred a kind of mosaic gene, wherein said dna molecule encode proporphyrinogen oxidase (protox), this kind of enzyme are from a kind of plant that is selected from following one group of plant: mouse ear mustard genus, sugarcane, soybean, barley, cotton, tobacco, beet, rape, corn, wheat, Chinese sorghum, rye, oat, mud grass and forage grass, millet, forage grass and rice.More preferably a kind of mosaic gene, wherein said dna molecule encode is from the proporphyrinogen oxidase (protox) of a kind of plant, and described plant is selected from soybean, cotton, tobacco, beet, rape, corn, wheat, Chinese sorghum, rye, oat, forage grass and rice.Preferred especially a kind of mosaic gene, wherein said dna molecule encode is from the proporphyrinogen oxidase (protox) of a kind of plant, and described plant is selected from the group that mouse ear mustard genus, soybean, cotton, beet, rape, corn, wheat, Chinese sorghum and rice are formed.

The present invention includes a kind of mosaic gene, it is included in has the active promotor that operationally is connected with dna molecular of the present invention in the plant, described dna molecule encode of the present invention comprises the proporphyrinogen oxidase (protox) of the modification with at least a amino acid modified eucaryon protox, wherein said amino acid modified characteristic with generation to the tolerance of protox inhibitor.

The present invention also comprises a kind of mosaic gene, it is included in has the active promotor that operationally is connected with dna molecular of the present invention in the plant, described dna molecule encode of the present invention comprises the proporphyrinogen oxidase (protox) of the modification of plant protox, and described plant protox has the replacement of first seed amino acid and second seed amino acid replaces; The replacement of first seed amino acid has the characteristic of generation to the tolerance of protox inhibitor; And the replacement of second seed amino acid has raising is replaced the tolerance that produces by first seed amino acid characteristic.The mosaic gene that preferably also comprises the signal sequence that operationally is connected with described dna molecular, wherein said signal sequence can be with protein directed importing chloroplast(id) or the plastosome by described dna molecule encode.

Mosaic gene of the present invention can further include a kind of signal sequence that operationally is connected with dna molecular of the present invention, and wherein this signal sequence can be with the directed importing of the protein chloroplast(id) by described dna molecule encode.Mosaic gene of the present invention can further include a kind of signal sequence that operationally is connected with described dna molecular, and wherein this signal sequence can be with the protein orientation lead-in wire plastochondria by described dna molecule encode.

The present invention also comprises any DNA sequence that can stably be integrated into host genome mentioned above.

The invention further relates to the derivative of equivalence on a kind of recombinant DNA molecules that comprises plant proporphyrinogen oxidase of the present invention or its function.

The invention further relates to a kind of recombinant DNA carrier that comprises recombinant DNA molecules of the present invention.

Another object of the present invention is a kind of recombinant vectors that comprises mosaic gene of the present invention, and wherein this carrier can be transformed into host cell with being stabilized.

Another object of the present invention is a kind of recombinant vectors that comprises mosaic gene of the present invention, and wherein this carrier can be transformed into plant, plant seed, plant tissue or vegetable cell with being stabilized.The recombinant vectors that preferably comprises mosaic gene of the present invention, wherein this carrier can be transformed into plant with being stabilized.Can express the dna molecular of coding proporphyrinogen oxidase (protox) with plant, plant seed, plant tissue or the vegetable cell of this carrier stable conversion.Preferred a kind of recombinant vectors, wherein can express the dna molecular of coding, but this kind of enzyme tolerance inhibitory phase is answered the weedicide under the concentration of enzyme of unmodified form from the proporphyrinogen oxidase (protox) of a kind of plant with plant, plant seed, plant tissue or the vegetable cell of this carrier stable conversion.

Preferably a kind of recombinant vectors that comprises mosaic gene of the present invention, described mosaic gene of the present invention comprises a kind of active promotor that operationally is connected with the allogeneic dna sequence DNA molecule that has in plant, described allogeneic dna sequence DNA molecule encoding is from the proporphyrinogen oxidase of sugarcane, it comprises the sequence of representing with SEQ IDNO:37, and wherein said carrier can be transformed into host cell with being stabilized.

Also preferred a kind of recombinant vectors, it comprises mosaic gene of the present invention, this mosaic gene comprises a kind of active promotor that operationally is connected with dna molecular that has in plant, described dna molecule encode comprises the proporphyrinogen oxidase of the modification of plant protox, and described plant protox has the replacement of first seed amino acid and second seed amino acid replaces; Described a kind of aminoacid replacement has the tolerific characteristic of protox inhibitor; And described second seed amino acid replacement has the characteristic of raising by the tolerance of first seed amino acid replacement generation, and wherein said carrier can be transformed into vegetable cell with being stabilized.

The present invention also comprises the host cell of a kind of usefulness carrier stable conversion of the present invention, and wherein this host cell can be expressed described dna molecular.Preferably a kind of host cell that is selected from the group of vegetable cell, bacterial cell, yeast cell and insect cell composition.

The invention further relates to plant and filial generation, plant tissue and the plant seed of herbicide-resistant, described weedicide can be suppressed at naturally occurring protox activity in these plants, and wherein said tolerance is produced by the gene of the protox enzyme of the anti-inhibitor of expressing the modification of instructing as this paper.Representational plant is included as described purpose and any plant that can use these weedicides to it.Preferably important farm crop, be angiosperm and gymnosperm, such as mouse ear mustard genus, sugarcane, soybean, barley, cotton, tobacco, beet, rape, corn, wheat, Chinese sorghum, rye, oat, tomato, potato, mud grass and forage grass, millet, forage grass and rice etc.More preferably important farm crop, i.e. angiosperm and gymnosperm are such as mouse ear mustard genus, cotton, soybean, rape, beet, corn, rice, wheat, barley, oat, rye, Chinese sorghum, millet, mud grass, forage grass, forage grass.Particularly preferably be important farm crop, i.e. angiosperm and gymnosperm are such as mouse ear mustard genus, soybean, cotton, beet, rape, corn, wheat, Chinese sorghum and rice.

Preferably a kind of coding that comprises contains the plant of dna molecular of proporphyrinogen oxidase (protox) of the modification of plant protox, and described plant protox has that first seed amino acid replaces and the replacement of second seed amino acid; Described a kind of aminoacid replacement has the tolerific characteristic of protox inhibitor; And described second seed amino acid replaces and has raising and replace the characteristic of the tolerance that produces by first seed amino acid, and wherein said dna molecular can be expressed and make thus described plant to produce tolerance to the weedicide that can suppress the active consumption of naturally occurring protox in described plant.A preferred kind of plant, wherein said dna molecular have replaced corresponding naturally occurring protox encoding sequence.The present invention includes a kind of plant and filial generation thereof that comprises mosaic gene of the present invention, wherein this mosaic gene makes described plant produce tolerance to the weedicide that can suppress the active consumption of naturally occurring protox thus.

The present invention includes transgenic plant tissue, comprise the tissue of plant and filial generation, seed and cultivation with at least a mosaic gene stable conversion of the present invention.The transgenic plant tissue of at least a mosaic gene stable conversion of preferred a kind of usefulness, comprise the tissue of plant, seed and cultivation, described mosaic gene comprises a kind of expression cassette, this expression cassette mainly comprises a kind of promotor and especially a kind ofly has an active promotor that operationally is connected with dna molecular in plant, described dna molecule encode proporphyrinogen oxidase (protox), this enzyme tolerates the weedicide under the concentration that can suppress the enzyme of corresponding unmodified form in the plant tissue.

The invention further relates to plant, plant tissue, plant seed and the vegetable cell of herbicide-resistant, described weedicide can suppress naturally occurring protox activity in these plants, and wherein said tolerance produces by the expression that increases wild-type herbicide sensitive protox.This result causes the level of protox enzyme in the vegetable cell to be enough to overcome the growth-inhibiting that is produced by weedicide at least.The level of expressed enzyme general at least 2 times to, preferred at least 5 times to and most preferably at least 10 times to the amount of natural expression.Express to increase may be since in the multiple copied of wild-type protox gene, gene in the repeatedly appearance (being gene amplification) of encoding sequence or the vegetable cell sudden change in the non-coding and regulating sequence of endogenous gene cause.Use method as known in the art, by (for example in plant, carrying out plant that orthoselection can obtain to have the gene activity that this class changes, referring to U.S. Patent number US 5,162,602 and U.S. Patent number US 4,761,373 reach the reference of wherein quoting).Also can obtain these plants by gene engineering as known in the art.Can also be by increasing herbicide sensitive protox expression of gene with reorganization or chimeric dna molecule stable conversion vegetable cell, described reorganization or chimeric dna molecule comprise the promotor that operationally is connected with the homology or the allos structure gene of coding protox enzyme that can start dependency structure genetic expression in the vegetable cell.

Can recombinant DNA molecules of the present invention be imported vegetable cell according to the mode of prior art.It will be understood by those skilled in the art that and to depend on to be converted into the vegetation type of purpose, be i.e. monocotyledons or dicotyledons the selection of method.The suitable method of transformed plant cells comprises microinjection (Crossway etc., biotechnology (BioTechniques) 4:320-334 (1986)), electroporation (Riggs etc., periodical (Proc.Natl.Acad.Sci.USA) 83:5602-5606 (1986) of institute of the state-run academy of sciences of the U.S., conversion (the Hinchee etc. of Agrobacterium mediation, biotechnology (Biotechnology) 6:915-921 (1988)), direct gene shifts (Paszkowski etc., EMBO is (1984) J.3:2717-2722), use is available from Agracetus, Inc., Madison, Wisconsin and Dupont, Inc., Wilmington, impinging particle that the device of Delaware carries out quickens that (for example, referring to Sanford etc., U.S.Patent 4,945,050; With McCabe etc., biotechnology (Biotechnology) 6:923-926 (1988)), protoplast transformation/method of reproduction is (referring to the U.S. Patent number US 5 of Ciba-Geigy Corp. in mandate on September 27th, 1994,350,689) and pollen transform (referring to U.S. Patent number US 5,629,183).In addition referring to Weissinger etc., heredity year summary (Annual Rev.Genet.) 22:421-477 (1988); Sanford etc., particulate science and technology (Particulate Science andTechnology) 5:27-37 (1987) (onion); Christou etc., plant physiology (PlantPhysiol.) 87:671-674 (1988) (soybean); McCabe etc., biology/technology (Bio/Technology) 6:923-926 (1988) (soybean); Datta etc., biology/technology (Bio/Technology) 8:736-740 (1990) (rice); Klein etc., periodical (Proc.Natl.Acad.Sci.USA) 85:4305-4309 (1988) (corn) of institute of the state-run academy of sciences of the U.S.; Klein etc., biology/technology (Bio/Technology) 6:559-563 (1988) (corn); Klein etc., plant physiology (Plant Physiol.) 91:440-444 (1988) (corn); Fromm etc., biotechnology (Bio/Technology) 8:833-839 (1990); Gordon-Kamm etc., vegetable cell (Plant Cell) 2:603-618 (1990) (corn); And U.S. Patent number 5,591,616 and 5,679,558 (rice).

Comprise transgenic plant in the scope of the invention, particularly the transgenosis that transforms by aforesaid method can be educated plant and vegetative propagation and/or sexual propagation filial generation, and they can tolerate or tolerate at least the restraining effect that is produced by the weedicide under the active concentration of naturally occurring protox in the common inhibition plant.Progeny plant also comprises the plant that has with the different genetic backgrounds of mother plant, and these plants produce because of the program of backcrossing and still comprise Herbicid resistant feature of the present invention in its genome.Especially particularly preferably be and tolerate or tolerate at least the inhibiting hybrid plant that produces by the weedicide under the active concentration of naturally occurring protox in the common inhibition plant.

Transgenic plant of the present invention can be dicotyledons or monocotyledons.Preferably the monocotyledons of Gramineae (Graminaceae) comprises lolium, Zea, Triticum, Triticale, Sorghum, saccharum, Brome, Oryza, Avena, Hordeum, Secale and millet.More preferably transgenic corns, wheat, barley, Chinese sorghum, rye, oat, sugarcane, forage grass and forage grass, millet and rice.Particularly preferably be corn, wheat, Chinese sorghum, rye, oat, forage grass and rice.

In dicotyledons, this paper is mouse ear mustard genus, soybean, cotton, beet, rape, tobacco, tomato, potato and Sunflower Receptacle more preferably.Especially preferred is soybean, cotton, tobacco, beet, tomato, potato and rape.

To express term " filial generation " and be interpreted as the transgenic plant filial generation that comprises that " vegetative propagation " and " sexual propagation " produces.This definition is also in order to comprise by selecting such known method to obtain such as for example cytogamy or mutant and still showing all mutant of characteristic properties of initial plant transformed and all hybridization and the fusion product of variant and plant transformed material.Also comprise the progeny plant that is produced by the program of backcrossing, condition is that this progeny plant still contains Herbicid resistant characteristic of the present invention.

Another object of the present invention relates to the breeding material of transgenic plant.The breeding material that will be referred to transgenic plant of the present invention is defined as can be in vivo or external any plant material of breeding by sexual propagation or vegetative propagation mode.Particularly preferably be protoplastis, cell, callus, tissue, organ, seed, embryo, pollen, ovum, zygote in the scope of the invention and available from any other breeding materials of transgenic plant.

Another object of the present invention relates to the position of plant, such as: for example derive from advance by the inventive method and transform and thus by flower, stem, fruit, leaf, the root of the transgenic plant of forming to the small part transgenic cell or its filial generation.

Another object of the present invention is a kind of plant, protoplastis, cell, callus, tissue, organ, seed, embryo, pollen, ovum, zygote and the production method of other breeding material, plant parts arbitrarily, wherein said plant parts is such as the flower, stem, fruit, leaf, the root that for example derive from advance by the inventive method transgenic plant transformed or its filial generation, and plant, the plant parts that this method has a DNA of the present invention by conversion thus produced the anti-inhibitor form of plant protox enzyme.Preferably a kind of production method that comprises the host cell of isolated DNA molecule, described isolated DNA molecule coding has the active protein of proporphyrinogen oxidase (protox) from eukaryotic, and this method comprises the step that transforms described host cell with recombinant carrier molecule of the present invention.Further preferably a kind of production method that comprises the vegetable cell of isolated DNA molecule, described isolated DNA molecule coding has the active protein of proporphyrinogen oxidase (protox) from eukaryotic, and this method comprises the step that transforms described vegetable cell with recombinant carrier molecule of the present invention.The production method of the transgenosis filial generation of preferably a kind of transgenosis mother plant that comprises isolated DNA molecule, described isolated DNA molecule coding has the active protein of proporphyrinogen oxidase (protox) from eukaryotic, and this method comprises with recombinant carrier molecule of the present invention and transforms described mother plant and the characteristic of herbicide-resistant is changed over to the step of the filial generation of the described transgenosis mother plant that relates to known plant propagation technology.

The production method of a kind of plant, plant tissue, plant seed and plant parts preferably, this method produces the anti-inhibitor form of plant protox enzyme, has wherein stably transformed described plant, plant tissue, plant seed and plant parts with the structure gene of the resistance protox enzyme of encoding.Particularly preferably be the production method of a kind of plant, plant tissue, plant seed and plant parts, wherein stably transformed described plant, plant tissue, plant seed and plant parts with DNA of the present invention.Especially preferred is the production method of a kind of plant, plant tissue, plant seed and plant parts, this method produces the anti-inhibitor form of plant protox enzyme, wherein prepared described plant, plant tissue, plant seed and plant parts, made the strain of herbicide-resistant obtain separation, qualitative and growth thus by the orthoselection technology.

By sexual propagation or nourish and grow will design hereditary property pass to above-mentioned transgenic seed and plant and can in progeny plant, keep and transmit these characteristics thus.In general, this keep and transmit made full use of known exploitation agricultural method to meet specific purpose, such as cultivating, sow or gathering in the crops.Can also use such as water planting or the such specific method of greenhouse technology.When the farm crop in the growth are subject to the competition of the erosion that caused by insect or infection and destruction and ruderal plant, take measures to improve output with control weeds, Plant diseases, insect, nematode and other unfavourable condition.These measures comprise such as tilling the land or removing the such mechanical measure of weeds and infection plant and use such as the such agrochemicals of weedicide, mycocide, gametocide, nematocides, growth regulator, maturing agent and sterilant.

The favourable hereditary property of transgenic plant of the present invention and seed can be further used in the plant cultivation, and its purpose is to develop to be had such as the output of anti-insect pest, herbicide-resistant or the withstand voltage nutritive value of compeling, improving, increase or hardly can be because of this plant of improving characteristic of the lodging or the damnous improvement structure of falling leaves.Various breeding steps are characterised in that the human intervention of fully determining, such as the direct pollination of the strain of selecting hybridization, parental line or select suitable progeny plant.According to the difference of desired characteristic, adopt different breeding measures.Correlation technique is well-known in the art and includes but not limited to hybridization, inbreeding, back cross breeding, multi-thread breeding, mutation mixing, species hybridization, aneuploid technology etc.Hybridization technique also comprises makes by machinery, chemistry or biochemical method that plant is sterile to be produced male or female sterile plants.The male sterile plants that has different strain pollen is carried out that cross-pollination can be guaranteed male sterile and female performance is educated the genome of plant and obtained two kinds of parental line characteristics equably.Therefore, transgenic seed of the present invention and plant can be used to breed the plant lines of improvement, these strains for example can increase such as the validity of weedicide or the such ordinary method of pesticide treatments or make that omitting described method because of the hereditary property of its change becomes possibility.On the other hand, because of optimizing heredity " equipment " can obtain to have the novel farm crop of the withstand voltage urgent characteristic of improvement, their make product of results have the quality that is better than tolerating quite bad developmental condition.

In the seed production process, the quality of germination and the homogeneity of seed are main product features, and unimportant by the quality and the homogeneity of the farmer results and the rudiment of selling.When being difficult to keep a kind of farm crop not contain other farm crop and weed seed, in order to control the disease that seed has and to produce well seeds germinated, seed producers has been developed quite extensively and the seed production measure of fully determining, these seed producers have the experience that makes purebred son growth in this area, regulates and sell.Therefore, be to buy the seed of the calibrating satisfy the extra fine quality standard rather than use the seed of from its oneself farm crop, gathering in the crops for the common measure of farmer.Usually will handle with the protective material coating that comprises weedicide, sterilant, mycocide, sterilant, nematocides, invertebrate poison or its mixture as the breeding material of seed.Comprise such as Vancide 89, carboxin, thiram (TMTD in the normally used protective material coating ^), methalaxyl (Apron ^) and pirimiphosmethyl (Actellic ^) such compound.If desired, these compounds adjuvant that normally used other carrier, tensio-active agent or promotion are used in formulation art is prepared so that the infringement that prevents to be caused by bacterium, fungi or animal pest.Can be by being coated with the coating protectant layer with liquid preparation infiltration breeding material or by wet or dry preparation with merging.Also can use other application process, such as directly bud or fruit being handled.

Therefore, another object of the present invention provides the plant propagation material that is used for cultivated plant, and the plant seed of especially handling with the seed protectant coating that is generally used for seed treatment.

Another aspect of the present invention provides such as the such novel agricultural method of above-mentioned typical method, it is characterized in that using transgenic plant of the present invention, transgenic plant material or transgenic seed.The present invention includes a kind of agricultural method, wherein use transgenic plant or its filial generation comprise mosaic gene of the present invention, the amount of described mosaic gene is enough to express the herbicidal target albumen of Herbicid resistant form in the plant and produces tolerance to weedicide.

For by the inventive method plant transformed breeding filial generation, can use:, make the maize plant described in the following embodiment be grown in the jar in the greenhouse or in the soil and it is bloomed as known in the art such as method described below.Pollen is available from ripe male flower fringe and be used for the pollination of the fringe of identical plant, plant born of the same parents or any required maize plant.Similarly can be by give the fringe pollination of growing on the plant available from the pollen of identical plant, plant born of the same parents or any required maize plant transforming.The filial generation that transforms by this method can be different from unconverted filial generation because of having the gene that imports and/or the DNA (genotype) of companion or the phenotype that produces.The filial generation that transforms similarly selfing or with other plant hybridization, as usually with the plant of carrying desired characteristic arbitrarily carry out the same.Similarly, the tobacco that is produced by present method or other plant transformed can selfings or are hybridized as known in the art and produce the filial generation with desired characteristic.Method is cultivated other transgenic organism by method known in the art and combination results of the present invention so that generation has the filial generation of desired characteristic as known in the art similarly.

The protox enzyme of the anti-inhibitor that the present invention modifies and naturally occurring counterpart (promptly in plant natural existence and not directly by DNA recombination method or indirect by manned inhibitor sensitive forms such as selection breedings) are compared has a kind of aminoacid replacement, interpolation or disappearance at least.Can change with the anti-inhibitor form that produces the protox enzyme or the amino acid position that improves the inhibitor resistance shown in the boldface letter among the table 1A, thus, plant protox-1 sequence is from mouse ear mustard genus, corn, soybean, cotton, beet, rape, rice, Chinese sorghum, wheat and sugarcane.One skilled in the art will realize that can be to having that any plant protox gene that fully is similar to the enzyme sequence of protox shown in this paper carries out identical change so that those amino acid of modifying according to the present invention are carried out the sequence contrast and identify, thereby produce the anti-inhibitor form of described enzyme.The standard technique described in disclosed obtains the protox gene of the additional plant of this class as WO95/346592 can to use the international application no PCT/IB95/0045 that submits to June 8 nineteen ninety-five, December 21 nineteen ninety-five, and the relevant portion of the document is incorporated herein by reference.

The dna molecular of the Herbicid resistant protox encoding sequence that the code book culture and education can be led carries out genetic modification so that carry out optimum expression in crop plants.This process can comprise the step of the encoding sequence that changes the resistance allele that is used for the variety of crops optimum expression of paying close attention to.Be used for changing the method that obtains the encoding sequence of optimum expression at specific variety of crops and be well-known (for example, referring to Perlak etc., periodical (Proc.Natl.Acad.Sci.USA) 88:3324 (1991) of institute of the state-run academy of sciences of the U.S.; Koziel etc., biotechnology (Bio/technol.) 11:194 (1993)).

The process that the protox encoding sequence that is used for optimum expression is carried out genetic modification can also comprise the step of the proper regulation sequence that is operably connected (being promotor, signal sequence, transcription terminator).The promotor example that can work in plant or vegetable cell (being that those can start in the vegetable cell promotor such as the such dependency structure expression of gene of protox) comprising: cauliflower mosaic virus (CaMV) 19S or 35S promoter and CaMV double-promoter; The nopaline synthase promoter; (PR) bak promoter relevant with pathology; The little subunit of carboxydismutase (ssuRUBISCO) promotor; Heat shock protein promotor (hsp80 promotor) from the Btassica of reference EPA 0 559 603; Mouse ear mustard belongs to actin promoter and with reference to the SuperMas promotor of WO 95/14098 etc.Preferred promotor is that those produce the promotor of high-level constitutive expression or more preferably those produce the promotor of specificity high level expression in the tissue that is subject to the weedicide infringement.Preferred promotor be the rice actin promoter (McElroy etc., molecular gene heredity (Mol.Gen.Genet.) 231:150 (1991)), (EP 0,342 926 for corn ubiqutin promotor; Taylor etc., vegetable cell report (Plant Cell Rep.) 12:491 (1993)) and from tobacco, mouse ear mustard belong to or the PR-1 promotor of corn (referring to the U.S. Patent number US 5 of Ryals etc., 614,395, with the full content of the document be incorporated herein the most with reference to).Can modify promotor self so that handle according to the step of prior art and increase protox expression promoter intensity.

The present inventor has found that also the another kind of preferred promotor that the protox encoding sequence with anti-inhibitor uses is that the promotor that is connected with natural protox gene (is the protox promotor; Not aesthetic state application number 08/808 referring to co-applications, 323, its title is " from the promotor of proporphyrinogen oxidase gene " (" Promoters from Protoporphyrinogen OxidaseGenes "), with the full content of the document be incorporated herein the most with reference to).The promoter sequence that belongs to the protox-1 gene from mouse ear mustard is represented with SEQ ID NO:13, promoter sequence from corn protox-1 gene is represented with SEQ ID NO:14, and is represented with SEQ ID NO:26 from the promoter sequence of beet protox-1 gene.

Because the protox promotor self is suitable for expressing the protox encoding sequence of anti-inhibitor, so directly the natural protox gene that is present in the vegetable cell genome is carried out the modification of this paper instruction and do not need to make up the mosaic gene with allos adjusting sequence.This class is modified can be by carrying out such as the such directed mutagenesis technology of homologous recombination and being that the basis is selected (for example, referring to embodiment 10 with the herbicide-resistant phenotype that produces; Pazkowski etc., EMBO are (1988) J.7:4021-4026; With U.S. Patent number 5,487,992, particularly 18-19 hurdle and embodiment 8).The attendant advantages of this means is that except that containing natural protox promotor the modifying factor of gained also comprises any other regulatory element that belongs to the part natural gene, such as the encoding sequence of signal peptide or transit peptides.

In the situation that transforms the nuclear gene group, the protox encoding sequence in signal peptide or transit peptides and the chimeric DNA construct of the present invention is merged so that the protox enzyme of expressing directly is transported to the position of required effect.The example of signal peptide comprises those and such protein natural signal peptide of being connected relevant with plant pathology such as for example PR-1, PR-2 etc.For example, referring to Payne etc., molecular biology of plants (Plant Mol.Biol.) 11:89-94 (1988).The example of transit peptides comprise such as those as Von Heijne etc. molecular biology of plants report (Plant Mol.Biol.Rep.) 9:104-126 (1991), Mazur etc. plant physiology (Plant Physiol.) 85:1110 (1987), Vorst etc. those chloroplast transit peptides described in gene (Gene) 65:59 (1988) and such as Boutry etc. at those mitochondrial transport peptides described in nature (Nature) 328:340-342 (1987).If the protox enzymic activity generally appears in plastosome and the chloroplast(id), what paid close attention to so is useful especially in the present invention chloroplast(id) and mitochondrial transport peptide.If pay close attention to restraining effect to protox enzymic activity in the chloroplast(id) and be the main basis of the effect of the weedicide that suppresses protox, that so most preferably use is chloroplast transit peptides (Witkowski and Halling, plant physiology (Plant Physiol.) 87:632 (1988); Lehnen etc., agricultural chemicals biochemical physiology (Pestic.Biochem.Physiol.) 37:239 (1990); Duke etc., WeedSci.39:465 (1991)).Also comprise and make encoded protein matter in sequence such as the so different cell cell positions of vacuole.For example, referring to: Neuhaus etc., institute of the state-run academy of sciences of the U.S. prints (Proc.Natl.Acad.Sci.USA) 88:10362-10366 (1991); And Chrispeels, Ann.Rev.Plant Physiol.Plant Mol.Biol.42:21-53 (1991).Whole disclosures that these public publications are relevant are incorporated herein by reference.

Mosaic gene of the present invention can multiple copied the protox structure gene that contains promotor or multiple copied.In addition, this construct can comprise the encoding sequence that is used for mark and be used for adding the encoding sequence of peptide class like this such as signal peptide or transit peptides that each in these peptides all is arranged in the suitable frame of other functional element with dna molecular.The preparation of this class construct belongs to those of ordinary skills' level.

Useful mark comprises the peptide class that can produce weedicide, microbiotic or drug resistance, such as, the peptide class of hygromycin resistance, kantlex, G418, gentamicin, lincomycin, methotrexate, glyphosate, phosphinothricin etc. for example.These marks can be used for selecting chimeric DNA construct cell transformed of the present invention from no transformed cells.Other useful mark is the peptase that can detect easily by the so visible reaction of for example color reaction, for example luciferase, β-glucuronidase or beta-galactosidase enzymes.

The positive system of selection of genetic transformation cell that will be described in WO 94/20627 is incorporated herein by reference, can import a kind of required nucleotide sequence in the cell of wherein said genetic transformation, this nucleotide sequence can produce the transformant with selectivity advantage.

If by crop plants or can be obtained Herbicid resistant protox allelotrope by the orthomutation of natural gene in the plant cell cultures of its crop plants of regenerating, this equipotential gene can be changed over to so in the commercial variety that uses traditional breeding technology to obtain does not need the encoding sequence of modifying is carried out genetic modification and it is transformed into plant so that cultivate a kind of farm crop of herbicide-resistant.On the other hand, herbicide resistance gene can be separated, genetic modification to be to be used for optimum expression and to be transformed into required kind then.

The gene of the protox of the inhibitor of anti-protox that coding can also be changed is as the selective marker in the vegetable cell method for transformation.For example, can also can be by the gene transformation of the protox of the change of expression of plants with transgenosis plant transformed, plant tissue or vegetable cell with coding.Change cell transformed thus over to contain the protox inhibitor substratum, wherein only cell transformed can be survived.The Protox inhibitor of being paid close attention to that is particularly useful as selective agent is: diphenyl ether for example acifluorfen, i.e. 5-[2-chloro-4-(trifluoromethyl) phenoxy group]-the 2-nitrobenzoic acid; Its methyl esters; Or oxyfluorfen, i.e. 2-chloro-1-(3-oxyethyl group-4-nitrophenoxy)-4-(trifluoro-benzene) }; Oxidiazoles (oxidiazon for example, i.e. 3-[2,4-two chloro-5-(1-methyl ethoxy) phenyl]-5-(1, the 1-dimethyl ethyl)-1,3,4-oxadiazole-2-(3H)-ketone); Cyclic imide class (S-23142 for example, i.e. N-(4-chloro-2-fluoro-5-propargyloxy phenyl)-3,4,5,6-tetrahydrochysene phthalimide; Chlorophthalim, i.e. N-(4-chloro-phenyl-)-3,4,5,6-tetrahydrochysene phthalimide); Phenyl pyrazoles (TNPP-ethyl for example, ethyl 2-[1-(2,3, the 4-trichlorophenyl)-4-nitropyrazole base-5-oxygen base] ethyl propionate; M﹠amp; B 39279); Pyridine derivate (for example LS 82-556); With phenopyrate and O-Phenylpyrrolidine thereof also-and similar thing of piperidino-(1-position only) carbamate and two ring triazolinones (as disclosed among International Patent Application WO 92/04827, the EP 532146).

This method be used for coding protox that can enough changes gene transformation any vegetable cell and can use with any transgenosis of paying close attention to.Start transgenosis and protox expression of gene by identical promoters that vegetable cell is worked or by independent promotor.

The protox enzyme tolerance of the anti-inhibitor of modification of the present invention can suppress the active weedicide of naturally occurring protox.The weedicide that suppresses protox comprises molecule (Duke etc., the Weed Sci.39:465 (1991) of many different types of structure; Nandihalli etc., agricultural chemicals biochemical physiology (Pesticide Biochem.Physiol.) 43:193 (1992); Matringe etc., FEBS Lett.245:35 (1989); Yanase and Andoh, agricultural chemicals biochemical physiology (Pesticide Biochem.Physiol.) 35:70 (1989)), comprise diphenyl ether for example acifluorfen, i.e. 5-[2-chloro-4-(trifluoromethyl) phenoxy group]-the 2-nitrobenzoic acid; Its methyl esters; Or oxyfluorfen, i.e. 2-chloro-1-(3-oxyethyl group-4-nitrophenoxy)-4-(trifluoro-benzene) }; Oxidiazoles (oxidiazon for example, i.e. 3-[2,4-two chloro-5-(1-methyl ethoxy) phenyl]-5-(1, the 1-dimethyl ethyl)-1,3,4-oxadiazole-2-(3H)-ketone); Cyclin imide class (S-23142 for example, i.e. N-(4-chloro-2-fluoro-5-propargyloxy phenyl)-3,4,5,6-tetrahydrochysene phthalimide; Chlorophthalim, i.e. N-(4-chloro-phenyl-)-3,4,5,6-tetrahydrochysene phthalimide); Phenyl pyrazoles (TNPP-ethyl for example, ethyl 2-[1-(2,3, the 4-trichlorophenyl)-4-nitropyrazole base-5-oxygen base] ethyl propionate; M﹠amp; B 39279); Pyridine derivate (for example LS82-556); With phenopyrate and O-Phenylpyrrolidine thereof also-and the similar thing of piperidino-(1-position only) carbamate.

The diphenyl ether of particularly important is those compounds with following general formula:

(general formula I) wherein R be-COONa (general formula I I) ,-CONHSO ₂CH ₃(general formula III) or-COOCH ₂COOC ₂H ₅(general formula I V; Referring to Maigrot etc., Brighton crop protection meeting-weeds (Brighton CropProtection Conference-Weeds): 47-51 (1989)).

Other diphenyl ether of being paid close attention to is that those R are the compound of following groups:

(general formula I Va; Referring to Hayashi etc., Brighton crop protection meeting-weeds (BrightonCrop Protection Conference-Weeds): 53-58 (1989)).

The another kind of phenyl ether of being paid close attention to is a kind of compound with following general formula: (general formula I Vb; Bifenox is referring to Dest etc., northeast weeds science proceeding (Proc.Northeast Weed Sci.Conf.) 27:31 (1973)).

The another kind of phenyl ether of being paid close attention to is a kind of compound with following general formula:

(general formula I Vc; Oxyfluorfen; Referring to Yih and Swithenbank, agricultural food product The Chemicals (J.Agric.Food Chem.), 23:592 (1975)).

The another kind of phenyl ether of being paid close attention to is a kind of compound with following general formula: (general formula I Vd; Lactofen referring to " The Pesticide Manual " the 623rd page, the 10th edition, is edited the Brighton crop protection council (British CropProtection Council), Surrey (1994) by C.Tomlin).

Other importantly is called the class weedicide with following general formula of acid imide:

(general formula V)

Wherein Q is: (general formula VI) or (general formula VII) or

(general formula VIII) or

(general formula I X) or

(general formula I Xa) or

(general formula I Xb) is (referring to (1995) such as Hemper described in " collection of thesis of the 8th international chemistry of pesticide meeting (Proceedings of the Eighth International Congress of PesticideChemistry) "; Ragdale etc., editor, american chemical association (Amer.Chem.Soc), Washington, D.C., 42-48 page or leaf (1994)); And R ₁Be H, Cl or F, R ₂Be Cl and R ₃Be to replace or not substituted ether, thioether, ester, amino or alkyl.On the other hand, R ₂And R ₃Form 5 or 6 yuan of heterocycles each other.The example of the imide weedicide of special concern is:

(general formula VIIa; Fluthiacet-methyl is referring to Miyazawa etc., Brighton crop protection meeting-weeds (Brighton Crop Protection Conference-Weeds), 23-28 page or leaf (1993))

(the general formula X sulfentrazone is referring to Van Saun etc., Brighton crop protection meeting-weeds (Brighton Crop Protection Conference-Weeds), 77-82 page or leaf (1991)).

(general formula X I)

(general formula X II) is (referring to Miura etc., Brighton crop protection meeting-weeds (Brighton Crop ProtectionConference-Weeds): 35-40 (1993)) (general formula X III) (general formula X IV)

(general formula X V) (general formula X VI)

The herbicidal activity of above-claimed cpd is described in the following document: 1991 Brighton crop protection proceedings (Proceedings of the 1991 Brighton Crop ProtectionConference), weeds (Weeds) (Britain crop protection association) (general formula X and XVI); 1993 Brighton crop protection meeting papers (Proceedings of the 1993 BrightonCrop Protection Conference), weeds (Weeds) (the Britain crop protection council) (general formula X II and XIII); U.S. Patent number US4,746,352 (general formula X I); With the 33rd in digest of U.S. weeds scientific institution (Abstracts of the Weed Science Societyof America vol.33), the 9th page of (1993) (general formula X IV).

Most preferred imide weedicide is those compounds that are categorized as aryi-uracile and have following general formula: (general formula X VII) wherein R represents as disclosed group (C among the U.S. Patent number US 5,183,492 _2-6-alkenyl oxy) carbonyl-C _1-4-alkyl is incorporated herein by reference the document.

Other importantly has the weedicide of following general formula:

(general formula X VIII; Thiadiazimin)

(referring to Weiler etc., Brighton crop protection meeting-weeds (BrightonCrop Protection Conference-Weeds), 29-34 page or leaf (1993));

(general formula X IX; Carfentrazone)

(referring to Van Saun etc., Brighton crop protection meeting-weeds (BrightonCrop Protection Conference-Weeds): 19-22 page or leaf (1993)); The pyrazoles that the N-of following general formula replaces:

(general formula X X) be R wherein ₁Be the C that can be replaced by one or more halogen atoms or do not replaced by them ₁-C ₄-alkyl;

R ₂Be hydrogen or C ₁-C ₄-alkoxyl group, they can be replaced by one or more halogen atoms separately or not replaced by them; Or

R ₁And R ₂Form group-(CH together ₂) _n-X-, wherein X is bonded to R ₂On;

R ₃It is hydrogen or halogen;

R ₄Be hydrogen or C ₁-C ₄-alkyl;

R ₅Be hydrogen, nitro, cyano group or group-COOR ₆Or-CONR ₇R ₈And

R ₆Be hydrogen, C ₁-C ₆-alkyl, C ₂-C ₆-alkenyl or C ₂-C ₆-alkynyl;

(referring to International Patent Publication No. WO 94/08999, WO 93/10100 and the U.S. Patent number US 5,405,829 that transfers Schering); The N-phenyl pyrazoles, such as:

(general formula X XI; Nipyraclofen)

(, the 9th edition, editing the Britain crop protection council (British CropProtection Council), Surrey (1991) by C.R.Worthing) referring to " agricultural chemicals handbook (The Pesticide Manual) " the 621st page; With 3-replace-2-aryl-4,5,6,7-tetrahydrochysene indazole class (Lyga etc., pesticide science (Pesticide Sci.) 42:29-36 (1994)).

(general formula X XIa; BAY 11340)

Other is the phenyl pyrazoles of type described in WO 96/01254 and the WO 97/00246 importantly, and the content of these two pieces of documents is incorporated herein by reference.(general formula X XII).

Other is importantly such as the pyridylpyrazole of following general formula:

(general formula X XIIIa) (general formula X XIIIb)

Other importantly has the phenyl pyrazoles of following general formula:

(general formula X XIV: fluorine chlorine grass ammonium) (referring to Prosch, S.D. etc., Brighton crop protection meeting-weeds (Brighton CropProtection Conference-Weeds): 45-50 page or leaf (1997), (Vol.1)).

Compound that other is important such as WO98/33927 and U.S. Pat 5,856 described in 495, are incorporated herein by reference the full content of these two pieces of documents.

Usually suppressing the active weedicide concentration of protox comprises known in the art and depends in part on applicating ratio such as the such external factor of environment, time and application process.For example, with regard to by the imide weedicide of general formula V-IX representative and more specifically say so with regard to those weedicides of general formula X-XVII representative, the ratio of using is in the scope of 0.0001-10kg/ha, preferred 0.005-2kg/ha.This dose ratio or weedicide concentration are can be with the difference of required effect and particularly compound used therefor different and can be measured by method as known in the art.

Another object of the present invention is a kind of method that is used to control unwanted vegetation growth, this method comprises the step of weedicide of plant population being used the inhibition protox of significant quantity, and described plant is selected from the group that mouse ear mustard genus, sugarcane, soybean, barley, cotton, tobacco, beet, rape, corn, wheat, Chinese sorghum, rye, barley, oat, mud grass and forage grass, millet, forage grass and rice etc. are formed.Preferably a kind of method of controlling unwanted vegetation growth, this method comprise the step of weedicide of the colony that is selected from soybean, cotton, tobacco, beet, rape, corn, wheat, Chinese sorghum, rye, barley, oat, forage grass and rice etc. being used the inhibition protox of significant quantity.Particularly preferably be a kind of method of controlling unwanted vegetation growth, this method comprises the step of weedicide of the colony that is selected from mouse ear mustard genus, soybean, cotton, beet, rape, corn, wheat, Chinese sorghum and rice etc. being used the inhibition protox of significant quantity.

The present invention further comprises a kind of mosaic gene, and it comprises the promotor that operationally is connected with dna molecular of the present invention that can express in plant plastid.Can expression promoter be the promotor of a kind of separation from 5 ' flanking region upstream, plastogene coding region in plant plastid preferably, described plastogene can derive from identical or different kind and general its natural product of finding in the most of plastid type that comprises those plastids that are present in the non-chlorenchyma.The example of this class promotor is the clpP gene promoter, (WO 97/06250 such as tobacco clpP gene promoter, the document is incorporated herein by reference) and mouse ear mustard genus clpP gene promoter (U. S. application US09/038,878, the document is incorporated herein by reference).Can express other promotor of dna molecular in the plant plastid and be promotor by viral rna polymerase identification.Preferred such promotor is that such as T7 gene 10 promotors, it is discerned by phage t7 DNA-RNA-dependent polysaccharase by the promotor of single subunit RNA polymerase identification.Can be in plant plastid the another kind of promotor of expressible dna molecule derive from the regulatory region (Harris etc. of plastid 16S ribosome-RNA(rRNA) operon, microbiology summary (Microbiol.Pev.) 58:700-754 (1994), Shinozaki etc., EMBO is (1986) J.5:2043-2049, and the content of these two pieces of documents is incorporated herein by reference).The gene transformation of T7 polysaccharase of preferably will encoding is gone into the nuclear gene group and is used plastid transit peptides to import described plastid with the T7 polysaccharase is directed.The expression of dna molecular in this plastid can be composing type maybe can be induction type.These different embodiments extensively are described among the WO98/11235, and the document is incorporated herein by reference.Described mosaic gene preferably further is included in 5 ' untranslated sequence (5 ' UTR) that works in the plant plastid and plastogene 3 ' the untranslated sequence (3 ' UTR) that operationally is connected with dna molecular of the present invention.Preferred 3 ' UTR is a kind of 3 ' untranslated sequence of plastid rps16 gene.In another embodiment, described mosaic gene comprises the poly--G sequence fragment rather than 3 ' the untranslated sequence.

The present invention also comprises a kind of plastid conversion carrier, and it comprises above-mentioned mosaic gene and is integrated into the flanking region of plastom by homologous recombination.This plastid conversion carrier can comprise at least a chloroplast(id) replication orgin or not comprise this replication orgin.The present invention also comprises this class plastid conversion carrier plant transformed plastid of a kind of usefulness, and wherein said dna molecular is effable in this plant plastid.The present invention also comprises a kind of plant or vegetable cell that comprises this plant plastid, comprises its filial generation.In a preferred embodiment, described plant is a homogeneity to the transgenosis plastid.Plant transformed can be monocotyledons or dicotyledons among the present invention.Preferred monocotyledons is that corn and preferred dicotyledons are tobaccos.Other preferred dicotyledons is tomato and potato.

By homologous recombination gene is inserted the plastid trans-utilization be present in some or all in ring-type plastom several thousand copies in each vegetable cell in the gene of nuclear expression the huge advantage of copy number make expression level can surpass 10% of total solubility vegetable-protein.In addition, because the characteristic of plastid coding is not that pollen transmits in most of plants, need plastid to transform; Therefore, got rid of the accidental potential hazard that fails into the wild correlative of transgenic plant of transgenosis.The plastid transformation technology extensively is described in the following document: U.S. Patent number US 5,451,513, US 5,545,817, US 5,545,818 and US 5,576,198; PCT application number WO 95/16783 and WO 97/32977; And McBride etc., periodical (Proc.Natl.Acad.Sci.USA) 91:7301-7305 (1994) of institute of the state-run academy of sciences of the U.S., all these documents are incorporated herein by reference.In single celled green alga Lei Shi chlamydomonas, carried out plastid conversion (Boynton etc. (1988) science (Science) 240:1534-1537 at first by biolistics, the document is incorporated herein by reference) and can select cis acting antibiotics resistance gene seat (spectinomycin/streptomycin resistance) or non-photosynthetic mutation type surface complementary means to extend to tobacco (Svab etc. (1990) Proc.Natl.Acad.Sci.USA 87:8526-8530 is incorporated herein by reference the document) rapidly this use.

Be used for basic fundamental that tobacco chloroplast transforms and comprise the picked-up in the plasmid DNA of protoplastis to the particle bombardment of leaf texture or PEG-mediation, wherein said plasmid DNA has the clone's who is positioned at selectivity antibiotics resistance mark flank plastid DNA district.The 1-1.5kb flanking region that is called " guiding peptide " helps using plastom to carry out homologous recombination and makes the specificity district of 156kb tobacco plastom take place to replace or change thus.At first will be to the point mutation in spectinomycin and/or Streptomycin sulphate tolerific chloroplast(id) 16S rDNA and the rps12 gene as the selective marker (Svab that transforms, Z., Hajdukiewicz, P., and Maliga, P. institute of the state-run academy of sciences of (1990) U.S. prints (Proc.Natl.Acad.Sci.USA) 87,8526-8530; Staub, J.M., and Maliga, P. (1992) vegetable cell (Plant Cell) 4,39-45 is incorporated herein by reference the content of these documents).This technology has produced stable homogeneity transformant, and frequency is that the every bombardment of target leaf is produced about 1 transformant 100 times.The cloning site that exists between these marks has caused generating the plastid targeting vector that is used to import foreign gene (P., EMBO be (1993) J.12:601-606 for Staub, J.M., and Maliga, and the content of the document is incorporated herein by reference).Significantly increased transformation frequency by replace recessive rRNA or r-albumen antibiotics resistance gene with dominant selectable marker, the described selective marker spectinomycin of promptly encoding is separated the bacterium aadA gene (Svab of toxenzyme aminoglycoside-3 '-adenylic acid (AMP) transferring enzyme, Z., and Maliga, P. institute of the state-run academy of sciences of (1993) U.S. prints (Proc.Natl.Acad.Sci.USA) 90,913-917 is incorporated herein by reference the content of the document).The high frequency that in advance this mark has been successfully used to green alga Lei Shi chlamydomonas plastom transforms (Goldschmidt-Clermont, M. (1991) nucleic acids research (Nucl.Acids Res.) 19,4083-4089 is incorporated herein by reference the content of the document).Recently used the DNA uptake assay of polyoxyethylene glycol (PEG) mediation realized the plastid of the protoplastis of tobacco and mosses exhibition leaf sword-like leave moss (Physcomitrella patens) transform (O ' (1993) plant magazine (Plant J.) 3:729-738 such as Neill; Koop etc. (1996) plants (Planta) 199:193-201 is incorporated herein by reference the content of these two pieces of documents).

In a preferred embodiment, the present invention includes a kind of mosaic gene, it comprise can in plant plastid, express operationally with separate the promotor that is connected from prokaryotic organism or Eukaryotic dna molecular, natural or the protox enzyme modified of described dna molecule encode, the dna molecular of or wheat, soybean, cotton, beet, rape, rice, Chinese sorghum or the sugarcane protox enzyme modified natural such as coding.This class dna molecular is included in the aforesaid plastid conversion carrier and has produced the transgenosis plastom is the plant of homogeneity.Expression in the plant plastid of the dna molecular of the protox enzyme that coding is modified preferably makes plant produce tolerance to the weedicide that suppresses the active consumption of naturally occurring protox thus.

In another preferred embodiment, the present invention includes a kind of mosaic gene, it comprises: (a) a kind of separation is from the dna molecular of plant, its polypeptide of comprising plastid transit peptides and by the maturing enzyme of the natural target of plastid transit peptides to plant plastid of encoding under native state wherein is modified into described dna molecular its not encoding function character body transit peptides; (b) a kind of promotor that can express dna molecular in the plastid, wherein this promotor operationally is connected with described dna molecular.In a preferred embodiment, described transit peptides is undergone mutation and it does not allow suitably to be transported to the required like this cell cell of all plastids as described by the enzyme of described dna molecule encode thus.In another preferred embodiment, from described dna molecular, remove part transit peptides encoding sequence or complete transit peptides encoding sequence, thereby prevent that the suitable target of described enzyme quilt is to required cell cell.

Above-mentioned mosaic gene insertion plastid conversion carrier and the present invention are also related to the plant that has the plastom that transforms with this class carrier thus, and described thus dna molecular is effable in plant plastid.Preferred this class plant is a homogeneity for the transgenosis plastid.

In a preferred embodiment, a kind of enzyme of the dna molecule encode of above-mentioned direct description, its wild-type form is subjected to weedicide and suppresses.In another preferred embodiment, a kind of wild-type form of described dna molecule encode is subjected to that weedicide suppresses and compares the enzyme that comprises at least a amino acid change with wild-type enzyme.This amino acid change makes the compound that the tolerance of described enzyme can natural inhibition wild-type enzyme.In another preferred embodiment, described dna molecule encode has the active enzyme of proporphyrinogen oxidase (protox).In another preferred embodiment, further as among the embodiment 37-42 illustrated from as described in remove transit peptides the dna molecular.For the weedicide of the transgenosis plastid of the present invention plant tolerance higher amount that is homogeneity, such as suppressing the active general formula X VII weedicide of naturally occurring protox (as further explaining among the embodiment 44).

In another preferred embodiment, make the transit peptides of the dna molecular of encoding 5-enolpyrul-shikimate acid-3-phosphate synthase (EPSP synthase) undergo mutation or be removed.The dna molecular that makes gained with can be in plant plastid expression promoter merge and obtained in plastom, to comprise the homogeneity plant of this class construct.These plants tolerate herbicidal compounds, the particularly glyphosate of natural inhibition EPSP synthase.In another preferred embodiment, make the transit peptides of the dna molecular of encoding acetolactate synthase (ALS) undergo mutation or be removed.The dna molecular that makes gained with can be in plant plastid expression promoter merge and obtained in plastom, to comprise the homogeneity plant of this class construct.These plants tolerate the herbicidal compounds of natural inhibition ALS, particularly sulfonylurea.In another preferred embodiment, make the transit peptides of dna molecular of coding acetoxyl group hydroxy acid synthase (AHAS) undergo mutation or be removed.The dna molecular that makes gained with can be in plant plastid expression promoter merge and obtained in plastom, to comprise the homogeneity plant of this class construct.These plants tolerate the herbicidal compounds of natural inhibition AHAS, particularly imidazolone and sulphonamide herbicides.In another preferred embodiment, make the transit peptides of dna molecular of coding acetyl-CoA carboxylase (ACCase) undergo mutation or be removed.The dna molecular that makes gained with can be in plant plastid expression promoter merge and obtained in plastom, to comprise the homogeneity plant of this class construct.These plants tolerate the herbicidal compounds of natural inhibition ACCase, particularly cyclohexanedione and aryl phenoxy propionic acid weedicide.In another preferred embodiment, make the transit peptides of dna molecular of coding glutamine synthase (GS) undergo mutation or be removed.The dna molecular that makes gained with can be in plant plastid expression promoter merge and obtained in plastom, to comprise the homogeneity plant of this class construct.These plants tolerate the herbicidal compounds of natural inhibition GS, particularly phosphinothricin and methionine(Met) sulfoximine.

The present invention also further relates to a kind of by transforming the method that plastom obtains herbicide resistant plants with mosaic gene, described mosaic gene comprises: (a) a kind of separation is from the dna molecular of plant, its polypeptide of comprising plastid transit peptides and by the maturing enzyme of the natural target of plastid transit peptides to plant plastid of encoding under native state wherein is modified into described dna molecular not encoding function character body transit peptides; (b) a kind of promotor that can express dna molecular in the plastid, wherein this promotor operationally is connected with described dna molecular.Directly quote the example of the enzyme that the invention described above uses, but the practicality of these class methods is not limited to the example quoted.

The invention further relates to a kind of novel method of selecting to change the plastom(e) vegetable cell, this method comprises the following steps: above-mentioned mosaic gene is imported the vegetable cell plastom(e); In the plastid of vegetable cell, express coded enzyme; With the cell of the herbicidal compounds of selecting the described enzymic activity of the natural inhibition of tolerance, consequent cell comprises the plastid of conversion.In a preferred embodiment, the described enzyme natural inhibition and the transgenic plant that are subjected to herbicidal compounds can grow under the herbicidal compounds consumption of the described enzymic activity of natural inhibition.In another preferred embodiment, described enzyme has proporphyrinogen oxidase (protox) activity and it is modified into the tolerance that can produce the protox inhibitor.

Another aspect of the present invention is a kind of method that plastid transforms resistance (recalcitrant) plant that is used for.The method that is used for plastid transformation of tobacco and lower plant kind at first depends on the antibiotic non-lethality of tolerance and selects, and described microbiotic preferentially influences plastid translation element and makes light heterotrophism transformant grow sooner than heterotrophic unconverted tissue thus.

Several factors may cause making the plastid conversion of monocotyledons and other dicotyledons to be met difficulty.For example, there is G in corn chloroplast(id) 16S ribosome-RNA(rRNA) (rRNA) and natural tolerance spectinomycin (Harris etc., 1994) on 1138 in corn 16S rDNA gene.Therefore, the application of 16s rRNA point mutation has produced spectinomycin and/or streptomycin resistance, successfully (Boynton and Gillham (1993) are at Wu as the selectivity chloroplast(id) mark in Chlamydomonas and the tobacco with this resistance, R.[Ed.] Enzymology method (Methods in Enzymology) the 217th volume. described in, Academic Press, San Diego, the 510-536 page or leaf; Institute of the state-run academy of sciences of Svab etc. (1990) U.S. prints (Proc.Natl.Acad.Sci.U.S.A.) 87:8526-8530), but this application is not practical concerning corn.Natural spectinomycin in the corn and streptomycin resistance have also avoided using coding aminoglycoside 3 "-the bacterium aadA gene of adenylic acid (AMP) transferring enzyme, this produces dominance spectinomycin and streptomycin resistance and makes the tobacco chloroplast transformation efficiency increase by 100 times (periodical (Proc.Natl.Acad.Sci.U.S.A.) 90:913-917 of institute of the state-run academy of sciences of Svab and Maliga (1993) U.S.).The application of kantlex (proof can be used for the unique another kind of microbiotic that chloroplast(id) transforms) also becomes query, and this is (Carrer etc. (1993) molecular gene genetics (Mol.Gen.Genet.) 241:49-56) that (about 50: 1) are caused because the ratio that bombards the resistance of back tobacco center and chloroplast(id) coding at the bacterium nptII gene to the coding neomycin phosphotransferase obviously is above standard.This result has been integrated into all higher being confirmed of frequency of nuclear gene group because of spontaneous nucleus resistant mutation and nptII.Because nptII still is a kind of selective marker efficiently that is used for the corn consideration conveyization, so have reason to estimate and observed similar background level in tobacco.With spontaneous resistance and obviously excessive selective marker go into the nuclear gene group by unconventional recombination and integration at random and non-homogeneous this process of chloroplast gene group that is integrated into can transform the body difficulty that become so that reclaim real chloroplast(id), but be not impossible.

A more basic reason of the difficulty that the plastid conversion in the plant variety of use except that tobacco runs into may be relevant with the non-photosynthetic properties that the plant tissue of many renewable cultivations, especially corn and mouse ear mustard belong to.Tobacco is that the reason of an exception is that the vegetation tissue of being cultivated is reproducible and contains belong to photosynthetic competent ripe differentiation chloroplast(id) in the presence of sucrose.So the system that is used to screen tobacco plastid transformant at present depends on the growth rate of the transformant that can use reduction and inorganic carbon source.In addition, cell transformed can not experience that to be suppressed the chloroplast membranes that causes in light impaired because of plastid albumen synthesizes.This expression by the selective marker that has highly active promoters driven in the differentiation chloroplast(id) of preferentially photosynthetic cells being worked can not be worked in the non-chlorenchyma that contains proplastid (corn I type callus, the somatic embryo of for example growing in the dark) or amyloplast/leukoplast (for example root culture of mouse ear mustard genus).Plastid in these plants transforms and need produce the strong selective marker of selecting in all plastid type.

A kind of preferred selective marker that is used for general plastid conversion: (1) only has active in to eliminate " decline " of consideration conveyization at plastid; (2) have and do not rely on the photosynthetic response ability or the mode of action of existence of the chloroplast(id) of differentiation fully; (3) have the resistance level that depends on adjustable external parameter (for example light) jointly but not only determine, make selective pressure in chosen process, to change so that help the separation of thousands of plastom copies by the bulk phase concentration of selective agent.

In a preferred embodiment, this class selectable marker gene relates to the application of mosaic gene, described mosaic gene comprises a kind of isolated DNA molecule, it is coded in the enzyme of the target plastid that has plastid transit peptides under its native state, wherein described dna molecular is modified into that transit peptides does not exist or can play a part described enzyme target to described plastid, wherein said dna molecular operationally with can in plant plastid, expression promoter be connected.In a preferred embodiment, the natural enzyme that is subjected to the weedicide inhibition of dna molecule encode of the present invention.In another preferred embodiment, described dna molecule encode protoporphyrinogen IX oxydase (" protox ").In a preferred embodiment, protoporphyrinogen IX oxidase gene is from mouse ear mustard; And at one more in the embodiment preferred, protoporphyrinogen IX oxidase gene is from mouse ear mustard and comprise at least a aminoacid replacement.Preferred amino acid replaces produces to the inhibiting tolerance of the weedicide of natural this enzymic activity of inhibition described enzyme.Think that the weedicide of lower concentration can kill and wound wild-type plant, this is to cause owing to constituted the sensitivity of light intermediate when the localized protox enzyme of plastid is suppressed.The generation of these sensitivity of light compounds does not need the chloroplast(id) that breaks up or photosynthesis initiatively, and this is that success is carried out renewable cultured tissue and had the key factor that the plastid of the plant of non-photosynthetic properties transforms.

Another key feature is the expression that has selectable marker gene in non-green plastid.In a preferred embodiment, the present invention includes the application that in the plastid of green and non-chlorenchyma, to express the promotor of the dna molecular that is operably connected.Particularly, a kind of such promotor derives from the regulatory region of plastid 16S ribosome-RNA(rRNA) operon.Another candidate is from the promotor of plastid clpP gene and 5 ' UTR.In from the plastid of all plant tissues that comprise those plant tissues that do not contain chloroplast(id), express clpP gene product (Shanklin (1995) vegetable cell (Plant Cell) 7:1713-22) in the composing type mode.

Can use aforesaid method that other dna molecular is imported plant plastid jointly.In a preferred embodiment, plastid conversion carrier of the present invention contains a kind of mosaic gene, and its require to select aforesaid transformant and other gene at least a and can expression promoter fusion in plant plastid.For example, other this genoid can produce to insect pest or to the tolerance of fungi or bacterial pathogens one or more characteristics that be worth to increase of maybe can encoding.

The concise and to the point description SEQ ID NO:1 of sequence in the sequence table: the proteic dna encoding sequence of mouse ear mustard protox-1.SEQ ID NO:2: the mouse ear mustard by SEQ ID NO:1 coding belongs to the protox-1 aminoacid sequence.SEQ ID NO:3: the proteic dna encoding sequence of mouse ear mustard protox-2.SEQID NO:4: the mouse ear mustard by SEQ ID NO:3 coding belongs to the protox-2 aminoacid sequence.SEQ ID NO:5: the proteic dna encoding sequence DNA of corn protox-1.SEQ ID NO:6: by the corn protox-1 aminoacid sequence of SEQ ID NO:5 coding.SEQ ID NO:7: the proteic dna encoding sequence DNA of corn protox-2.SEQ ID NO:8: by the corn protox-2 aminoacid sequence of SEQ ID NO:7 coding.SEQID NO:9: the proteic part dna encoding of wheat protox-1 sequence.SEQ ID NO:10: by the part wheat protox-1 aminoacid sequence of SEQ ID NO:9 coding.SEQ ID NO:11: the proteic dna encoding sequence of soybean protox-1.SEQ ID NO:12: by the soybean protox-1 albumen of SEQ ID NO:11 coding.SEQ ID NO:13: from the promoter sequence of mouse ear mustard protox-1 gene.SEQ ID NO:14: from the promoter sequence of corn protox-1 gene.SEQ ID NO:15: the proteic dna encoding sequence of cotton protox-1.SEQ ID NO:16: by the cotton protox-1 aminoacid sequence of SEQ ID NO:15 coding.SEQ ID NO:17: the proteic dna encoding sequence of beet protox-1.SEQ ID NO:18: by the beet protox-1 aminoacid sequence of SEQ ID NO:17 coding.SEQ ID NO:19: the proteic dna encoding sequence of rape protox-1.SEQ ID NO:20: by the rape protox-1 aminoacid sequence of SEQ ID NO:19 coding.SEQ ID NO:21: the proteic part dna encoding of rice protox-1 sequence.SEQ ID NO:22: by the part rice protox-1 aminoacid sequence of SEQ ID NO:21 coding.SEQ ID NO:23: the proteic part dna encoding of Chinese sorghum protox-1 sequence.SEQ ID NO:24: by the part Chinese sorghum protox-1 aminoacid sequence of SEQ ID NO:23 coding.SEQ ID NO:25: corn protox-1 intron sequences.SEQ ID NO:26: from the promoter sequence of beet protox-1 gene.SEQ ID NO:27:Pclp_P1a-plastid clpP gene promoter cochain PCR primer.Chain PCR primer under the SEQ ID NO:28:Pclp_P1b-plastid clpP gene promoter.Chain PCR primer under the SEQ ID NO:29:Pclp_P2b-plastid clpP gene promoter.SEQ ID NO:30:Trps16_P1a-plastid rps16 gene cochain PCR primer.Chain PCR primer under the SEQ ID NO:31:Trps16_p1b-plastid rps16 gene.Strand primer on the SEQ ID NO:32:minpsb_U-plastid psbA gene.Strand primer under the SEQ ID NO:33:minpsb_L-plastid psbA gene.SEQ ID NO:34:APRTXP1a-cochain PCR primer.Chain PCR primer under the SEQ ID NO:35:APRTXP1b-.SEQ ID NO:36: the proteic part dna encoding of sugarcane protox-1 sequence.SEQ ID NO:37: by the part sugarcane protox-1 aminoacid sequence of SEQ ID NO:36 coding.1. deposit

Following carrier molecule has been deposited with patent culture collection center, northern regional study center, (the Agricultural Research Service of farming research service organization, Patent CultureCollection (NRRL), Northern Regional Research Center), 1815North University Street, Peoria, Illinois 61604, U.S.A, relevant data is as follows:

Wheat protox-1a in the pBluescript SK carrier was deposited into pWDC-13 (NRRL #B21545) on March 19th, 1996.

Soybean protox-1 in the pBluescript SK carrier was deposited into pWDC-12 (NRRL #B-21516) December 15 nineteen ninety-five.

Cotton protox-1 in the pBluescript SK carrier was deposited into pWDC-15 (NRRL #B-21594) on July 1st, 1996.

Beet protox-1 in the pBluescript SK carrier was deposited into pWDC-16 (NRRL #B-21595N) on July 29th, 1996.

Rape protox-1 in the pBluescript SK carrier was deposited into pWDC-17 (NRRL #B-21615) on August 23rd, 1996.

Rice protox-1 in the pBluescript SK carrier was deposited into pWDC-18 (NRRL #B-21648) on December 6th, 1996.

Chinese sorghum protox-1 in the pBluescript SK carrier was deposited into pWDC-19 (NRRL #B-21649) on December 6th, 1996.

Resistant mutants pAraC-2Cys in the pMut-1 plasmid was deposited with farming research culture collection center (Agricultural Research CultureCollection), called after pWDC-7 and gives its deposit numbers NRRL #21339N on November 14th, 1994.

The AraPT1Pro that will contain mouse ear mustard genus protox-1 promotor deposited into pWDC-11 (NRRL #B-21515) December 15 nineteen ninety-five.

To contain the corn protox-1 promoter plasmid that merges with corn protox-1 encoding sequence remainder deposited into pWDC-14 (NRRL #B-21546) on March 19th, 1996.

The plasmid that will contain beet protox-1 promotor was deposited into pWDC-20 (NRRL #B-21650) on December 6th, 1996.

Embodiment

By further describing the present invention with reference to following specific embodiment.It only is purpose in order to explain that these embodiment are provided, and except as otherwise noted, and they have not been used for the qualification effect.Standard recombinant dna used herein and molecule clone technology are well-known in the art and are described in the following document: Ausubel (editor), up-to-date experimental methods of molecular biology compilation (CurrentProtocols in Molecular Biology), John Wiley and Sons, Inc. (1994); T.Maniatis, E.F.Fritsch and J.Sambrook, molecular cloning (MolecularCloning): laboratory manual (A Laboratory Manual), cold spring harbor laboratory (ColdSpring Harbor laboratory), cold spring port (Cold Spring Harbor), New York (NY) (1989); And T.J.Silhavy, M.L.Berman, and L.W.Enquist, gene fusion experiment (Experiments with Gene Fusions), cold spring harbor laboratory (ColdSpring Harbor Laboratory), cold spring port (Cold Spring Harbor), New York (NY) (1984).

The A part. the separation and the characterization embodiment 1 of plant proporphyrinogen oxidase (Protox) gene: based on the wheat Protox-1 of corn Protox-1 encoding sequence homologous sequence

The separation of cDNA

To submit to Clontech to be used for the structure in the conventional cDNA of λ Uni-Zap carrier library by total RNA of common wheat (cv Kanzler) preparation.With about 50, the cDNA library of 000pfu is with about 5, and the density flat board of 000pfu/10cm is fixed on the culture dish and the bipartite thing that leaches is placed (Schleicher and Schuell) on the nitrocellulose membrane.Use is by guiding corn protox-1 cDNA (the SEQ ID NO:5 of method (LifeTechnologies) by the 32P-dCTP mark at random; Referring to the embodiment 2 among the international application no PCT/IB95/00452 that submits to June 8 nineteen ninety-five, the December 21 nineteen ninety-five disclosed WO 95/34659) survey plaque and leach thing.Hybridization conditions: 7% sodium lauryl sulphate (SDS) under 50 ℃, 0.5M NaPO4 pH7.0,1mM EDTA.Wash conditions: 2X SSC, 1%SDS under 50 ℃.(Church and Gilbert, institute of the state-run academy of sciences of U.S. periodical (Ptoc.Natl.Acad.Sci.USA) 81:1991-1995 (1984) are incorporated herein by reference the full content of the document).Forward is hybridized plaque to carry out purifying and is cut into the pBluescript plasmid in vivo.(Applied Biosystems, Inc.) chain termination method of the dideoxy terminator of mark is measured cDNA and is inserted fragments sequence by fluorescence dye by using.The length of the longest wheat protox-1 cDNA of the called after of attempting available from initial screening " wheat protox-1 " is 1489-bp.Wheat protox-1 lacks the encoding sequence of transit peptides and based on about 126 amino acid of the ripe encoding sequence that compares with other known plants protox plant peptide sequence.

Carry out programmed screening so that obtain long wheat protox cDNA.With regard to current screening, use λ Uni-Zap carrier to prepare common wheat (cv Kanzler) cDNA library in inside.Screen as mentioned above about 200, the cDNA library of 000pfu, but do not comprise with wheat protox-1cDNA as probe and hybridization and wash conditions be at 65 ℃ down rather than under 50 ℃.The length of the longest wheat protox-1 cDNA of the called after of attempting available from initial screening " wheat protox-1a " is 1811-bp.The nucleotide sequence of this cDNA and coded aminoacid sequence thereof are respectively with SEQID NOs:9 and 10 expressions.Based on other known plants protox peptide sequence and with corresponding genome sequence relatively, this cDNA is codon total length or that only lacked several transit peptides (table 1A).This wheat protein sequence have 91% with the corn protox-1 protein sequence of representing with SEQ ID NO:6 identical (95% is similar).

Embodiment 2: based on belonging to the soybean of Protox-1 encoding sequence homologous sequence with mouse ear mustard

The separation of Protox-1 cDNA

λ Uni-Zap cDNA library by soybean (v Williams 82, epicotyl) preparation is purchased from Stratagene.With about 50, this library of 000pfu is with about 5, and the density flat board of 000pfu/10cm is fixed on the culture dish and the bipartite thing that leaches is placed (NEN Dupont) on bacterium colony/plaque screening film.Use belongs to protox-1 cDNA (SEQ ID NO:1 by guiding method (Life Technologies) at random by the mouse ear mustard of 32P-dCTP mark; Referring to the embodiment 1 among the international application no PCT/IB95/00452 that submits to June 8 nineteen ninety-five, the December 21 nineteen ninety-five disclosed WO95/34659) survey plaque and leach thing.Hybridization conditions: 7% sodium lauryl sulphate (SDS) under 50 ℃, 0.5M NaPO4 pH7.0,1mM EDTA.Wash conditions: 2X SSC, 1%SDS under 50 ℃.(Church and Gilbert (1984)).Forward is hybridized plaque to carry out purifying and is cut into the pBluescript plasmid in vivo.(Applied Biosystems, Inc.) chain termination method of the dideoxy terminator of mark is measured cDNA and is inserted fragments sequence by fluorescence dye by using.Be the basis to compare with other known plants protox peptide sequence (table 1A), the beans cDNA that grows up most of the called after of acquisition " soybean protox-1 " is a total length.The length of soybean protox-1 is the protein of 1847-bp and coding 58.8kDa.The nucleotide sequence of this cDNA and coded aminoacid sequence thereof are respectively with SEQ ID NOs:11 and 12 expressions.This soybean protein has 78% to belong to protox-1 albumen identical (87% is similar) with mouse ear mustard.

Soybean protox-1 in the pBluescript SK carrier was deposited into pWDC-12 (NRRL #B-21516) December 15 nineteen ninety-five.Embodiment 3: based on the cotton Protox-1 of corn Protox-1 encoding sequence homologous sequence

The separation of cDNA

Dick doctor Trelease (Ni W. and Trelease R.N., Arch.Biochem.Biophys.289:237-243 (1991)) that the λ Uni-Zap cDNA library that is prepared by upland cotton (dicotyledons that grew in the dark in 72 hours) available from Arizona national university (Arizona State University) phytology is.With about 50, this library of 000pfu is with about 5, and the density flat board of 000pfu/10cm is fixed on the culture dish and the bipartite thing that leaches is placed (NEN Dupont) on bacterium colony/plaque screening film.Use leaches thing by guiding method (Life Technologies) at random by corn protox-1 cDNA (SEQ ID NO:5) the detection plaque of 32P-dCTP mark.Hybridization conditions: 7% sodium lauryl sulphate (SDS) under 50 ℃, 0.5M NaPO4 pH7.0,1mM EDTA.Wash conditions: 2X SSC, 1%SDS under 50 ℃.(Church and Gilbert (1984)).Forward is hybridized plaque to carry out purifying and is cut into the pBluescript plasmid in vivo.(Applied Biosystems, Inc.) chain termination method of the dideoxy terminator of mark is measured cDNA and is inserted fragments sequence by fluorescence dye by using.Be the basis to compare, seem that the longest cotton cDNA of the called after " cotton protox-1 " that obtains is a total length with other known plants protox peptide sequence (table 1A).The length of cotton protox-1 is the protein of 1826-bp and coding 58.2kDa.The nucleotide sequence of this cDNA and coded aminoacid sequence thereof are respectively with SEQID NOs:13 and 14 expressions.This albumen that grows cotton has 77% identical with corn protox-1 albumen (86% is similar).

Embodiment 4: based on belonging to the beet of Protox-1 encoding sequence homologous sequence with mouse ear mustard

The separation of Protox-1 cDNA

By the λ Uni-Zap cDNA library of beet preparation available from Philadelphia, Philip doctor Rea (the Yongcheol Kim of the PA plant science research institute plant department of the Chinese Academy of Sciences, Eugene J.Kim and Philip A.Rea, plant physiology (Plant Physiol.) 106:375-382 (1994)).With about 50, this library of 000pfu is with about 5, and the density flat board of 000pfu/10cm is fixed on the culture dish and the bipartite thing that leaches is placed (Schleicher and Schuell) on the nitrocellulose membrane.Use leaches thing by guiding method (Life Technologies) at random by mouse ear mustard genus protox-1 cDNA (SEQ ID NO:1) the detection plaque of 32P-dCTP mark.Hybridization conditions: 7% sodium lauryl sulphate (SDS) under 50 ℃, 0.5M NaPO4 pH7.0,1mM EDTA.Wash conditions: 2X SSC, 1%SDS under 50 ℃.(Church and Gilbert (1984)).Forward is hybridized plaque to carry out purifying and is cut into the pBluescript plasmid in vivo.(Applied Biosystems, Inc.) chain termination method of the dideoxy terminator of mark is measured cDNA and is inserted fragments sequence by fluorescence dye by using.Be the basis to compare with other known plants protox peptide sequence (table 1A), the longest beet cDNA of the called after of acquisition " beet protox-1 " is a total length.The length of beet protox-1 is the protein of 1910-bp and coding 60kDa.The nucleotide sequence of this cDNA and coded aminoacid sequence thereof are respectively with SEQ ID NOs:15 and 16 expressions.This beet albumen has 73% to belong to protox-1 albumen identical (82% is similar) with mouse ear mustard.

Embodiment 5: based on belonging to the rape of Protox-1 encoding sequence homologous sequence with mouse ear mustard

The separation of Protox-1 cDNA

By the λ Uni-Zap cDNA library of colea (the ripe green leaf in 3-4 week) preparation available from Guenther doctor Ochsb, Institut Fuer Allgemeine Botanik, Johannes Gutenberg-Universitaet Mainz, Germany (G ü nther Ochs, Gerald Schock, with Aloysius Wild, plant physiology (Plant Physiol.) 103:303-304 (1993)).With about 50, this library of 000pfu is with about 5, and the density flat board of 000pfu/10cm is fixed on the culture dish and the bipartite thing that leaches is placed (Schleicher and Schuell) on the nitrocellulose membrane.Use leaches thing by guiding method (Life Technologies) at random by mouse ear mustard genus protox-1 cDNA (SEQ ID NO:1) the detection plaque of 32P-dCTP mark.Hybridization conditions: 7% sodium lauryl sulphate (SDS) under 50 ℃, 0.5M NaPO4pH7.0,1mM EDTA.Wash conditions: 2X SSC, 1%SDS under 50 ℃.(Church and Gilbert (1984)).Forward is hybridized plaque to carry out purifying and is cut into the pBluescript plasmid in vivo.(Applied Biosystems, Inc.) chain termination method of the dideoxy terminator of mark is measured cDNA and is inserted fragments sequence by fluorescence dye by using.Be the basis to compare with other known plants protox peptide sequence (table 1A), the long oil dish cDNA of the called after of acquisition " rape protox-1 " is a total length.The length of rape protox-1 is the protein of 1784-bp and coding 57.3kDa.The nucleotide sequence of this cDNA and coded aminoacid sequence thereof are respectively with SEQ ID NOs:17 and 18 expressions.This rape albumen has 87% to belong to protox-1 albumen identical (92% is similar) with mouse ear mustard.

Rape protox-1 in the pBluescript SK carrier was deposited into pWDC-17 (NRRL #B-21615) on August 23rd, 1996.Embodiment 6: based on the rice Protox-1 of corn Protox-1 encoding sequence homologous sequence

The separation of cDNA

λ gt11 cDNA library by rice (branch that bleached in 5th) preparation is purchased from Clontech.With about 50, this library of 000pfu is with about 5, and the density flat board of 000pfu/10cm is fixed on the culture dish and the bipartite thing that leaches is placed (Schleicher and Schuell) on the nitrocellulose membrane.Use leaches thing by guiding method (Life Technologies) at random by corn protox-1 cDNA (SEQ ID NO:5) the detection plaque of 32P-dCTP mark.Hybridization conditions: 7% sodium lauryl sulphate (SDS) under 50 ℃, 0.5M NaPO4 pH7.0,1mM EDTA.Wash conditions: 2X SSC, 1%SDS under 50 ℃.(Church and Gilbert (1984)).Forward is hybridized plaque to carry out purifying and uses Wizard λ-Prep test kit (Promega) preparation λ DNA.Use standard technique that cDNA is inserted fragment and go into pBluescript SK carrier as EcoRI fragment subclone.(Applied Biosystems, Inc.) chain termination method of the dideoxy terminator of mark is measured cDNA and is inserted fragments sequence by fluorescence dye by using.The length of the longest rice protox-1 cDNA of the called after " rice protox-1 " that obtains is 1224-bp.Rice protox-1 lacks the encoding sequence of transit peptides and based on about 172 amino acid of the ripe encoding sequence that compares with other known plants protox peptide sequence (table 1A).The nucleotide sequence of this Partial cDNA and coded aminoacid sequence thereof are respectively with SEQ ID NOs:19 and 20 expressions.

Rice protox-1 in the pBluescript SK carrier was deposited into pWDC-18 (NRRL #B-21648) on December 6th, 1996.Embodiment 7: based on the Chinese sorghum Protox-1 of corn Protox-1 encoding sequence homologous sequence

The separation of cDNA

By the λ Zap II cDNA library of dichromatism chinese sorghum (3-6 day green seedling) preparation available from Stuttgart, Germany university cytobiology and immunology research institute (Institute of CellBiology and Immunology, University of Stuttgart, Germany) Klaus doctor Pfizenmaier (Harald Wajant, Karl-Wolfgang Mundry, with Klaus Pfizenmaier, molecular biology of plants (Plant Mol.Biol.) 26:735-746 (1994)).With about 50, this library of 000pfu is with about 5, and the density flat board of 000pfu/10cm is fixed on the culture dish and the bipartite thing that leaches is placed (Schleicher and Schuell) on the nitrocellulose membrane.Use leaches thing by guiding method (Life Technologies) at random by corn protox-1 cDNA (SEQ ID NO:5) the detection plaque of 32P-dCTP mark.Hybridization conditions: 7% sodium lauryl sulphate (SDS) under 50 ℃, 0.5M NaPO4 pH7.0,1mM EDTA.Wash conditions: 2X SSC, 1%SDS under 50 ℃.(Church and Gilbert (1984)).Forward is hybridized plaque to carry out purifying and is cut into the pBluescript plasmid in vivo.(Applied Biosystems, Inc.) chain termination method of the dideoxy terminator of mark is measured cDNA and is inserted fragments sequence by fluorescence dye by using.The length of the fine strain of millet protox-1 cDNA that grows tall most of the called after " Chinese sorghum protox-1 " that obtains is 1590-bp.Chinese sorghum protox-1 lacks the encoding sequence of transit peptides and based on about 44 amino acid of the ripe encoding sequence that compares with other known plants protox peptide sequence (table 1A).The nucleotide sequence of this Partial cDNA and coded aminoacid sequence thereof are respectively with SEQ ID NOs:21 and 22 expressions.

Chinese sorghum protox-1 in the pBluescript SK carrier was deposited into pWDC-19 (NRRL #B-21649) on December 6th, 1996.Embodiment 8: based on the sugarcane Protox-1 of corn Protox-1 encoding sequence homologous sequence

The separation of cDNA

By the λ Zap II cDNA library of sugarcane preparation Henrik Albert available from farming research center, Hawaii (HawaiiAgricultural Research Center) USDA/ARS.With about 50, this library of 000pfu is with about 5, and the density flat board of 000pfu/10cm is fixed on the culture dish and the bipartite thing that leaches is placed (Schleicher and Schuell) on the nitrocellulose membrane.Use leaches thing by guiding method (Life Technologies) at random by corn protox-1 cDNA (SEQ ID NO:5) the detection plaque of 32P-dCTP mark.Hybridization conditions: 7% sodium lauryl sulphate (SDS) under 50 ℃, 0.5M NaPO4 pH7.0,1mM EDTA.Wash conditions: 2X SSC, 1%SDS under 50 ℃.(Church and Gilbert (1984)).Forward is hybridized plaque to carry out purifying and is cut into the pBluescript plasmid in vivo.(Applied Biosystems, Inc.) chain termination method of the dideoxy terminator of mark is measured cDNA and is inserted fragments sequence by fluorescence dye by using.The length of the longest sugarcane protox-1 cDNA of the called after " sugarcane protox-1 " that obtains is 633-bp.Sugarcane protox-1 lacks the encoding sequence of transit peptides and based on about 382 amino acid of the ripe encoding sequence that compares with other known plants protox peptide sequence (table 1A).The nucleotide sequence of this Partial cDNA and coded aminoacid sequence thereof are respectively with SEQ ID NOs:36 and 37 expressions.Embodiment 9: plant Protox clone is to the sensitivity of Protox inhibition weedicide in the bacterial system

The confirmation of property

Make the liquid culture of protox-1/SASX38, protox-2/SASX38 and pBluescript/XL1-Blue be grown in Lamp ¹⁰⁰In.The aliquot sample flat board of 100 each culture of microlitre is fixed in the substratum of protox inhibition aryi-uracile (aryluracil) weedicide of the general formula X VII that contains different concns (1.0nM-10mM).Down duplicate one group flat board is incubated 18 hours at 37 ℃.

Protox ⁺Coli strain XL1-Blue does not all demonstrate the susceptibility to described weedicide under any concentration, this result is consistent to the tolerance of similar weedicide with the natural bacteria enzyme of being reported.Protox-1/SASX38 obviously has susceptibility, and wherein the lawn of bacterium the inhibitor that almost is low to moderate 10nM concentration is eliminated fully.Protox-2/SASX38 also has susceptibility, but is only eliminated by the weedicide of higher concentration (10 μ M).Weedicide even also effective to almost completely keeping adusk flat board.By in flat board, adding the toxicity that 20 Therewith/ml protoferriheme can be eliminated described weedicide fully.

Different herbicide tolerant between the two kind of plant protox bacterial strains may be because the expression of these two kinds of plasmids is variant rather than because the result of any inherent difference in the enzyme susceptibility.Protox-1/SASX38 grows slowly more than protox-2/SASX38 in the substratum of any shortage protoheme.In addition, the Mzprotox-2/SASX38 bacterial strain with growth rate suitable with Arab protox-1/SASX38 is also very responsive to the weedicide under lower (10-100nM) concentration.B part: the evaluation that Protox inhibition weedicide is had the plant Protox gene of tolerance

With characterization embodiment 10: the plant Protox of the inhibition of anti-Protox weedicide in the escherichia expression system

The screening of gene

Mouse ear mustard (Landsberg) cDNA library (Minet etc., plant magazine (Plant J.) 2:417-422 (1992) among acquisition and the amplification plasmid vector pFL61.Obtain intestinal bacteria hemG mutant SASX38 (Sasarman etc., hereditary JOURNAL OF MICROBIOLOGY (J.Gen.Microbiol.) 113:297 (1979)) and it is maintained in the L substratum that contains 20ug/ml protoferriheme (United StatesBiochemicals).Carry out electroporation and change this plasmid library over to SASX38 by the condition of using Bio-Rad gene pulse producer and manufacturers.With the cell of electroporation be tiled on the L agar that contains 100 μ g/ml penbritins, density is about 500,000 transformant/10cm flat board.Then under the lower condition of 37 ℃ and light with cell insulation 40 hours and under the condition of not adding exogenous protoheme, select energy for growth.With 400/10 ⁷Frequency from the pFL61 library, reclaim protoheme prototroph.The sequential analysis of 22 complementation clonings is shown that 9 expectations that belong to called after " protox-1 " can express the protox gene type of chloroplast(id) protox enzyme.

The pFL61 library is the yeast expression library that a kind of mouse ear mustard with two-way insertion belongs to cDNAs.Can also in bacterium, express these cDNAs.The protox cDNAs obviously interior ATG of the frame from about 10 amino acid 5 ' of yeast PGK 3 ' sequence in the carrier begins to the NotI cloning site and by the lacZ promotor of the above upstream of 300bp or by undetermined hiding bacterium promoter expression.Can suppress the growth of intestinal bacteria SASX38 bacterial strain because comprise the protox-1 cDNAs of the integral part of chloroplast transit sequence, so select the clone who has the minimum chloroplast transit sequence that connects to be used for mutagenesis/weedicide selection experiment.This clone pSLV19 only contains 17 amino acid of inferring chloroplast transit peptides, and wherein dna sequence dna starts from the bp-151 that mouse ear mustard belongs to protox-1 cDNA (SEQ IDNO:1).

With plasmid pSLV19 change over to random mutagenesis strain X L1-Red (Stratagene, La Jolla, CA).The transformant flat board is fixed in the L substratum that contains the 50ug/ml penbritin and at 37 ℃ to descend to be incubated 48 hours.Scrape the lawn of getting transformant and use WizardMegaprep test kit (Promega, Madison, WI) preparation plasmid DNA from described flat board.Expectation is separating in the plasmid DNA of this mutant strain, per 2000 Nucleotide contain at least have an appointment one at random sequence change (referring to Greener etc., strategy (Strategies) 7 (2): 32-34 (1994).

(Sasarman etc., hereditary microorganism magazine (J.Gen.Microbiol.) 113:297 (1979) and flat board are fixed in the L substratum of the weedicide (general formula X VII) that contains different concns inhibition protox to change the plasmid DNA of suddenling change over to hemG mutant SASX38.Under 37 ℃, should be incubated 2 days by flat board.Plasmid DNA is separated comfortable all bacterium colonies of growing under the weedicide concentration of wild type strain that effectively kill.Then separated DNA is changed over to SASX38 and dull and stereotyped once more being fixed on the weedicide to guarantee that viewed resistance is that plasmid has.By NotI digestion will from the protox encoding sequence excision of the plasmid by this screening, with its be cloned into again carrier of not mutagenesis and once more test produce the ability of herbicide tolerant.Then measure the dna sequence dna of the protoxcDNAs that produces Herbicid resistant and compare and identify sudden change by belonging to protox-1 sequence (SEQ ID NO:1) with wild-type mice ear mustard.

From the experiment of mutagenesis first, reclaim single sequence encoding mutant body.This mutant only improves weedicide " resistance " by increasing growth rate.Contain the sudden change of C → A on the 197th Nucleotide of its SEQ ID NO:1 in the pSLV19 of brachymemma chloroplast transit sequence, thereby on the 56th amino acids of SEQ ID NO:2, the ACG codon of Threonine is changed into the AAG codon of Methionin and makes bacteria variants obtain better complementation.This plasmid also contains reticent sequence encoding mutant on the 1059th Nucleotide, wherein AGT (Ser) changes over AGC (Ser).With this plasmid called after pMut-1.

As mentioned above the pMut-1 plasmid is changed over to mutant strain XL1-Red then and separate on the weedicide of the DNA of sudden change and the concentration that the dull and stereotyped pMut-1 protox gene that is fixed on not mutagenesis belongs to lethality.Separate the herbicide tolerant bacterium colony after following 2 days and analyze as mentioned above at 37 ℃.Show that many plasmids contain Herbicid resistant protox encoding sequence.Sequential analysis shows that resistant gene belongs to two classes.A kind of resistant mutation of being identified is that the C on the 689th Nucleotide of the mouse ear mustard genus protox-1 sequence of representing with SEQ ID NO:1 changes over T.This change the GCT codon of L-Ala on SEQ ID NO:2 the 220th amino acids is transformed into the GTT codon of Xie Ansuan and with its called after pAraC-1Val (referring to table 1B; Subsequence 3).

The second class Herbicid resistant mutant belongs in the protox-1 sequence change that contains A → G on the 1307th Nucleotide in mouse ear mustard.This change the TAC codon of tyrosine on the 426th amino acids is transformed into the TGC codon of halfcystine and with its called after pAraC-2Cys (referring to table 1B; Subsequence 7).

The third resistant mutants belongs in the protox-1 sequence change that contains G → A on the 691st Nucleotide in mouse ear mustard.This sudden change is transformed into the GGT codon of glycine on the 221st amino acids AGT codon of the Serine on the codon position of the sudden change adjacency among coding and the pAraC-1.With this plasmid called after pAraC-3Ser (referring to the table 1B; Subsequence 4).

With the resistant mutants pAraC-2Cys in the pMut-1 plasmid on November 14th, 1994 be deposited with farming research culture collection center (Agricultural Research CultureCollection), give its name be called pWDC-7 and give its deposit numbers NRRL #21339N.Embodiment 11: the getting of locational other Herbicid resistant codon of identifying in random screening

Generation

In random screening, be accredited as amino acid on the Herbicid resistant site by other aminoacid replacement and test its function and the herbicide tolerant in bacterial system.(Clontech, Palo Alto CA) carry out the oligonucleotide directed mutagenesis that mouse ear mustard belongs to the protox-1 sequence to use transformer site-directed mutagenesis test kit.After confirming amino acid change, change the plasmid of sudden change over to SASX38 and flat board is fixed on L-amp by sequential analysis ¹⁰⁰On the substratum so that test its function and suppress its tolerance of test under the concentration of weedicide of protox in difference.

Be used to make mouse ear mustard to belong to L-Ala codon on protox-1 sequence (SEQ ID NO:1) the 688-690 position Nucleotide and the tyrosine codon on the Nucleotide of 1306-1308 position this method.Result's proof can change over the L-Ala codon on the Nucleotide of 688-690 position the codon (pAraC-1Ile) of the codon (pAraC-1Val) of Xie Ansuan, the codon (pAraC-1Thr) of Threonine, the codon (pAraC-1Cys) of halfcystine, leucic codon (pAraC-1Leu) or Isoleucine to produce a kind of Herbicid resistant protox enzyme of function that keeps (referring to table 1B; Subsequence 3).The further proof of this result can change over the tyrosine codon on the Nucleotide of 1306-1308 position the codon (pAraC-2Cys) of halfcystine, the codon of Isoleucine (pAraC-2Ile), leucic codon (pAraC-2Leu), the codon of Threonine (pAraC-2Thr), the codon of methionine(Met) (pAraC-2Met), the codon (pAraC-2Ala) of codon of Xie Ansuan (pAraC-2Val) or L-Ala is to produce a kind of Herbicid resistant protox enzyme of function that keeps (referring to table 1B; Subsequence 7).Embodiment 12: its of the enzyme function of the resistant mutants that increase is identified in advance and/or Herbicid resistant

The separation of its sudden change

The plasmid that will contain Herbicid resistant protox gene changes mutant strain XL1-Red over to and separates the DNA that suddenlys change as mentioned above.The plasmid of sudden change is changed over to SASX38 and is being enough to suppress to screen mutant under weedicide (general formula X VII) concentration of original " resistance " mutant.The separation resistance bacterium colony also is accredited as higher tolerogenic phenotype as mentioned above and belongs to the encoding sequence dependency.Measure the sequence of these mutant and identify sudden change by comparing with my late grandfather's sequence.

This method is used for aforesaid pAraC-1Val mutant.Result's proof can change over the leucine codon with the Serine codon on the 305th amino acids (SEQ ID NO:2) so that produce the enzyme that the tolerance that the weedicide that suppresses protox is had is higher than independent pAraC-1Val mutant.With this second kind of point mutation called after AraC305Leu (referring to the table 1B; Subsequence 13).Threonine codon on the 249th amino acids has been proved identical result, wherein changed over Isoleucine or change over this result of L-Ala and produced the enzyme that has more tolerance (referring to table 1B; Subsequence 12).Respectively these are changed called after AraC249Ile and AraC249Ala.

This method is used for above-mentioned pAraC-2Cys mutant.Result's proof can change over the leucine codon with the proline(Pro) codon on the 118th amino acids (SEQ ID NO:2) so that produce the enzyme that the tolerance that the weedicide that suppresses protox is had is higher than independent pAraC-1Cys mutant.With this sudden change called after AraC118Leu (referring to the table 1B; Subsequence 11).Serine codon on the 305th amino acids has been proved identical result, wherein changed over this result of leucine and produced the pAraC-2Cys enzyme that has more tolerance (referring to table 1B; Subsequence 13).Also use the pAraC-1Val mutant to separate this change as mentioned above and with its called after AraC305Leu.Other sudden change that improves the Herbicid resistant of pAraC-2Cys mutant comprises the l-asparagine on the 425th amino acids is changed over Serine, called after AraC425Ser (table 1B; Subsequence 14) and with the tyrosine on the 498th amino acids change over halfcystine, called after AraC498Cys (table 1B; Subsequence 15).

With these changes (table 1B; Subsequence 11-15) be called " second site " sudden change, because they are not enough to produce separately herbicide tolerant, but the function and/or the herbicide tolerant of the enzyme that the obvious raising of meeting has suddenlyd change.This result is not precluded within the possibility that other aminoacid replacement on these sites is enough to produce the enzyme of Herbicid resistant, because detailed test is not carried out in all possible replacement.Embodiment 13: the resistant mutation of identifying is combined with second site mutation of evaluation and produce and possess

Higher functionality/than the Protox enzyme of height endurability

Find the function/Herbicid resistant of above-mentioned AraC305Leu sudden change can the raising AraC-1Val and AraC-2Cys mutant plasmid.In the trial of the general application of testing this second site mutation, it and AraC-2Leu, AraC-2Val and AraC-2Ile abrupt junction are merged the test herbicide tolerant.In each case, AraC305Leu changes the growth rate that has significantly increased resistance protox mutant in the presence of the weedicide that suppresses protox.AraC-2Ile resistant mutants and the second site mutation body AraC249Ile or AraC118Leu combination also produced possessed the more mutant protox enzyme of height endurability.The AraC249Ile sudden change shows that second site mutation that is accredited as increase AraC-1 (subsequence 3) mutant can also increase the tolerance of AraC-2 (subsequence 7) mutant.Also confirm to contain AraC-2Ile, AraC305Leu and AraC249Ile (table 1B; Subsequence 7,13 and 12) plasmid of three kinds of sudden changes has produced the protox-1 enzyme that possesses higher functionality, higher herbicide tolerant.Embodiment 14: can suddenly change in the corn Protox-1 gene and produce the mirror in the site of Herbicid resistant

Fixed

It is very effective when being used for mutagenesis/screening experiment that above-mentioned mouse ear mustard belongs to protox-1 plasmid pMut-1, and promptly opposite with isolating common synergy promoter mutation body when using other plasmid, this plasmid produces high-frequency true sequence encoding mutant body.Be used for the trial of effective plasmid selective system of corn protox-1 in generation, with corn cDNA be transformed into the pMut-1 carrier, in that to belong to the sequence of cDNA identical with mouse ear mustard basically aspect the sequence.The overlapping PCR fusion method of use standard merges the 5 ' end that pMut-1 mouse ear mustard belongs to clone's (17 amino acid that comprise the chloroplast transit peptides that has aforesaid a kind of missense mutation) with the corn protox-1 cDNA sequence that begins from corn sequence (SEQ ID NO:6) the 14th amino acids.3 ' the terminal not change of corn cDNA.The NotI restriction site is placed the two ends of this syzygy and mosaic gene is cloned into pFL61 plasmid main chain from pMut-1.Sequential analysis has shown the mononucleotide PCR deutero-silent mutation that the ACG codon on the 745-747 position Nucleotide (SEQ ID NO:5) is transformed into the ACT codon, the equal codified Threonine of wherein said ACG codon and ACT codon.With this chimeric Arab-corn protox-1 plasmid called after pMut-3.

As mentioned above plasmid pMut-3 is changed over to mutant strain XL1-Red and separate the DNA of sudden change and be under the lethal weedicide concentration (general formula X VII) that it is dull and stereotyped fixing at pMut-3 corn protox gene for not mutagenesis.Separate the herbicide tolerant bacterium colony after following 2 days and analyze as mentioned above at 37 ℃.This analysis shows that a plurality of plasmids contain Herbicid resistant protox encoding sequence.Sequential analysis demonstrates 5 single sequence changes that produce herbicide tolerant corn protox-1 enzyme separately.Tolerific amino acid change on three kinds of same source positions that are equivalent to be presented in advance in the mouse ear mustard genus protox-1 gene in these sudden changes.Two kinds in three kinds of sudden changes is pMzC-1Val and pMzC-1Thr, wherein the L-Ala (GCT) on the 164th amino acids (SEQ ID NO:6) is transformed into Xie Ansuan (GAT) or has been transformed into Threonine (ACT).This position is equivalent to above-mentioned pAraC-1 sudden change (referring to table 1B; Subsequence 3).The third similarly changes pMzC-3Ser the glycine on the 165th amino acids (GGT) has been transformed into Serine (AGT), and it is equivalent to above-mentioned AraC-3Ser sudden change (referring to table 1B; Subsequence 4).These results are used for verifying that the sudden change at the herbicide-resistant that a kind of plant protox gene is identified also can produce the such prediction of herbicide tolerant in from the identical plant protox gene of another kind.

The amino acid that the two kind sudden changes of separation in the sudden change of corn protox-1 screening cause not being accredited as in advance on the residue in Herbicid resistant site changes.A kind of change (Mz159Phe) is that the halfcystine (TGC) on corn protox-1 sequence (SEQ ID NO:6) the 159th amino acids is transformed into phenylalanine (TTC) (referring to table 1B; Subsequence 2).Second kind of change (Mz419Thr) is that the Isoleucine on the 419th amino acids (ATA) is transformed into Threonine (ACA) (referring to table 1B; Subsequence 9).

Carry out testing on other aminoacid replacement and in maize mutant position point three.Be changed into leucine (Mz159Leu) or be changed into Methionin (Mz159Lys) when 165 glycine are changed into leucine (pMzC-3Leu) or work as 159 halfcystines (referring to table 1B; Subsequence 4 and 2) time, tolerance is confirmed.By 419 Isoleucines being changed over Histidine (Mz419His), glycine (Mz419Gly) or l-asparagine (Mz419Asn) (referring to table 1B; Subsequence 9) also can produce the tolerance enzyme.

Each amino acid change that will produce the mouse ear mustard genus protox-1 enzyme of height herbicide tolerant by aforesaid site-directed mutagenesis is introduced corn protox-1 gene.The bacterium test shows changes over this result of leucine (CTT) with the L-Ala (GCT) on 164 amino acids (SEQ ID NO:6) can produce high resistance corn enzyme (pMzC-1Leu) (referring to table 1B; Subsequence 3).In the corn random screening, do not isolate and AraC-2 site (table 1B; Subsequence 7) similarly sudden change.Yet, 370 tyrosine on this site in the corn enzyme (SEQ ID NO:6) are changed over Isoleucine (pMzC-2Ile) or this result of methionine(Met) (pMzC-2Met) have produced the herbicide tolerant enzyme really (referring to table 1B; Subsequence 7).

As described in the previous section of present embodiment, carry out other screening mutant, but use general formula X XIIIa and XXIIIb rather than general formula X VII, these Screening and Identification produce tolerance protox enzyme other three seed amino acids change.A kind of screening of using general formula X XIIIb to carry out shows that the arginine (CGT) on the 88th amino acids (SEQ ID NO:6) is changed over this result of halfcystine (TGT) has produced high resistance corn enzyme (Mz88Cys) (referring to table 1B; Subsequence 1).The another kind screening of using general formula I Vc to carry out shows that the arginine (CGT) on the 88th amino acids (SEQ ID NO:6) is changed over this result of leucine (CTT) has produced high resistance corn enzyme (Mz88Cys) (referring to table 1B; Subsequence 1).The another kind screening of using general formula X XIIIa to carry out shows the leucine (TTA) on the 347th amino acids (SEQ ID NO:6) changed over Serine (TCA) and the L-Ala (GCA) on the 453rd amino acids (SEQ ID NO:6) is changed over this result of Threonine (ACA) and has produced high resistance corn enzyme (Mz347Ser453Thr) (referring to table 1B; Subsequence 16 and 17).Different with second kind of point mutation of the enzyme function of the above-mentioned resistant mutants that improves above-mentioned evaluation and/or herbicide tolerant, Mz347Ser453Thr a kind ofly requires two kinds of sudden changes to have " double mutant " because of herbicide tolerant.

Two kinds of other amino acid changes that can produce tolerance protox enzyme have been identified as the present embodiment other screening mutant that carries out noted earlier.A kind of screening of using general formula X XIV to carry out shows the L-Ala (GCA) on the 175th amino acids (SEQ ID NO:6) changed over Xie Ansuan (GTA) or change over this result of Threonine (ACA) and has produced high resistance corn enzyme (being respectively Mz175Val and Mz175Thr) (referring to table 1B; Subsequence 18).The another kind screening of using general formula I Vc to carry out shows that the leucine (TTG) on the 337th amino acids (SEQ ID NO:6) is changed over this result of Serine (TCG) has produced high resistance corn enzyme (Mz337Ser) (referring to table 1B; Subsequence 19).Embodiment 15: can suddenly change in the wheat Protox-1 gene and the site that produces Herbicid resistant

Identify

In order to generate the effective plasmid screening system that is used for wheat protox-1, as mentioned above wheat cDNA is imported the pMut-1 carrier of corn cDNA.With this chimeric Arab-wheat protox-1 plasmid called after pMut-4.PMut-4 DNA is undergone mutation and screen herbicide tolerant.This analysis has shown a plurality of plasmids that contain Herbicid resistant protox encoding sequence.Sequential analysis demonstrates 7 single sequence changes that produce herbicide tolerant wheat protox-1 enzyme separately.Tolerific amino acid change on four kinds of same source positions that are equivalent to be presented in advance in mouse ear mustard genus and/or the corn protox-1 gene in these sudden changes.Two kinds of sudden changes pWhtC-1Val and pWhtC-1Thr are transformed into the L-Ala (GCT) on the 211st amino acids (SEQ ID NO:10) Xie Ansuan (GAT) or have been transformed into Threonine (ACT) respectively.This position is equivalent to above-mentioned pAraC-1 sudden change (referring to table 1B; Subsequence 3).The third similarly changes pWhtC-3Ser the glycine on the 212nd amino acids (GGT) has been transformed into Serine (AGT), and it is equivalent to above-mentioned AraC-3Ser sudden change (referring to table 1B; Subsequence 4).The 4th kind similarly changes Wht466Thr the Isoleucine on the 466th amino acids (ATA) has been transformed into Threonine (ACA), and it is equivalent to Mz419Thr mutant from corn (referring to table 1B; Subsequence 9).

The amino acid that separation causes not being accredited as in advance from three kinds of sudden changes of wheat protox-1 screening on the residue in Herbicid resistant site changes.A kind of change (Wht356Leu) is that the Xie Ansuan (GTT) on wheat protox-1 sequence (SEQ ID NO:10) the 356th amino acids has been changed over leucine (CTT) (referring to table 1B; Subsequence 6).Second kind of change (Wht421Pro) is that the Serine (TCT) on the 421st is transformed into proline(Pro) (CCT) (referring to table 1B; Subsequence 8).The third change (Wht502Ala) is that the Xie Ansuan on the 502nd amino acids (GTT) is transformed into L-Ala (GCT) (referring to table 1B; Subsequence 10).Embodiment 16: can suddenly change in the soybean Protox-1 gene and produce the mirror in the site of Herbicid resistant

Fixed

In order to generate the effective plasmid screening system that is used for soybean protox-1, as mentioned above soybean cDNA is imported the pMut-1 carrier of corn cDNA.With this chimeric Arab-soybean protox-1 plasmid called after pMut-5.PMut-5 DNA is undergone mutation and screen herbicide tolerant.This analysis has shown a plurality of plasmids that contain Herbicid resistant protox encoding sequence.Sequential analysis demonstrates 4 single sequence changes that produce herbicide tolerant soybean protox-1 enzyme separately.Tolerific amino acid change on two kinds of same source positions that are equivalent to be presented in advance in mouse ear mustard genus and/or the wheat protox-1 gene in these sudden changes.A kind of sudden change pSoyC-1Thr has been transformed into Threonine (ACA) with the L-Ala (GCT) on the 226th amino acids (SEQ ID NO:12).This position is equivalent to above-mentioned pAraC-1Thr sudden change (referring to table 1B; Subsequence 3).Second kind of similar Soy517Ala that changes has been transformed into L-Ala (GCT) with the Xie Ansuan on the 517th amino acids (GTT), and it is equivalent to suddenly change (referring to table 1B from the Wht502Ala of wheat; Subsequence 10).

The amino acid that separation two kinds in the sudden change of soybean protox-1 screening cause not being accredited as in advance on the residue in Herbicid resistant site changes.A kind of change (Soy369Ser) is that the proline(Pro) (CCT) on soybean protox-1 sequence (SEQ ID NO:12) the 369th amino acids has been transformed into Serine (TCT) (referring to table 1B; Subsequence 5).Second kind of change (Soy369His) is to go up identical proline(Pro) (CCT) with the 369th to be transformed into Histidine (CAT) (referring to table 1B; Subsequence 5).

Each amino acid change that will produce the mouse ear mustard genus protox-1 enzyme of height herbicide tolerant by aforesaid site-directed mutagenesis is introduced soybean protox-1 gene.The bacterium test shows changes over this result of leucine (pSoyC-1Leu) with the L-Ala (GCA) on 226 amino acids (SEQ ID NO:12) and has produced tolerance soybean enzyme (referring to table 1B; Subsequence 3).Tyrosine on the 432nd amino acids (TAC) is changed over leucine (pSoyC-2Leu) or this result of Isoleucine (pSoyC-2Ile) also produced the herbicide tolerant enzyme (referring to table 1B; Subsequence 7).Embodiment 17: can suddenly change in the beet Protox-1 gene and produce the mirror in the site of Herbicid resistant

Fixed

In order to generate the effective plasmid screening system that is used for beet protox-1, as mentioned above beet cDNA is imported the pMut-1 carrier of corn cDNA.With this chimeric Arab-beet protox-1 plasmid called after pMut-6.PMut-6 DNA is undergone mutation and screen herbicide tolerant.This analysis has shown a plurality of plasmids that contain Herbicid resistant protox encoding sequence.Sequential analysis demonstrates the single sequence change that produces herbicide tolerant beet protox-1 enzyme.This change (pSugC-2Cys) has been transformed into halfcystine (TGC) and similar (referring to table 1B with the AraC-2 sudden change in the mouse ear mustard genus with the tyrosine on the 449th amino acids (TAC); Subsequence 7).

Each amino acid change that will produce the mouse ear mustard genus protox-1 enzyme of height herbicide tolerant by aforesaid site-directed mutagenesis is introduced beet protox-1 gene.The bacterium test shows changes over leucine (pSugC-2Leu), Isoleucine (pSugC-2Ile), Xie Ansuan (pSugC-2Val) or this result of methionine(Met) (pSugC-2Met) with the tyrosine on 449 amino acids (TAC) and has produced herbicide tolerant beet enzyme (referring to table 1B; Subsequence 7).Embodiment 18: can suddenly change in the cotton Protox-1 gene and produce the mirror in the site of Herbicid resistant

Fixed

Be used for the trial of effective plasmid screening system of cotton protox-1 in generation, as mentioned above cotton cDNA imported the pMut-1 carrier of corn cDNA.With this chimeric Arab-cotton protox-1 plasmid called after pMut-7.PMut-7 DNA is undergone mutation and screen herbicide tolerant.This analysis has shown a plurality of plasmids that contain Herbicid resistant protox encoding sequence.Sequential analysis demonstrates 3 single sequence changes that produce herbicide tolerant beet protox-1 enzyme separately.Three kinds of mutant pCotC-2Cys, pCotC-2His and pCotC-2Arg have been transformed into the tyrosine (TAC) on the 428th amino acids (SEQ ID NO:16) halfcystine (TGC), Histidine (CAC) and arginine (CGC) respectively (referring to table 1B; Subsequence 7).Arginine be a kind of on AraC-2 (subsequence 7) site of identifying in advance tolerific new substituted.The third sudden change (Cot365Ser) is that the proline(Pro) on the 365th amino acids (CCT) has been transformed into Serine (TCT).This change is equivalent to soybean mutant Soy369Ser (referring to table 1B; Subsequence 5).Embodiment 19: resistant mutation is to the confirmation of the cross resistance of the compound of difference inhibition Protox

At a series of other protox inhibition compounds tests at first based on resistant mutants plasmid to the tolerance of single protox inhibition weedicide.In order to carry out this test, the SASX38 bacterial strain flat board that will contain wild plasmid is fixed on the lethality concentration of measuring on every kind of compound of a series of agricultural degree every kind of bacterial strain.Carry out the resistant mutants plasmid among the SASX38 dull and stereotyped fixing and its survival ability under each compound concentrations is given a mark, described each compound concentration is higher than at least the SASX38 bacterial strain that contains wild plasmid is belonged to 10 times of lethality concentration.

As show to explain among 3A and the 3B from bacterium cross resistance result of experiment show each in the evaluation sudden change all can produce tolerances to different protox inhibition compounds.2.C part: Herbicid resistant Protox expression of gene embodiment 20 in the transgenic plant: by homologous recombination or genetic modification transformation planting to the weedicide that suppresses Protox

The thing tolerance

Because described mutant code sequence can produce herbicide tolerant when being expressed effectively under natural protox promotor control, so the directed change representative of the protox encoding sequence on its natural dyeing body position is produced the another kind of mode of herbicide-resistant plant and vegetable cell.Method that can be by any several prior aries (for example Agrobacterium conversion method, directly with method, the microprojectile bombardment methods of transgenosis to protoplastis) imports and contain the transformant that required sudden change lacks the protox dna fragmentation of himself expression signal (promotor or 3 ' the untranslated district) and selects herbicide-resistant.The dna fragmentation that is imported also has a diagnosis and imports and can not change other sequence polymorphism of coded aminoacid sequence (being silent mutation) with restriction enzyme sites or by external site-directed mutagenesis.As in the past to various selective markers and herbicide tolerant gene reported (for example, referring to EMBO such as Paszkowski J.7:4021-4026 (1988); Lee etc., vegetable cell (Plant Cell2): 415-425 (1990); Risseeuw etc., plant magazine (Plant J.) 7:109-119 (1995)).Find that some transformant produces because of the mutant sequence that the mutant dna homology is integrated into protox chromogene seat or changes into importing because of natural proto chromosome sequence.By existing diagnosis to discern these transformant with restriction enzyme sites in conjunction with the phenotype of herbicide-resistant with on its protox chromogene seat.3. embodiment 21: the structure of plant conversion carrier

A large amount of conversion carriers are used for Plant Transformation and gene of the present invention can be used with any this class carrier.Preferred transformation technology and the target kind that is used to transform are depended in the selection of used carrier.With regard to some target kind, microbiotic or weedicide selective marker that can be preferably different.Normally used selective marker comprises nptII gene (the Messing ﹠amp that can produce resistance to kantlex and associated antibiotic in conversion; Vierra, gene (Gene) 19:259-268 (1982); Bevan etc., nature 304:184-187 (1983)), the weedicide phosphinothricin is produced the bar gene (White etc. of resistance, nucleic acids research (Nucl Acids Res) 18:1062 (1990), Theor Appl Genet 79:625-631 (1990) such as Spencer), antibiotic hygromycin is produced the hph gene (Blochinger of resistance; Diggelmann, molecular cytobiology (Mol CellBiol) 4:2929-2931) and methotrexate is produced the dhfr gene of resistance (Bourouis etc., EMBO are (7) J.2: 1099-1104 (1983)).I. the structure that is suitable for the carrier of Agrobacterium conversion

Many carriers are used to the conversion of using Agrobacterium tumefaciens to carry out.They generally carry at least a T-DNA edge sequence and comprise such as pBIN19 (Bevan, nucleic acids research (Nucl.AcidsRes.) (1984)) and the such carrier of pXYZ.The structure of two class carriers is described below.

The structure of pCIB200 and pCIB2001: the binary vector of pCIB200 and pCIB2001 is used for Agrobacterium recombinant vectors structure and make up according to following manner.By to pTJS75 (Schmidhauser ﹠amp; Helinski, bacteriology magazine (J Bacteriol.) 164:446-455 (1985)) carries out NarI digestion and downcut tetracycline resistance gene, insert from carrying NPTII (Messing ﹠amp subsequently; Vierra, gene (Gene) 19:259-268 (1982); Bevan etc., natural 304:184-187 (1983); McBride etc., molecular biology of plants (PlantMolecular Biology) 14:266-276 (1990)) the AccI fragment of pUC4K generates pTJS75kan.The XhoI joint is connected with the EcoRV fragment of pCIB7, it contains the left side of T-DNA and right side edge, the selectable nos/nptII mosaic gene of plant and pUC polylinker (Rothstein etc., gene (Gene) 53:153-161 (1987)) and the fragment cloning of XhoI-digestion is gone into the pTJS75kan of SalI-digestion and generated pCIB200 (in addition referring to EP 0 332104, embodiment 19).PCIB200 contains the polylinker restriction site of following uniqueness: EcoRI, SstI, KpnI, BglII, XbaI and salI.PCIB2001 is the derivative of a kind of pCIB200 that produces by the polylinker that inserts other restriction site.Restriction site unique in the pCIB2001 polylinker is EcoRI, SstI, KpnI, BglII, XbaI, SalI, MluI, BclI, AvrII, ApaI, HpaI and StuI.PCIB2001 also has plant transformed and the bacterium kantlex selectivity that is used for Agrobacterium mediation except that containing these unique restriction sites, left side and T-DNA edge, right side, be used for the RK2-deutero-trfA function and other OriT and the OriV function from RK2 that shift between intestinal bacteria and other host.The pCIB2001 polylinker is suitable for cloning the expression of plants box that contains himself conditioning signal.

The structure of pCIB10 and Totomycin selective derivatization thing thereof: binary vector pCIB10 contains in the coded plant gene of kalamycin resistance, T-DNA right side and the left side edge sequence of selecting usefulness and has merged sequence from promiscuous plasmid pRK252, makes it to duplicate in intestinal bacteria and Agrobacterium.It makes up as Rothstein etc. described in gene (Gene) 53:153-161 (1987).As Gritz etc. at gene (Gene) 25:179-188 (1983)) described in made up the various derivatives of the pCIB10 that is associated with the gene that is used for hygromycin B phosphotransferase.These derivatives can only be selected transgenic plant cells under the situation that Totomycin (pCIB743) or Totomycin and kantlex (pCIB715, pCIB717) arranged.II. the structure that is suitable for the carrier of non-Agrobacterium conversion

Do not use the conversions of Agrobacterium tumefaciens to satisfy in the conversion carrier of selecting, can also use the carrier that lacks these sequences to the demand of T-DNA sequence and thus except that the carrier such such as the above-mentioned T-DNA of containing sequence.The transformation technology that does not rely on Agrobacterium comprises the conversion of being undertaken by particle bombardment, protoplastis picked-up (for example PEG and electroporation) and microinjection.The preferential selection of depending on to a great extent the kind that is transformed that is chosen in to carrier.The structure of some typical carriers is described below.

The structure of pCIB3064: pCIB3064 is a kind of pUC-deutero-carrier that is suitable for the direct gene transfer techniques of selecting by weedicide Glufosinate (or phosphinothricin) to be used in combination.Plasmid pCIB246 comprises operationally the CaMV 35S promoter that merges with the intestinal bacteria gus gene and CaMV 35S transcription terminator and is described in PCT and openly applies among the WO 93/07278.The 35S promoter of this carrier contains two ATG sequences 5 ' in the initiation site.Use standard round pcr is undergone mutation these sites, removes ATG ' s in such a way and produces restriction site SspI and PvuII.New restriction site is apart from unique SalI site 96 and 37-bp and actual initiation site 101 and the 42-bp of distance.PCIB246 derivative called after pCIB3025 with gained.Then by digesting with SSalI and SacI and from pCIB3025, downcutting gus gene, make terminal blunt endization and connect into plasmid pCIB3060 again.Plasmid pJIT82 is available from John Innes Centre, Norwich and downcut the HpaI site (Thompson etc., EMBO J 6:2519-2523 (1987)) of containing the 400-bp SmaI fragment of bar gene and inserting pCIB3060 from streptomyces viridochromogenes.This step produces pCIB3064, it be included in the control of CaMV 35S promoter down the bar gene and be used for terminator, the ampicillin resistance gene (being used for selecting) of weedicide selection and have the polylinker of unique site SphI, PstI, HindIII and BamHI intestinal bacteria.This carrier is suitable for cloning the expression of plants box that contains himself conditioning signal.

The structure of pSOG19 and pSOG35: pSOG35 is the conversion carrier of a kind of use bacillus coli gene Tetrahydrofolate dehydrogenase (DHFR) as the selective marker that produces the methotrexate resistance.With PCR be used to increase 35S promoter (～800-bp), from the intron 6 of corn Adh1 gene (～550-bp) and 18-bp from the untranslated leader sequence of the GUS of pSOG10.The 250-bp fragment by pcr amplification coding intestinal bacteria Tetrahydrofolate dehydrogenase II type gene and come free pUC19 carrier main chains and nopaline synthase to stop the SacI-PstI fragment of molecular pBI221 (Clontech) for these two kinds of PCR fragments assemblings also.Assemble these fragments and produced pSOG19, it contains 35S promoter, GUS leader, DHFR gene and the nopaline synthase terminator that merges with intron 6 sequences.The GUS leader that is replaced among the pSOG19 by the leader sequence from corn chlorotic mottle poison (MCMV) has produced carrier pSOG35.PSOG19 and pSOG35 carry the pUC gene that is used for amicillin resistance and have HindIII, SphI, PstI and the EcoRI site that can be used for cloning exogenous array.4. embodiment 22: the structure of expression of plants box

At first will be used for gene order that transgenic plant express and be fitted into expression cassette after suitable promotor and the suitable transcription terminator upstream.These can be expressed cartridge clip then and change plant conversion carrier in the foregoing description 21 easily over to.I. promotor is selected

Selection to used promotor in the expression cassette will determine genetically modified room and time expression pattern in the transgenic plant.Transgenosis and this selection that the promotor of selecting will be expressed in particular cell types (such as cell, mesophyll cell, the root tegumental cell of leaf epidermis) or particular organization or the organ (for example root, leaf or flower) reflect the transgene expression desired position.On the other hand, selected promotor can start the expression of described gene under promotor control photoinduced or that regulate At All Other Times.The another kind of selection is to regulate selected promotor with chemical mode.It is only handled at needs with chemical inducer the possibility of inducing transgene expression is provided when producing.II. transcription terminator

Various transcription terminators are used for expression cassette.They be stop except that transgenosis transcribe and correct polyadenylation requisite.Suitable transcription terminator is those known terminators that work in plant and the terminator (i.e. " protox terminator ") that comprises CaMV 35S terminator, tml terminator, nopaline synthase terminator, pea rbcS E9 terminator and natural and plant protox gene-correlation.They can be used for monocotyledons and dicotyledons.III. be used to the enhancing expressed or the sequence of adjusting

Have been found that many sequences can promote from the genetic expression in the transcription unit and these sequences can be used with gene of the present invention so that increase its expression in transgenic plant.

Confirmed that different intron sequences can promote to express, the especially expression in the monocot plant cell.For example, the intron that has been found that corn Adh1 gene can significantly promote the expression of wild type gene under its connection promotor control when being imported into maize cell.Find introne 1 with the fusion constructs of chloramphenicol acetyl transferasegene in expression especially effectively and obtain increasing gene exploitation (Genes Develop.) 1:1183-1200 (1987) such as () Callis.In identical experimental system, has similar effect (Callis etc., document is the same) aspect the expression strengthening from the intron of corn bronzel gene.Usually intron sequences imported plant conversion carrier, generally be its untranslated leader.

The also known many untranslated leader sequence that derives from virus can promote to express and they are effective especially in the dicotyledons cell.Verified from tobacco mosaic virus (TMV) (TMV, " W-sequence "), the leader sequence of corn chlorotic mottle poison (MCMV) and alfalfa mosaic virus (AMV) strengthening aspect the expression especially effectively (Gallie etc. for example, nucleic acids research (Nucl.Acids Res.) 15:8693-8711 (1987); Skuzeski etc., molecular biology of plants (Plant Molec.Biol.) 15:65-79 (1990)).IV. the target of gene product in the cell

There are the various mechanism of target gene product in the known plants and will have controlled the sequence that these mechanism work to a certain extent and carry out concrete characterization.For example, by find at the range protein N-terminal and in the chloroplast(id) absorption process that produces mature protein the control of cracked signal sequence with the gene product target to chloroplast(id) (for example Comai etc., journal of biological chemistry (J.Biol.Chem.) 263:15104-15109 (1988)).These signal sequences and heterologous gene products can be merged the allos product is imported chloroplast(id) (van den Broeck etc., nature (Nature) 313:358-363 (1985)).Can separate the DNA of coding appropriate signal sequence by many other proteic 5 ' the end cDNAs from coding RUBISCO albumen, CAB albumen, EPSP synthase, GS2 albumen and known locations in chloroplast(id).

Other gene product is positioned such as plastosome and the such organoid (for example Unger et al. molecular biology of plants (Plant Molec.Biol.) 13:411-418 (1989)) of peroxisome.Can also handle the coding these products cDNAs so that the heterologous gene products target to these organoids.The example of this class sequence is the adenosine triphosphatase and the mitochondrial specificity aspartic transaminase isotype of nuclear coding.Rogers etc. have described the target of pair cell proteoplast in periodical (Proc.Natl.Acad.Sci.USA) 82:6512-6516 of institute of the state-run academy of sciences of the U.S. (1985).

In addition, will make gene product target to the sequence of other cell cell carry out characterization.To be target carry out requisite (the Koehler ﹠amp of exocytosis to ER, apoplast with from the aleurone cell to the N-terminal sequence; Ho, vegetable cell (Plant Cell) 2:769-783 (1990)).In addition, the N-terminal sequence with the C-terminal sequence together with the vacuole target that is gene product requisite (Shinshi etc., molecular biology of plants (Plant Molec.Biol.) 14:357-368 (1990)).

By being merged, above-mentioned suitable guide peptide and purpose transgenic sequence can be directed to organoid or cell cell arbitrarily by the render transgenic product.For example, with regard to the target of chloroplast(id), make chloroplast(id) signal sequence and genetically modified N-terminal ATG carry out the frame endomixis from RUBISCO gene, CABEPSP synthase gene or GS2 gene.Selected signal sequence should comprise the arbitrary amino acid after known cleavage site and constructed syzygy should be taken into account the required cleavage site of cutting.In these situations, can satisfy this demand by some amino acid that between described cleavage site and transgenosis ATG, adds a small amount of amino acid or replace on the other hand in this transgenic sequence.Can be by at the structure of external translation in-vitro transcription, use the described technology of document in the bracket (Bartlett etc.: Edelmann etc. (Eds.) chloroplast(id) molecular biology method (Methods in ChloroplastMolecular Biology), Elsevier.pp.1081-1091 (1982) subsequently; Wasmann etc., molecular gene genetics (Mol.Gen.Genet.) 205:446-453 (1986)) the picked-up situation of carrying out external chloroplast(id) is tested to importing the chloroplast(id) picked-up effect of the syzygy that chloroplast(id) makes up.These constructing technologies are well-known in the art and are equally applicable to plastosome and peroxysome.Cellular localization as the required precursor of the initiation site of given approach is depended in the selection of the required target of express transgenic.It is kytoplasm location or chloroplast(id) location normally, but also can be plastosome location or peroxysome location in some cases.The product of transgene expression does not need target to ER, apoplast or vacuole usually.

The used mechanism of above-mentioned cell-targeting not only can use with its connection promotor, can also use so that be issued to specific cell-targeting purpose in a kind of transcriptional regulatory of promotor with allogeneic promoter, and described promotor has the expression pattern that is different from the promotor that targeting signal originates.5. embodiment 23: the conversion of dicotyledons

The transformation technology of dicotyledons is well-known in the art and comprises based on the technology of Agrobacterium and do not need the technology of Agrobacterium.The technology of non-Agrobacterium comprises the step of directly passing through protoplastis or the exogenous genetic material of cellular uptake.This step is finished by the picked-up of PEG or electroporation mediation, the transhipment or the microinjection of particle bombardment mediation.The example of these technology by Paszkowski etc. at EMBO J 3:2717-2722 (1984), Potrykus etc. at molecular gene genetics (Mol.Gen.Genet.) 199:169-177 (1985), Reich et al., biotechnology (Biotechnology) 4:1001-1004 (1986) and Klein etc. describe in nature (Nature) 327:70-73 (1987).In each case, use standard technique as known in the art to make the complete plant of institute's cell transformed regeneration.

The conversion of Agrobacterium mediation is because of the high-level efficiency of its conversion and be used for many different types of extensive uses and become a kind of preferred technology that transforms dicotyledons.Usually can comprise tobacco, tomato, Sunflower Receptacle, cotton, rape, potato, soybean, clover and willow (EP 0 317 511 (cotton) by many varieties of crops that Agrobacterium transforms; EP 0 249 432 (tomato, Calgene); WO 87/07299 (Btassica, Calgene); US 4,795,855 (willows)).

With the reorganization Agrobacterium transform this process of target floristics generally include with Agrobacterium with from the common step of cultivating of the explant of plant and implement scheme well known in the art subsequently.Make being organized in of conversion of carrying the microbiotic that is present between the binary plasmid T-DNA edge or Herbicid resistant mark select to regenerate on the substratum.6. embodiment 24: monocotyledonous conversion

The conversion of most of monocotyledons kinds has also become conventional at present.Preferred technology comprises uses PEG or electroporation technology that gene directly is transferred to the method for protoplastis and the method that callus is gone in particle bombardment.Conversion can be used for unique DNA kind or many DNA kind (being cotransformation) and this two technology and all be applicable to the present invention.Cotransformation has avoids complex carrier to make up and the advantage that produces the transgenic plant that are not connected locus and selective marker that have goal gene, thereby can remove described selective marker in the generation subsequently, should regard this respect as needed.Yet, use the shortcoming of cotransformation to be that each DNA kind is integrated into genomic frequency and is lower than 100% biotechnology (Biotechnology) 4:1093-1096 (1986) such as () Schocher.

Described among patent application EP 0 292 435 (Ciba-Geigy), EP 0 392 225 (Ciba-Geigy) and the WO 93/07278 (Ciba-Geigy) by corn original seed inbred lines prepare callus and protoplastis technology, use PEG or electroporation to transform the technology of protoplastis and by the technology of the protoplast regeneration maize plant that transforms.Gordon-Kamm etc. in vegetable cell (Plant Cell) 2:603-618 (1990) and Fromm etc. the technology that makes alpha bombardment transform A188 deutero-corn is disclosed in biotechnology (Biotechnology) 8:833-839 (1990).In addition, WO 93/07278 application (Ciba-Geigy) and Koziel etc. have described the technology by the original seed inbred lines of particle bombardment maize transformation in biotechnology (Biotechnology) 11:194-200 (1993).This technology is used the long prematurity maize and the PDS-1000He Biolistics device that bombards usefulness of 1.5-2.5mm that downcuts from 14-15 days the mealie in back of pollinating.

Can also transform rice by technology or the particle bombardment of using protoplastis directly to carry out transgenosis.Described Japanese class and India's class have been carried out conversion (Zhang etc., the Plant Cell Rep 7:379-384 (1988) that protoplastis mediates; Shimamoto etc., nature (Nature) 338:274-277 (1989); Datta etc., biotechnology (Biotechnology) 8:736-740 (1990)).Usually also make alpha bombardment transform two types (Christou etc., biotechnology (Biotechnology) 9:957-962 (1991)).

Described among the patent application EP 0 332 581 (Ciba-Geigy) and be used to produce, transform and the technology of the Pooideae protoplastis of regenerating.These technology allow to transform orchardgrass and wheat.In addition, Vasil etc. have described in biotechnology (Biotechnology) 10:667-674 (1992) and have made alpha bombardment go into conversion and this technology that long-term reproducible C type callus cell carries out wheat also to be described in biotechnology (Biotechnology) 11:1553-1558 (1993) by Vasil etc.) in, and Weeks etc. have described in plant physiology (Plant Physiol.) 102:1077-1084 (1993) and make alpha bombardment immature embryo and immature embryo deutero-callus carry out the wheat transformed technology.Yet the optimization technique that is used for the wheat conversion comprises by immature embryo being carried out particle bombardment coming the step of transformed wheat and comprising high-sucrose or the high malt sugar step before gene delivery.Before bombarding, the embryo of any amount (0.75-1mm is long) flat board is fixed on and contains 3% sucrose (Murashige ﹠amp; Skoog, Physiologia Plantarum 15:473-497 (1962)) and 3mg/l 2, be used for the inductor somatic embryo on the MS substratum of 4-D, require this step to carry out in the dark.On the selected same day of bombarding, from inducing culture, take out embryo and flat board and be fixed on the osmoticum (promptly contain desired concn and be generally 15% sucrose or the inducing culture of maltose).Described embryo was carried out plasmolysis 2-3 hour and bombard then.In general each target flat board 20 embryos are arranged, but it is not crucial.Use standard step that the plasmid (such as pCIB3064 or pSG35) that carries suitable gene is deposited on the gold particle of micron size.Use the explosion pressure of application～1000psi and the DuPont Biolistics helium device of 80 mesh sieves to bombard each dull and stereotyped embryo.After the bombardment, these embryos are put back into dark sentence and just recover about 24 hours (still on osmoticum).After 24 hours, from osmoticum, take out these embryos and be put back on the inducing culture, they were stablized about 1 month.After about 1 month, the embryo explants that will have developmental embryo generation callus change over to also contain suitable selective agent (with regard to regard to the pCIB3064 be the 10mg/l Glufosinate and be the 2mg/l methotrexate with regard to pSOG35) regeneration culture medium (MS+1mg/ rises NAA, and 5mg/ rises GA) on.After about 1 month, the seedling of growing is changed in the big sterile chamber that is called " GA7s ", contain the selective agent of MS, 2% sucrose and the same concentrations of half intensity in this container.Described among the patent application WO 94/13822 and be used for method that wheat transforms and the document is incorporated herein by reference.

Embodiment 25: the separation of mouse ear mustard Protox-1 promoter sequence

λ Zap II genome dna library by mouse ear mustard (Columbia, complete plant) preparation is purchased from Stratagene.With about 25, the density flat board of 000pfu/15cm is fixed on the culture dish and the bipartite thing that leaches is placed on bacterium colony/plaque screening film on (NEN Dupont) with this libraries of about 125,000 phages.Use leaches thing by guiding method (Life Technologies) at random by mouse ear mustard genus protox-1 cDNA (SEQ ID NO:1) the detection plaque of 32P-dCTP mark.Hybridization and wash conditions are Church and Gilbert prints 65 ℃ described in (Proc.Natl.Acad.Sci.USA) 81:1991-1995 (1984) in institute of the state-run academy of sciences of the U.S..Forward is hybridized plaque to carry out purifying and is cut into the pBluescript plasmid in vivo.(Applied Biosystems, Inc.) chain termination method of the dideoxy terminator of mark is measured from described genomic dna and is inserted fragments sequence by fluorescence dye by using.After measured, the 580-bp mouse ear mustard that contains in the upstream of the initial methionine (ATG) of protox-1 albumen coded sequence of a kind of AraPT1Pro of clone belongs to sequence.This clone also contains the encoding sequence and the intron of the protox-1 cDNA sequence that extends to 1241bp.The non-encode fragment of 580-bp 5 ' is that mouse ear mustard genus protox-1 promotor and this sequence of inferring are represented with SEQ ID NO:13.

AraPT1Pro was deposited into pWDC-11 (NRRL #B-21515) December 15 nineteen ninety-five.Embodiment 26: express natural mouse ear mustard and belong to the Protox-1 base that changes after the Protox-1 promotor

The structure of the plant conversion carrier of cause

The full-length cDNA that the mouse ear mustard of appropriate change is belonged to protox-1 cDNA is separated into EcoRI-XhoI part digestion fragment and is cloned into plant expression vector pCGN1761ENX (referring to the embodiment 9 of the international application no PCT/IB95/00452 that submits to June 8 nineteen ninety-five, December nineteen ninety-five disclosed WO 95/34659 on the 21st).This plasmid is produced with NcoI and BamHI digestion by complete protox-1 cDNA with from the molecular fragment of Transcription Termination of 3 ' the untranslated sequence of Agrobacterium tumefaciens tml gene.Above-mentioned AraPT1Pro plasmid is digested to the mouse ear mustard genus protox-1 that is inferred by pBluescript and 580-bp with NcoI and BamHI starts molecular fragment.Connect these two fragments and the syzygy of mutagenic protox cDNA and natural protox promotor.Contain the expression cassette of protox-1 promotor/protox-1cDNA/tml terminator syzygy and it is cloned into binary vector pCIB200 by downcutting with KpnI digestion.Import Agrobacterium and use the vacuum infiltration method to penetrate mouse ear mustard then by electroporation and belong to and transform binary plasmid (Bechtold etc., C.R. Acad.Sci.Paris 316:1194-1199 (1993).On the weedicide of the inhibition protox of kantlex or different concns, select to express the transformant of the protox gene that changes.

Embodiment 27: melt by the Protox-1 that expresses natural Protox-1 promotor/change

Zoarium prepares the herbicide tolerant plant

Use above-mentioned steps will contain TAC → ATG (tyrosine → methionine(Met)) change on protox-1 sequence (SEQ ID NO:1) 1306-1308 position Nucleotide mouse ear mustard to belong to protox-1cDNA and the fusion of natural protox-1 promoter fragment and change mouse ear mustard over to.Verified when testing in above-mentioned bacterial expression system, the protox-1 enzyme (AraC-2Met) of this change is higher more than 10 times than the enzyme of natural appearance to the tolerance of the weedicide of various inhibition protox.Collect from the seed of the plant that vacuum is infiltrated and its flat board is fixed on protox inhibition aryi-uracile (aryluracil) weedicide of general formula X VII of certain limit (10.0nM-1.0uM).The repeatedly experiment of using wild-type mice ear mustard genus to carry out has proved that this compound of 10.0nM concentration is enough to prevent that normal seedling from germinateing.Express the AraC-2Met transgenic seed with enzyme natural protox-1 promotor fusion that change and under weedicide concentration, produced normal mouse ear mustard genus seedling, thereby the herbicide tolerant that shows it is higher more than 50 times than the herbicide tolerant that wild-type mice ear mustard belongs at least up to 500nM.The protox enzyme syzygy of this promotor/change is used from the effect of effective selective marker of Plant Transformation thus.To be transplanted in the soil at the several plant that 100.0nM suppresses to germinate on the weedicide of protox, make its growth 2-3 week and in a kind of spray testing the weedicide with the inhibition protox of different concns test.When comparing with empty carrier contrast transformant, the AraPT1Pro/AraC-2Met transgenic plant transform height more than 10 times to the tolerance of the weedicide of sprinkling than the empty carrier contrast.Embodiment 28: resistant mutation belongs to the chemical combination of inhibition protox different in the germination test to mouse ear mustard

The proof of the cross resistance of thing

Use above-mentioned steps containing mouse ear mustard genus protox-1 cDNA and the fusion of natural protox-1 promoter fragment that contains TCA → TTA (Serine → leucine) change on TAC → ATC (tyrosine → Isoleucine) change and the 945-947 position Nucleotide on protox-1 sequence (SEQ ID NO:1) the 1306-1308 position Nucleotide and to change mouse ear mustard over to.Verified when testing in a kind of bacterial expression system, the tolerance of the protox inhibition weedicide of protox-1 enzyme (AraC-2Ile+AraC305Leu) the mutual-through type XVII of this change is higher more than 10 times (referring to embodiment 9-13) than naturally occurring enzyme.Produce the homozygosity mouse ear mustard that contains this syzygy and belong to strain by in aforesaid seedling germination test, demonstrating the transformant that the weedicide that suppresses protox is had a height endurability.By repeating described germination test under the inhibited compound concentration that wild-type mice ear mustard belong to germinateed and test demonstrating from a kind of seed of strain cross resistance to different protox inhibition compounds.The result who draws from these experiments is as shown in table 4.7. embodiment 29: the separation of corn Protox-1 promoter sequence

Corn in the λ FIX II carrier (seedling that Missouri 17 inbreeding bleach) genome dna library is purchased from Stratagene.With about 250, this library of 000pfu is fixed on the culture dish with the density flat board of 50,000 phages/15cm and the bipartite thing that leaches is placed (NEN Dupont) on bacterium colony/plaque screening film.Use leaches thing by guiding method (Life Technologies) at random by corn protox-1 cDNA (SEQ ID NO:5) the detection plaque of 32P-dCTP mark.Hybridization and wash conditions are Church and Gilbert prints 65 ℃ described in (Proc.Natl.Acad.Sci.USA) 81:1991-1995 (1984) in institute of the state-run academy of sciences of the U.S..Use Wizard λ Preps dna purification system (Promega) from three forward hybridization phages, to separate lambda bacteriophage dna.Identified the λ clone who contains the about 3.5kb corn gene group DNA that is positioned at the corn protox-1 encoding sequence 5 ' that is separated into the cDNA clone in advance by restrictive diges-tion analysis, crossing pattern and dna sequence analysis.This fragment comprises corn protox-1 promotor.This fragments sequence is represented with SEQ ID NO:14.This sequence of from 1 to 3532 Nucleotide is made up of 5 ' non-coding sequence.Proteic 5 ' the end of this sequence encoding corn protox-1 of from 3533 to 3848 Nucleotide.

The plasmid that will contain the SEQ ID NO:14 sequence that merges with corn protox-1 encoding sequence remainder was deposited into pWDC-14 (NRRL #B-21546) on March 19th, 1996.

Embodiment 30: express the protox-1 base that changes after the natural corn Protox-1 promotor

The structure of the plant conversion carrier of cause

From isolating lambda particles phage clone, downcut as SalI-KpnI part digestion product 3848-bp corn gene group fragment (SEQ ID NO:14) and with derive from the KpnI-NotI fragment that contains the change corn protox-1cDNA that L-Ala → leucine changes in the 164th amino acids (SEQ ID NO:6) and be connected.This step produces a kind of natural corn protox-1 promotor and the verified syzygy (embodiment 9-14) that can produce the full-length cDNA of herbicide tolerant in bacterial system.This syzygy is cloned into the pUC18 deutero-carrier that contains CaMV 35S terminator sequence and the protox cDNA/ terminator box that generates a kind of protox promotor/change.The plasmid called after pWCo-1 that will contain this box.

Import again by first kind of intron finding in will encoding sequence and to generate the second kind of construct that is used for the corn conversion among the corn cDNA from corn gene group clone.The overlapping PCR integration technology of use standard is carried out this inserting step.Intron (SEQ ID NO:25) length is 93-bp and appears under natural surroundings among the λ clone described in the embodiment 29 as it, is inserted between the 203rd and 204 Nucleotide of SEQ IDNO:6.The expression cassette called after pWCo-2 that will contain this intron form.8. embodiment 31: the proof of corn Protox-1 promoter activity in the rotaring gene corn plant

Transgenosis is had pcr analysis that specific primer carries out identify maize plant by adopting with the protox syzygy conversion of corn protox promotor/changes.Prepare total RNA and under the condition of being advised, use Superscript M-MLV (LifeTechnologies) that it is carried out reverse transcription by forward PCR plant.The reverse transcription reaction thing of 2 microlitres is used for being designed to that the protox sequence that changes is had specific PCR reaction.Although unconverted reference substance does not produce product in this reaction, about 85% produced positive findings with the pWCo-1 plant transformed, thereby show that existence derives from genetically modified mRNA.There is the certain activity level in this result's proof to corn protox promotor.Use radiolabeled corn protox cDNA fragment is carried out standard to the RNA from rotaring gene corn plant as probe the careful engram analysis of promise that passes through in addition from SEQ ID NO:6.In some rotaring gene corn plant, detected the Protox-1 mRNA level that is significantly higher than those unconverted reference substances.According to the mRNA level of inferring this rising is the result of the protox-1 mRNA that changes of the corn protox promoter expression by the clone.9. embodiment 32: the separation of beet Protox-1 promoter sequence

In λ Fix II carrier, prepare the beet genomic library by Stratagene.With about 300, this library of 000pfu is dull and stereotyped fixing and with among the embodiment 29 the described garden beet protox-1 of corn cDNA sequence (SEQ ID NO:17) being surveyed.Identified the λ clone who contains the about 7kb beet genomic dna that is positioned at the beet protox-1 encoding sequence 5 ' that is separated into the cDNA clone in advance by restrictive diges-tion analysis, crossing pattern and dna sequence analysis.The fragment of the PstI-SalI of 2606-bp is cloned into the pBluescript carrier from the λ clone Central Asia.This fragment contains the 5 ' non-coding sequence of 2068-bp and comprises beet protox-1 promoter sequence.First intron of 85-bp that contains in the protox-1 encoding sequence of 453-bp and this encoding sequence before it also comprises.This fragments sequence is represented with SEQ ID NO:26.

The plasmid that will contain SEQ ID NO:26 sequence was deposited into pWDC-20 (NRRL #B-21650) on December 6th, 1996.

Embodiment 33: express the beet that changes after the natural beet Protox-1 promotor

The structure of the plant conversion carrier of Protox-1 gene

Downcut beet genomic fragment (SEQ IDNO:26) as the SacI-BsrGI fragment from the genome subclone described in the embodiment 32, it comprises the 5 ' non-coding sequence of 2068-bp and the beet protox-1 encoding sequence of preceding 300-bp.This fragment is connected with the BsrGI-NotI fragment that derives from the change beet protox-1cDNA that contains tyrosine → methionine(Met) change on the 449th amino acids (SEQ ID NO:18).This step produces a kind of natural beet protox-1 promotor and the verified syzygy (embodiment 9-14) that can produce the full-length cDNA of herbicide tolerant in bacterial system.This syzygy is cloned into the pUC18 deutero-carrier that contains CaMV 35S terminator sequence and the protox cDNA/ terminator box that generates a kind of protox promotor/change.The plasmid called after pWCo-3 that will contain this box.

Embodiment 34: by expressing the beet of natural beet Protox-1 promotor/change

The Protox-1 syzygy prepares the herbicide tolerant plant

To change beet over to from the expression cassette of pWCo-3 by any method for transformation that comprises Agrobacterium, protoplastis and biolistic transformation technology that is applied to dicotyledons.Identify the transgenic beet of expressing the protox-1 enzyme that changes and for unconverted beet be its tolerance of test under the lethality concentration by RNA-PCR the weedicide of inhibition protox.

D part: Protox expression of gene embodiment 35 in the plant plastid: contain the tobacco plastid clpP gene promoter and natural clpP 5 ' the untranslated preface that merge with GUS reporter gene and plastid rps16 gene 3 ' the untranslated sequence in the plastid conversion carrier

The preparation I. tobacco plastid clpP gene promoter of the mosaic gene of row and the amplification of complete 5 ' the untranslated RNA (5 ' UTR)

To carry out the template of PCR as a use left side → right side " cochain " primer and the right side → left side " chain down " primer from total DNA of tobacco (N.tabacum c.v.) " Xanthi NC ", described " cochain " primer is included in 197 EcoRI restriction site (primer Pclp_P1a:5 '-GCG that go up introducing for the ATG initiator codon of expressing plastid clpP gene in the composing type mode GAATTCATACTTATTTATCATTAGAAAG-3 ' (SEQ ID NO:27); Underline for the EcoRI restriction site); Described " down chain " primer with for the ATG initiator codon of the clpP promotor of the NcoI restriction site of having introduced on having merged the translation initiation position, belong to-regional homology (the primer Pclp_P2b:5 '-GCG of 21-1 position CCATGGTAAATGAAAGAAAGAACTAAA-3 ' (SEQ ID NO:28); Underline for the NcoI restriction site).In Perkin Elmer thermal cycler 480, use the heat-stable archaeal dna polymerase (Stratagene of Pfu, La Jolla CA), according to manufacturer's recommendation (PerkinElmer/Roche, Branchburg NJ) carries out this PCR reaction, and condition is as follows: 95 ℃ following 7 minutes; Be subsequently 95 ℃ following 1 minute/43 ℃ following 2 minutes/72 ℃ following 1 minute, amount to 4 circulations; Be then 95 ℃ following 1 minute/55 ℃ following 2 minutes/72 ℃ following 1 minute, amount to 25 circulations.Use standard step will comprise described promotor and on left end, contain the EcoRI site and contain at right-hand member the NcoI site the clpP gene 5 ' non-translational region and be equivalent to the 213-bp amplified production (Shinozaki etc. of the 74700-74505 position Nucleotide of tobacco plastid DNA sequence, EMBO is (1986) J.5:2043-2049) carry out gel-purified and digest with EcoRI and NcoI that (all restriction enzymes all are purchased the Biolabs from New England, Beverly, MA).II. the amplification of tobacco plastid rps16 gene 3 ' the untranslated RNA sequence (3 ' UTR)

As mentioned above will be from total DNA of tobacco (N.tabacum c.v.) " Xanthi NC " as using a left side → right side " cochain " primer and the right side → left side " under chain " primer carry out the template of PCR, described " cochain " primer comprises XbaI restriction site (primer rps16P_1a (the 5 '-GCG that follows introducing after the TAA terminator codon of the plastid rps16 gene of the ribosomal protein S16 that encodes closely TCTAGATCAACCGAAATTCAATTAAGG-3 ' (SEQ ID NO:30); Underline for the XbaI restriction site); Described " down chain " primer with for the TAA terminator codon of 3 ' the terminal rps16 that goes up the HindIII restriction site of introducing that has merged rps163 ' UTR, belong to+regional homology (primer rps16P_1b (5 '-CGC of 134-+151 AAGCTTCAATGGAAGCAATGATAA-3 ' (SEQ ID NO:31); Underline for the HindIII restriction site).Contain the XbaI site on the left end and carry out gel-purified and with XbaI and HindIII digestion being included at the 169-bp amplified production (Shinozaki etal., 1986) of 3 ' non-translational region of rps16 gene that right-hand member contains the HindIII site and contains the district of the 4943-5093 position Nucleotide that is equivalent to tobacco plastid DNA sequence.III.GUS reporter gene fragment and clpP gene promoter with 5 ' with being connected of 3 ' UTR ' s

By digest 1864-bp β-glucuronidase (GUS) reporter gene fragment of producing the plasmid pRAJ275 (Clontech) that derives from the NcoI restriction site that contains on the ATG initiator codon and the XbaI site behind natural 3 ' UTR with NcoI and XbaI.This fragment is connected so that make up plasmid pPH138 with 201-bp EcoRI/NcoI clpP promoter fragment, 157-bpXbaI/HindIII rps16 3 ' UTR fragment with from the 3148-bp EcoRI/HindIII fragment of cloning vector pGEM3Zf (-) (Promega, Madison WI) in the reaction of 4 kinds of modes.Make up plastid conversion carrier pPH140 by being connected with the 2222-bp EcoRI/HindIII fragment of pPH138 with EcoRI and HindIII digested plasmid pPRV111a (Zoubenko etc., 1994) and with the 7287-bp fragment of gained.Embodiment 36: contain the tobacco plastid clpP gene promoter and tobacco plastid psbA minimum 5 ' non-the turning over of merging with GUS reporter gene and plastid rps16 gene 3 ' non-translated sequence in the plastid conversion carrier

Translate the preparation of the mosaic gene of sequence

The amplification of 5 ' untranslatable rna of tobacco plastid clpP gene promoter and brachymemma (5 ' UTR): as mentioned above will be from total DNA of tobacco (N.tabacum c.v.) " Xanthi NC " as using a left side → right side " cochain " primer Pclp_P1a (SEQ ID NO:27) and a right side → left side " under chain " primer carry out the template of PCR, regional homology (the primer Pclp_P1b:5 '-GCG of described " under chain " primer and for the ATG initiator codon of the clpP promotor that has merged-11 XbaI restriction sites of upward introducing among the clpP5 ' UTR, belonging to-3--11 TCTAGAAAGAACTAAATACTATATTTCAC-3 ' (SEQ ID NO:29); Underline for the XbaI restriction site).To comprise described promotor and carry out gel-purified and digest with XbaI at the 202-bp amplified production of 5 ' UTR of brachymemma that contains the EcoRI site on the left end and on right-hand member, contain the clpP gene in XbaI site.Subsequently the XbaI site is filled up with Ke Lienuo archaeal dna polymerase (New England Biolabs) and this fragment is digested with EcoRI.It is connected so that make up plasmid pPH139 with last 38 Nucleotide that are equivalent to tobacco plastid psbA gene 5 ' UTR (overhang that has the NcoI restriction site of introducing the ATG initiator codon) and the double chain DNA fragment of ATG initiator codon in 5 kinds of modes are reacted, wherein said tobacco plastid psbA gene 5 ' UTR by make synthetic oligonucleotide minpsb_U (cochain: 5 '-GGGAGTCCCTGATGATTAAATAAACCAAGATTTTAC-3 ' (SEQ ID NO:32)) and minpsb_L (following chain: 5 '- CATGGTAAAATCTTGGTTTATTTAATCATCAGGGACTCCC-3 ' (SEQ ID NO:33); Underline for NcoI restriction site 5 ' overhang), above-mentioned NcoI/XbaI GUS reporter gene fragment, above-mentioned XbaI/HindIII rps163 ' UTR fragment and the annealing of above-mentioned EcoRI/HindIII pGEM3Zf (-) fragment and generate.Make up plasmid conversion carrier pPH144 by being connected with the 2251-bp EcoRI/HindIII fragment of pPH139 with EcoRI and HindIII digested plasmid pPRV111a (Zoubenko etc., nucleic acids research (Nucleic Acids Res) 22:3819-3824 (1994)) and with the 7287-bp fragment of gained.Embodiment 37: be used for carrier that the tobacco plastid transforms and contain the tobacco plastid clpP gene promoter that merges with mouse ear mustard Protox-1 encoding sequence and plastid rps16 gene 3 ' non-translated sequence and intact

The preparation of the mosaic gene of whole 5 ' non-translated sequence

To insert the DNA of a small amount of preparation of segmental plasmid AraC-2Met as the template of aforesaid PCR from carrying mouse ear mustard NotI, described mouse ear mustard NotI inserts fragment and comprises from encoding part N-terminal plastid transit peptides, the cDNA sequence of protoporphyrinogen IX oxydase (" the protox ") gene of full-length cDNA and part 3 ' non-translational region, wherein used PCR uses a left side → right side " cochain " primer (be the nucleotide homology of+172-+194 for the ATG initiator codon of total length precursor protein) and the right side → left side primer that " descends chain ", and described " cochain " primer is included in the NcoI restriction site introduced on the zero position of inferring of ripe protox albumen coded sequence and new ATG initiator codon (primer APRTXP1a:5 '-GGGA CCATGGATTGTGTGATTGTCGGCGGAGG-3 ' (SEQ ID NO:34); Underline for the NcoI restriction site); And described " down chain " primer with for the natural A TG initiator codon of protox precursor protein, be the nucleotide homology (primer APRTXP1b:5 '-CTCCGCTCTCCAGCTTAGTGATAC-3 ' (SEQ ID NO:35)) of+917-+940.With the product of 778-bp with NcoI and SfuI digestion and the 682-bp fragment of gained and 844-bp are comprised that the NcoI/NotI fragment of the SfuI/NotI dna fragmentation of protox encoding sequence 3 ' AraC-2Met partly and 2978-bp cloning vector pGEM5Zf (+) (Promega, Madison WI) connects make up plasmid pPH141.Make up the plastid conversion carrier pPH143 that contains the clpP promotor that can start the general formula X VII-resistance AraC-2Met protox gene that has rps163 ' UTR through the following steps: with NcoI and SspI digestion pPH141 and separate the 1491-bp fragment that contains complete protox encoding sequence, digest above-mentioned rps16P_1a with HindIII and be connected with the 7436-bp NcoI/HindIII fragment of pPH140 with the rps16P_1bPCR product and with them.Example 38: be used for carrier that the tobacco plastid transforms contain the tobacco plastid clpP gene promoter that merges with mouse ear mustard Protox-1 encoding sequence and plastid rps16 gene 3 ' non-translated sequence and

The preparation of the mosaic gene of tobacco plastid psbA gene minimum 5 ' non-translated sequence

Make up the plastid conversion carrier pPH145 that contains clpP promotor/psbA 5 ' UTR syzygy that can start the general formula X VII-resistance AraC-2Met protox gene that has rps16 3 ' UTR through the following steps: with NcoI and SspI digestion pPH141 and separate the 1491-bp fragment that contains complete protox encoding sequence, digest above-mentioned rps16P_1a with HindIII and be connected with the 7465-bp NcoI/HindIII fragment of pPH144 with the rps16P_1bPCR product and with them.Embodiment 39: be used for carrier that the tobacco plastid transforms and contain the tobacco plastid clpP gene promoter that merges with EPSP synthase coding sequence and plastid rps16 gene 3 ' non-translated sequence and 5 ' non-

The preparation of the mosaic gene of translation sequences

Screening is used for the cDNA library (U.S. Patent number US 5,310,667, US 5,312,910 and US 5,633,435 are incorporated herein by reference these documents) of 5-enolpyrul-3-shikimic acid synthase (EPSP synthase) gene.Molecule clone technology by standard separates the plasmid clone that contains total length epsp synthase gene cDNA.Design PCR primer is used to increase from the big or small EPSP synthase coding sequence of the maturation of this plasmid, strand primer and following strand primer in this PCR use, the wherein said strand primer of going up has to contain and is working the 5 ' extension of lighting the NcoI restriction site that inserts on the 1st amino acids, generation ATG initiator codon on this position thus from the maturation protein of inferring; And described strand primer down has the 5 ' extension in the XbaI restriction site downstream of the terminator codon that contains the ripe encoding sequence of EPSP in amplification PCR products.Use is carried out this pcr amplification according to the primer and the plasmid DNA template of standard scheme design.The product of clonal expansion and order-checking and by limiting digestion with NcoI and XbaI, carrying out the 0.8%TAE agarose gel electrophoresis and the band of cutting-out is carried out phenol extraction and separates the NcoI-XbaIDNA fragment that contains complete ripe EPSP synthase coding sequence.Make up through the following steps and contain the plastid conversion carrier of clpP promotor that orientation is transcribed the epsp synthase gene of the ripe size that has rps163 ' UTR: contain the fragment that described carrier main chain, the integration of 5 ' and 3 ' plastid guide peptide, aadA selective marker box and clpP promotor/rps16 3 ' UTR expressed sequence with NcoI and XbaI digestion pPH140 and purifying.This product is connected with the NcoI-XbaI dna fragmentation of the EPSP synthase coding sequence that contains isolating as mentioned above ripe size in the dual mode reaction.

Embodiment 40: the carrier that is used for the conversion of tobacco plastid contains the tobacco plastid clpP gene promoter and 5 ' non-the turning over of merging with ALS encoding sequence and plastid rps16 gene 3 ' non-translated sequence

Translate the preparation of the mosaic gene of sequence

Screening is used for the cDNA library (U.S. Patent number US5,013,659) of acetolactate synthase (ALS) gene.Molecule clone technology by standard separates the plasmid clone that contains total length als gene cDNA.Design PCR primer is used to increase from the big or small ALS encoding sequence of the maturation of this plasmid, strand primer and following strand primer in this PCR use, the wherein said strand primer of going up has to contain and is inserted in from 5 ' extension of the NcoI restriction site on the amino acid-1 of the maturation protein starting point of inferring, generation ATG initiator codon on this position thus; And described strand primer down has the 5 ' extension in the XbaI restriction site downstream of the terminator codon that contains the ripe encoding sequence of ALS in amplification PCR products.Use is carried out this pcr amplification according to the primer and the plasmid DNA template of standard scheme design.The product of clonal expansion and order-checking and by limiting digestion with NcoI and XbaI, carrying out the 0.8%TAE agarose gel electrophoresis and the band of cutting-out is carried out phenol extraction and separates the NcoI-XbaI dna fragmentation that contains complete ripe ALS encoding sequence.

Make up the plastid conversion carrier of the clpP promotor contain the als gene that starts the ripe size that has rps16 3 ' UTR through the following steps: contain the fragment that carrier main chain, the integration of 5 ' and 3 ' plastid guide peptide, aadA selective marker cartridge clip and clpP promotor/rps16 3 ' UTR expressed sequence with NcoI and XbaI digestion pPH140 and purifying.This product is connected with the NcoI-XbaI dna fragmentation of the ALS encoding sequence that contains isolating as mentioned above ripe size in the dual mode reaction.

Embodiment 41: the carrier that is used for the conversion of tobacco plastid contains the tobacco plastid clpP gene promoter and 5 ' non-the turning over of merging with AHAS encoding sequence and plastid rps16 gene 3 ' non-translated sequence

Translate the preparation of the mosaic gene of sequence

Screening is used for the cDNA library (U.S. Patent number US4,761,373) of acetohydroxy acid synthetase (AHAS) gene.Molecule clone technology by standard separates the plasmid clone that contains total length AHAS gene cDNA.Design PCR primer is used to increase from the big or small AHAS encoding sequence of the maturation of this plasmid, strand primer and following strand primer in this PCR use, the wherein said strand primer of going up has to contain and is inserted in from 5 ' extension of the NcoI restriction site on the amino acid-1 of the maturation protein starting point of inferring, generation ATG initiator codon on this position thus; And described strand primer down has the 5 ' extension in the XbaI restriction site downstream of the terminator codon that contains the ripe encoding sequence of AHAS in amplification PCR products.Use is carried out this pcr amplification according to the primer and the plasmid DNA template of standard scheme design.The product of clonal expansion and order-checking and by limiting digestion with NcoI and XbaI, carrying out the 0.8%TAE agarose gel electrophoresis and the band of cutting-out is carried out phenol extraction and separates the NcoI-XbaI dna fragmentation that contains complete ripe AHAS encoding sequence.

Make up the plastid conversion carrier of the clpP promotor contain the AHAS gene that starts the ripe size that has rps16 3 ' UTR through the following steps: contain the fragment that carrier main chain, the integration of 5 ' and 3 ' plastid guide peptide, aadA selective marker box and clpP promotor/rps16 3 ' UTR expressed sequence with NcoI and XbaI digestion pPH140 and purifying.This product is connected with the NcoI-XbaIDNA fragment of the AHAS encoding sequence that contains isolating as mentioned above ripe size in the dual mode reaction.Embodiment 42: the carrier that is used for the conversion of tobacco plastid contains the tobacco plastid clpP gene promoter and 5 ' non-the turning over of merging with ACCase encoding sequence and plastid rps16 gene 3 ' non-translated sequence

Translate the preparation of the mosaic gene of sequence

Screening is used for the cDNA library (U.S. Patent number US 5,162,602) of acetyl-CoA carboxylase (ACCase) gene.Molecule clone technology by standard separates the plasmid clone that contains total length ACCase gene cDNA.Design PCR primer is used to increase from the big or small ACCase encoding sequence of the maturation of this plasmid, strand primer and following strand primer in this PCR use, the wherein said strand primer of going up has to contain and is inserted in from 5 ' extension of the NcoI restriction site on the amino acid-1 of the maturation protein starting point of inferring, generation ATG initiator codon on this position thus; And described strand primer down has the 5 ' extension in the XbaI restriction site downstream of the terminator codon that contains the ripe encoding sequence of ACCase in amplification PCR products.Use is carried out this pcr amplification according to the primer and the plasmid DNA template of standard scheme design.The product of clonal expansion and order-checking and by limiting digestion with NcoI and XbaI, carrying out the 0.8%TAE agarose gel electrophoresis and the band of cutting-out is carried out phenol extraction and separates the NcoI-XbaI dna fragmentation that contains complete ripe ACCase encoding sequence.

Make up the plastid conversion carrier of the clpP promotor contain the ACCase gene that starts the ripe size that has rps16 3 ' UTR through the following steps: contain the fragment that carrier main chain, the integration of 5 ' and 3 ' plastid guide peptide, aadA selective marker box and clpP promotor/rps16 3 ' UTR expressed sequence with NcoI and XbaI digestion pPH140 and purifying.This product is connected with the NcoI-XbaIDNA fragment of the ACCase encoding sequence that contains isolating as mentioned above ripe size in the dual mode reaction.10. embodiment 43: the Biolistic of tobacco plastom transforms

Make tobacco (Nicotiana tabacum c.v.) ' seed of Xanthi nc ' with 7/dull and stereotyped ratio on the T nutrient agar with 1 " mode of circular permutation germinate and after planting 12-14 days with bag by from main as Svab; Z. and Maliga are at P. (1993) PNAS90; (M10; Biorad; Hercules CA) bombards 1 μ m tungsten particle of the DNA of pPH143 of plasmid described in the 913-917 and pPH145.Seedling incubation 2 days on the T substratum with bombardment, after this downcut leaf and make shaft at side under Mingguang City (350-500 μ mol photon/m2/s) contain 500 μ g/ml, two hydrochloric acid spectinomycin (Sigma, St.Louis, MO) RMOP substratum (Svab, Z., Hajdukiewicz, P. and Maliga, P. (1990) PNAS 87 is 8526-8530) on the flat board.Subclone bombardment back grows the resistance seedling at the adaxial surface of the leaf that bleaches, makes it to form callus and separates secondary seedling and carry out subclone in 3-8 week on identical selection substratum.Standard technique (Sambrook etc. by southern blotting technique, (1989) molecular cloning (Molecular Cloning): lab guide (A Laboratory Manual), cold spring harbor laboratory (Cold Spring HarborLaboratory), cold spring port (Cold Spring Harbor)) estimate the complete separation case of the plastom copy (homogeneity (homoplasmicity)) that transforms in the subclone independently.Total cell dna (the Mettler that on 1%Tris-borate (TBE) sepharose, separates BamHI/EcoRI-digestion, I.J. (1987) molecular biology of plants reporter gene (Plant Mol BiolReporter) 5,346-349), forward it to nylon membrane (Amersham) and go up and use ³²The random primer dna sequence dna of P-mark is surveyed, and is described ³²The random primer dna sequence dna of P-mark is equivalent to the 0.7kb BamHI/HindIIIDNA fragment from the pC8 that contains part rps7/12 plastid guiding peptide.The seedling rooting that makes homogeneity with sterile manner contain the MS/IBA substratum of spectinomycin (McBride, K.E. etc. (1994) PNAS 91,7301-7305) in and change the greenhouse over to.

Embodiment 44:Nt_pPH143 and Nt_pPH145 plastid transform herbicide tolerant in the strain

The evaluation of property

The elementary homogeneity transformant product that the pPH143 (strain Nt_pPH143) that obtains or pPH145 (strain Nt_pPH145) are transformed tie up to and grow to maturation in the greenhouse.Flower is carried out: (a) self-pollination; (b) with wild-type tobacco (c.v.Xanthi nc) pollination; Or (c) as the pollen donor so that make the Hua Nengyu of the castration of wild-type Xanthi plant.Check the plastid separation case of the spectinomycin resistance marker that is connected by using 50 seeds/derive from selfing or the minimum quantity in the selection storehouse of the bud capsule of backcrossing at each female character inheritance of single parent that transforms the spectinomycin resistant phenotype that carries out in strain.Other selfing or wild-type (Xanthi pollen parent) seed of backcrossing is germinateed in soil.36 strain wild-type Xanthi plant-growths of genetic contrast product such as the 36 strain plants (143 1B-1,143 1B-4,143 4A-2,143 4A-5,145 7A-5,145 7A-6,145 8A-3) that make each strain and conduct are in the controlled environment cell independently 6 " in the clay jar.For the tolerance of the protox inhibitor of estimating mutual-through type XVII, the plant that makes Xanthi and 7 transformant strains are distributed into flat the going up (every type 2 strain plant/flat on) of 8 identical 16-jars.Spray general formula X VII for flat going up, up to 0,0.5,2.5,5,10,25,50 or one of the concentration that rises of 100mg general formula X VII/ under overflow till.Utilize 4g/ to rise or 40g/ rises the mode water that general formula X VII is dissolved in the resulting mother liquor of methyl-sulphoxide (DMSO) and makes solution and use immediately after preparation.In the herbicide solution of each 200ml volume, add the wetting agent Silwett of 20 microlitres so that final concentration is 0.01%.Make it dried overnight afternoon to spraying on flat and changing over to the growth cell.Relatively the infringement and the withered degree of leaf and unconverted Xanthi reference substance leaf are estimated tolerance by will use back 0,18 hour, 48 hours and 6 days the time.Under all concentration more than the 0.5mg/l, on the Xanthi plant, obviously there are 7 kinds of infringements, fall and withered fully/accent when 2.5mg/l is above, occurs.Even promptly grow the spot that bleaches (outward appearance of Xanthi is identical with the outward appearance of Nt_pPH143 1B-1 under 100mg/ rises basically under 0.5mg/ rises, thereby the tolerance that obtains the latter is about 200 times result of the former tolerance) soon only occurring mild damage and this plant under the maximum concentration (100mg/ liter) on the Nt_pPH143 plant.Embodiment 45: the plastid of corn transforms

(Green etc. (1983) are at A.Fazelahmad from the cultivation of the I type embryo generation callus that begins to carry out patented genotype CG00526 and CG00714 from the long immature embryo of the 1.5-2.5mm of greenhouse growth substance, K.Downey, J.Schultz, described in the progress of the gene engineering that R.W.Voellmy edits (Advances in Gene Technology): the molecular genetics of plant and animal, Miami's winter, committee talked book series (Molecular Genetics ofPlants and Animals.Miami Winter Symposium Series), roll up 20. scholarly publications, New York (Academic Press, N.Y.)).Downcutting embryo with sterile manner from the fringe of surface sterilization in about 14 days after the pollination.The embryo of CG00526 is placed (Duncan etc. (1985) plants (Planta) 165:322-332) on the initial substratum of D callus that contains 2% sucrose and 5mg/L chloramben, simultaneously the embryo of those CG00714 is placed and contain 3% sucrose and 0.75mg/L2, on the initial substratum of KM callus of 4-d (Kao and Michayluk (1975) plant (Planta) 126,105-110).Culturing embryo and embryogenesis culture in the dark subsequently.After～14 days, from explant, remove the embryo thing that reacts.The CG00526 reactant placed contain 2% sucrose and 0.5mg/L 2, the D callus of 4-d is kept on the substratum, and the reactant with CG00714 places the KM callus that contains 2% sucrose and 5mg/L dicamba 98 to keep on the substratum simultaneously.Selectivity goes down to posterity and is cultured to the fresh substratum of keeping and carries out 3-8 after week weekly, has set up high-quality intensive embryogenesis culture.The embryo generation callus sheet of active growth is elected to be the target tissue of gene delivery.Gene delivery precontract 4 hours callus sheet flat board is fixed on the target flat board, comprises the substratum of keeping that contains 12% sucrose in this target flat board.According to circular arrangement, is 8 and 10mm apart from the dull and stereotyped radius centered of target with these callus sheets wherein.Plasmid DNA is deposited on the golden microcarrier described in the DuPontBiolistics guide.In the microcarrier preparation of per 6 bombardments, use every kind of plasmid of 2-3 μ g.Use PDS-1000He Biolistics device with gene delivery to the target tissue cell.Carrying out following setting on the Biolistics device: be 8mm between rupture disk and the larger vector; Between larger vector and the filling sieve is 10mm; And filling between sieve and the target thing is 7cm.Use the 650psi rupture disk with twice of the dull and stereotyped bombardment of each target.Fill place between sieve and the target tissue 200 * 200 stainless steel meshs (McMaster-Carr, New Brunswick, NJ).

After 5 days, will change over to and contain 2% sucrose and 0.5mg/L 2 through the callus sheet of bombardment, 4-d but do not conform to amino acid and contain 750 or 1000nM general formula X VII keep substratum.At 4-5 after the transfer hour or the callus sheet is placed illumination last 1 hour of frame and the dark 27 ℃ under preservation 5-6 week at the 2nd day.After the first choice phase in 5-6 week, will forward to by the tissue that yellow becomes white contain replenished 500 or the fresh flat board of the same medium of 750nM general formula X VII on.After transfer 4-5 hour or, tissue is placed illumination last 1 hour of frame and the preservation 3-4 week of the dark 27 ℃ under at second day., after the choice phase this tissue is forwarded on the flat board that contains the same medium of having replenished 500 general formula X VII at the secondary in 3-4 week.The tissue of healthy growth placed illumination last 1 hour of frame and be kept at dark under 27 ℃.Per 2 weeks it is gone down to posterity and cultivates 1 time, up to colony greatly till be enough to regeneration.Move to the MS substratum (Murashige and Skoog (1962) plant physiology (Physiol.Plant) 15:473-497) that contains 3% sucrose (MS3S) but do not contain the modification of selective agent with colony and go up and place under the illumination this moment.With regard to CG00526, in this substratum, add 0.25mg/L ancymidol and 0.5mg/L phytokinin and germinate, and with regard to CG00714, add the 2mg/L benzyladenine to induce embryo.After 2 weeks, with regard to CG00526 or CG00714, respectively the regenerated colony is moved on the MS3S substratum that does not contain ancymidol and phytokinin or benzyladenine.The regrowth that is with or without root changed in the case that contains the MS3S substratum and final the recovery has the plantlet of root and it is changed in the soil in greenhouse.Table 1A

Belong to (" Arabpt-1 " from mouse ear mustard; SEQ ID NO:2), corn (" Mzpt-1 "; SEQ ID NO:6), wheat (" Wtpt-1 "; SEQ ID NO:10), soybean (" Soybeanpt-1 "; SEQ ID NO:12), cotton (" Cottonpt-1 "; SEQ IDNO:16), beet (" Sugpt-1 "; SEQ ID NO:18), rape (" Rapept-1 "; SEQID NO:20), rice (" Ricept-1 "; SEQ ID NO:22), Chinese sorghum (" Sorghumpt-1 "; SEQ ID NO:24) and sugarcane (" Scpt-1 "; The sequence contrast of total length SEQ ID NO:37) and part protox-1 aminoacid sequence.(Madison WI) carries out the sequence contrast for GCG bag, University of Wisconsin to use the PileUp program.Can modify with the position that produces or improve the inhibitor tolerance shown in boldface letter according to the instruction of this paper.

1???????????????????????????????????????????????50???Rapept-1?..........?..........?MDLSLLRP..?QPFLSPFSNP?FPRSRPYKPL???Arabpt-1?..........?..........?MELSLLRPTT?QSLLPSFSKP?NLRLNVYKPLSorghumpt-1?..........?..........?..........?..........?..........

Mzpt-1?..........?..........?..........?..........?..........

Wtpt-1?..........?..........?.........M?ATATVAAASP?LRGRVTGRPH???Ricept-1?..........?..........?..........?..........?..........??Cottonpt-1?..........?......MTAL?IDLSLLRSSP?SVSPFSIPHH?QHPPRFRKPF?Soybeanpt1?........MV?SVFNEILFPP?NQTLLRPSLH?SPTSFFTSPT?RKFPRSRPNP

Sugpt-1?MKSMALSNCI?PQTQCMPLRS?SGHYRGNCIM?LSIPCSLIGR?RGYYSHKKRR

Scpt-1?..........?..........?..........?..........?..........

51?????????????????????????????????????????????100???Rapept-1?NLRCSVSGGS?VVGSSTIEGG?GGGKTVTADC?VIVGGGISGL?CIAQALVTKH???Arabpt-1?RLRCSVAGGP?TVGSSKIEGG?GGT.TITTDC?VIVGGGISGL?CIAQALATKHSorghumpt-1?..........?..........?..........?..........?..........

Mzpt-1?..........?..........?.......ADC?VVVGGGISGL?CTAQALATRH

Wtpt-1?RVRPRCATAS?SATETPAAPG?VRL...SAEC?VIVGAGISGL?CTAQALATRY???Ricept-1?..........?..........?..........?..........?..........?Cottonpt-1?KLRCSLAEGP?TISSSKIDGG?ESS...IADC?VIVGGGISGL?CIAQALATKH?Soybeanpt1?ILRCSIAEES?TASPPKTR..?DSA...PVDC?VVVGGGVSGL?CIAQALATKH

Sugpt-1?MSMSCSTSSG?SKSAVKEAGS?GSGAGGLLDC?VIVGGGISGL?CIAQALCTKH

Scpt-1?..........?..........?..........?..........?..........

101????????????????????????????????????????????150???Rapept-1?PDA..AKNVM?VTEAKDRVGG?NIIT..REEQ?GFLWEEGPNS?FQPSDPMLTM???Arabpt-1?PDA..APNLI?VTEAKDRVGG?NIIT..REEN?GFLWEEGPNS?FQPSDPMLTMSorghumpt-1?..........?..........?..STVERPEE?GYLWEEGPNS?FQPSDPVLSM

Mzpt-1?..G..VGDVL?VTEARARPGG?NITTVERPEE?GYLWEEGPNS?FQPSDPVLTM

Wtpt-1?..G..VSDLL?VTEARDRPGG?NITTVERPDE?GYLWEEGPNS?FQPSDPVLTM???Ricept-1?..........?..........?..........?..........?..........??Cottonpt-1?RDV..ASNVI?VTEARDRVGG?NITTVER..D?GYLWEEGPNS?FQPSDPILTM?Soybeanpt1?..A..NANVV?VTEARDRVGG?NITTMER..D?GYLWEEGPNS?FQPSDPMLTM

Sugpt-1?SSSSLSPNFI?VTEAKDRVGG?NIVTVE..AD?GYIWEEGPNS?FQPSDAVLTM

Scpt-1?..........?..........?..........?..........?..........

151????????????????????????????????????????????200???Rapept-1?VVDSGLKDDL?VLGDPTAPRF?VLWNGKLRPV?PSKLTDLPFF?DLMSIGGKIR???Arabpt-1?VVDSGLKDDL?VLGDPTAPRF?VLWNGKLRPV?PSKLTDLPFF?DLMSIGGKIRSorghumpt-1?AVDSGLKDDL?VFGDPNAPRF?VLWEGKLRPV?PSKPADLPFF?DLMSIPGKLR

Mzpt-1?AVDSGLKDDL?VFGDPNAPRF?VLWEGKLRPV?PSKPADLPFF?DLMSIPGKLR

Wtpt-1?AVDSGLKDDL?VFGDPNAPRF?VLWEGKLRPV?PSKPGDLPFF?SLMSIPGKLR???Ricept-1?..........?..........?..........?..........?..........?Cottonpt-1?AVDSGLKDDL?VLGDPNAPRF?VLWEGKLRPV?PSKPTDLPFF?DLMSIAGKLR?Soybeanpt1?VVDSGLKDEL?VLGDPDAPRF?VLWNRKLRPV?PGKLTDLPFF?DLMSIGGKIR

Sugpt-1?AVDSGLKDEL?VLGDPNAPRF?VLWNDKLRPV?PSSLTDLPFF?DLMTIPGKIR

Scpt-1?..........?..........?..........?..........?..........

201????????????????????????????????????????????250???Rapept-1?AGFGAIGIRP?SPPGREESVE?EFVRRNLGDE?VFERLIEPFC?SGVYAGDPAK???Arabpt-1?AGFGALGIRP?SPPGREESVE?EFVRRNLGDE?VFERLIEPFC?SGVYAGDPSKSorghumpt-1?AGLGALGIRP?PAPGREESVE?EFVRRNLGAE?VFERLIEPFC?SGVYAGDPSK

Mzpt-1?AGLGALGIRP?PPPGREESVE?EFVRRNLGAE?VFERLIEPFC?SGVYAGDPSK

Wtpt-1?AGLGALGIRP?PPPGREESVE?EFVRRNLGAE?VFERLIEPFC?SGVYAGDPSK???Ricept-1?..........?..........?..........?..........?..........??Cottonpt-1?AGFGAIGIRP?PPPGYEESVE?EFVRRNLGAE?VFERFIEPFC?SGVYAGDPSK?Soybeanpt1?AGFGALGIRP?PPPGHEESVE?EFVRRNLGDE?VFERLIEPFC?SGVYAGDPSK

Sugpt-1?AALGALGFRP?SPPPHEESVE?HFVRRNLGDE?VFERLIEPFC?SGVYAGDPAK

Scpt-1?..........?..........?..........?..........?..........

251????????????????????????????????????????????300???Rapept-1?LSMKAAFGKV?WKLEENGGSI?IGGAFKAIQA?KNKAPKTTRD?PRLPKPKGQT???Arabpt-1?LSMKAAFGKV?WKLEQNGGSI?IGGTFKAIQE?RKNAPKAERD?PRLPKPQGQTSorghumpt-1?LSMKAAFGKV?WRLEEAGGSI?IGGTIKTIQE?RGKNPKPPRD?PRLPKPKGQT

Mzpt-1?LSMKAAFGKV?WRLEETGGSI?IGGTIKTIQE?RSKNPKPPRD?ARLPKPKGQT

Wtpt-1?LSMKAAFGKV?WRLEEIGGSI?IGGTIKAIQD?KGKNPKPPRD?PRLPAPKGQT???Ricept-1?RALKAAFGKV?WRLEDTGGSI?IGGTIKTIQE?RGKNPKPPRD?PRLPTPKGQT?Cottonpt-1?LSMKAAFGRV?WKLEEIGGSI?IGGTFKTIQE?RNKTPKPPRD?PRLPKPKGQT?Soybeanpt1?LSMKAAFGKV?WKLEKNGGSI?IGGTFKAIQE?RNGASKPPRD?PRLPKPKGQT

Sugpt-1?LSMKAAFGKV?WKLEQKGGSI?IGGTLKAIQE?RGSNPKPPRD?QRLPKPKGQT

Scpt-1?..........?..........?..........?..........?..........

301????????????????????????????????????????????350???Rapept-1?VGSFRKGLTM?LPEAISARLG?DKVKVSWKLS?SITKLASGEY?SLTYETPEGI???Arabpt-1?VGSFRKGLRM?LPEAISARLG?SKVKLSWKLS?GITKLESGGY?NLTYETPDGLSorghumpt-1?VASFRKGLAM?LPNAITSSLG?SKVKLSWKLT?SMTKSDGKGY?VLEYETPEGV

Mzpt-1?VASFRKGLAM?LPNAITSSLG?SKVKLSWKLT?SITKSDDKGY?VLEYETPEGV

Wtpt-1?VASFRKGLAM?LPNAIASRLG?SKVKLSWKLT?SITKADNQGY?VLGYETPEGL???Ricept-1?VASFRKGLTM?LPDAITSRLG?SKVKLSWKLT?SITKSDNKGY?ALVYETPEGV?Cottonpt-1?VGSFRKGLTM?LPEAIANSLG?SNVKLSWKLS?SITKLGNGGY?NLTFETPEGM?Soybeanpt1?VGSFRKGLTM?LPDAISARLG?NKVKLSWKLS?SISKLDSGEY?SLTYETPEGV

Sugpt-1?VGSFRKGLVM?LPTAISARLG?SRVKLSWTLS?SIVKSLNGEY?SLTYDTPDGL

Scpt-1?..........?..........?..........?..........?..........

351????????????????????????????????????????????400???Rapept-1?VTVQSKSVVM?TVPSHVASSL?LRPLSDSAAE?ALSKLYYPPV?AAVSISYAKE???Arabpt-1?VSVQSKSVVM?TVPSHVASGL?LRPLSESAAN?ALSKLYYPPV?AAVSISYPKESorghumpt-1?VLVQAKSVIM?TIPSYVASDI?LRPLSGDAAD?VLSRFYYPPV?AAVTVSYPKE

Mzpt-1?VSVQAKSVIM?TIPSYVASNI?LRPLSSDAAD?ALSRFYYPPV?AAVTVSYPKE

Wtpt-1?VSVQAKSVIM?TIPSYVASDI?LRPLSIDAAD?ALSKFYYPPV?AAVTVSYPKE???Ricept-1?VSVQAKTVVM?TIPSYVASDI?LRPLSSDAAD?ALSIFYYPPV?AAVTVSYPKE?Cottonpt-1?VSLQSRSVVM?TIPSHVASNL?LHPLSAAAAD?ALSQFYYPPV?ASVTVSYPKE?Soybeanpt1?VSLQCKTVVL?TIPSYVASTL?LRPLSAAAAD?ALSKFYYPPV?AAVSISYPKE

Sugpt-1?VSVRTKSVVM?TVPSYVASRL?LRPLSDSAAD?SLSKFYYPPV?AAVSLSYPKE

Scpt-1?..........?..........?..........?..........?..........

401????????????????????????????????????????????450???Rapept-1?AIRSECLIDG?ELKGFGQLHP?RTQKVETLGT?IYSSSLFPNR?APPGRVLLLN???Arabpt-1?AIRTECLIDG?ELKGFGQLHP?RTQGVETLGT?IYSSSLFPNR?APPGRILLLNSorghumpt-1?AIRKECLIDG?ELQGFGQLHP?RSQGVETLGT?IYSSSLFPNR?APAGRVLLLN

Mzpt-1?AIRKECLIDG?ELQGFGQLHP?RSQGVETLGT?IYSSSLFPNR?APDGRVLLLN

Wtpt-1?AIRKECLIDG?ELQGFGQLHP?RSQGVETLGT?IYSSSLFPNR?APAGRVLLLN???Ricept-1?AIRKECLIDG?ELQGFGQLHP?RSQGVETLGT?IYSSSLFPNR?APAGRVLLLN?Cottonpt-1?AIRKECLIDG?ELKGFGQLHP?RSQGIETLGT?IYSSSLFPNR?APSGRVLLLN?Soybeanpt1?AIRSECLIDG?ELKGFGQLHP?RSQGVETLGT?IYSSSLFPNR?APPGRVLLLN

Sugpt-1?AIRSECLING?ELQGFGQLHP?RSQGVETLGT?IYSSSLFPGR?APPGRILILS

Scpt-1?..........?..........?..........?..........?..........

451????????????????????????????????????????????500???Rapept-1?YIGGATNTGI?LSKSEGELVE?AVDRDLRKML?IKPSSTDPLV?LGVKLWPQAI???Arabpt-1?YIGGSTNTGI?LSKSEGELVE?AVDRDLRKML?IKPNSTDPLK?LGVRVWPQAISorghumpt-1?YIGGATNTGI?VSKTESELVE?AVDRDLRKML?INPTAVDPLV?LGVRVWPQAI

Mzpt-1?YIGGATNTGI?VSKTESELVE?AVDRDLRKML?INSTAVDPLV?LGVRVWPQAI

Wtpt-1?YIGGSTNTGI?VSKTESDLVG?AVDRDLRKML?INPRAADPLA?LGVRVWPQAI???Ricept-1?YIGGSTNTGI?VSKTESELVE?AVDRDLRKML?INPRAVDPLV?LGVRVWPQAI?Cottonpt-1?YIGGATNTGI?LSKTEGELVE?AVDRDLRKML?INPNAKDPLV?LGVRVWPKAI?Soybeanpt1?YIGGATNTGI?LSKTDSELVE?TVDRDLRKIL?INPNAQDPFV?VGVRLWPQAI

Sugpt-1?YIGGAKNPGI?LNKSKDELAK?TVDKDLRRML?INPDAKLPRV?LGVRVWPQAI

Scpt-1?..........?.SKTESELVE?AVDRDLRKML?INPTAVDPLV?LGVRVWPQAI

501????????????????????????????????????????????550???Rapept-1?PQFLIGHIDL?VDAAKASLSS?SGHEGLFLGG?NYVAGVALGR?CVEGAYETAT???Arabpt-1?PQFLVGHFDI?LDTAKSSLTS?SGYEGLFLGG?NYVAGVALGR?CVEGAYETAISorghumpt-1?PQFLVGHLDL?LEAAKSALDQ?GGYNGLFLGG?NYVAGVALGR?CIEGAYESAA

Mzpt-1?PQFLVGHLDL?LEAAKAALDR?GGYDGLFLGG?NYVAGVALGR?CVEGAYESAS

Wtpt-1?PQFLIGHLDR?LAAAKSALGQ?GGYDGLFLGG?KYVAGVALGR?CIEGAYESAS???Ricept-1?PQFLIGHLDH?LEAAKSALGK?GGYDGLFLGG?NYVAGVALGR?CVEGAYESAS?Cottonpt-1?PQFLVGHLDL?LDSAKMALRD?SGFHGLFLGG?NYVSGVALGR?CVEGAYEVAA?Soybeanpt1?PQFLVGHLDL?LDVAKASIRN?TGFEGLFLGG?NYVSGVALGR?CVEGAYEVAA

Sugpt-1?PQFSIGHFDL?LDAAKAALTD?TGVKGLFLGG?NYVSGVALGR?CIEGAYESAA

Scpt-1?PQFLVGHLDL?LEAAKSALDR?GGYDGLFLGG?NYVAGVALGR?CVEGAYESAS551???????563???Rapept-1?QVNDFMSRYA?YK*???Arabpt-1?EVNNFMSRYA?YK*Sorghumpt-1?QIYDFLTKYA?YK*

Mzpt-1?QISDFLTKYA?YK*

Wtpt-1?QVSDFLTKYA?YK*???Ricept-1?QISDYLTKYA?YK*?Cottonpt-1?EVKEFLSQYA?YK*?Soybeanpt1?EVNDFLTNRV?YK*

Sugpt-1?EVVDFLSQYS?DK*

Scpt-1?QIYDFLTKYA?YK*

Subsequence #	Subsequence AA sequence	Δ _nThe AA wild-type	?Δ _nAA replaces	Δ among the table 1A _nCorresponding AA position	Typical mutant
Subsequence #	Subsequence AA sequence	Δ _nThe AA wild-type	?Δ _nAA replaces	Δ among the table 1A _nCorresponding AA position	Typical mutant	?1	?APΔ ₁F	?R	?C，L	?169	?Mz88Cys ?Mz88Leu
?2	?FΔ ₂S	?C	?F，L，K	?240	?Mz159Phe ?Mz159Leu ?Mz159Lys	?1	?APΔ ₁F	?R	?C，L	?169	?Mz88Cys ?Mz88Leu
?2	?FΔ ₂S	?C	?F，L，K	?240	?Mz159Phe ?Mz159Leu ?Mz159Lys	?3	?YΔ ₃G	?A	?V，T，L， ?C，I	?245	?pAraC-1Val ?pAraC-1Thr ?pAraC-1Leu ?pAraC-1Cys ?pAraC-1Ile ?pMzC-1Val ?pMzC-1Thr ?pMzC-1Leu ?pWhtC-1Val ?pWhtC-1Thr ?pSoyC-1Thr ?pSoyC-1Leu
?4	?AΔ ₄D	?G	?S，L	?246	?pAraC-3Ser ?pMzC-3Ser ?pMzC-3Leu ?pWhtC-3Ser	?3	?YΔ ₃G	?A	?V，T，L， ?C，I	?245
?4	?AΔ ₄D	?G	?S，L	?246	?pAraC-3Ser ?pMzC-3Ser ?pMzC-3Leu ?pWhtC-3Ser	?5	?YΔ ₅P	?P	?S，H	?388	?Soy369Ser ?Soy369His ?Cot365Ser
?6	?PΔ ₆A	?V	?L	?390	?Wht356Leu	?5	?YΔ ₅P	?P	?S，H	?388	?Soy369Ser ?Soy369His ?Cot365Ser
?6	?PΔ ₆A	?V	?L	?390	?Wht356Leu	?7	?Δ ₇IG	?Y	?C，I，L， ?T，M，V， ?A，H，R	?451	?pAraC-2Cys ?pAraC-2Ile ?pAraC-2Leu

									?pAraC-2Thr ?pAraC-2Met ?pAraC-2Val ?pAraC-2Ala ?pMzC-2Ile ?pMzC-2Met ?pSoyC-2Leu ?pSoyC-2Ile ?pSugC-2Cys ?pSugC-2Leu ?pSugC-2Ile ?pSugC-2Val ?pSugC-2Met ?pCotC-2Cys ?pCotC-2His ?pCotC-2Arg
											?8	?YIGGΔ ₈		?A，S		?P		?455		?Wht421Pro
?9	?AΔ ₉P		?I		?T，H，G， ?N		?500		?Mz419Thr ?Mz419His ?Mz419Gly ?Mz419Asn ?Wht466Thr		?8	?YIGGΔ ₈		?A，S		?P		?455		?Wht421Pro
?9	?AΔ ₉P		?I		?T，H，G， ?N		?500		?Mz419Thr ?Mz419His ?Mz419Gly ?Mz419Asn ?Wht466Thr		?10	?GΔ ₁₀A		?V		?A		?536		?Wht502Ala ?Soy517Ala
?18	?KA□ ₁₈F		?A		?T，V		?256		?Mz175Thr ?Mz175Val		?10	?GΔ ₁₀A		?V		?A		?536		?Wht502Ala ?Soy517Ala
?18	?KA□ ₁₈F		?A		?T，V		?256		?Mz175Thr ?Mz175Val		?19	?Q□ ₁₉H		?L		?S		?418		?Mz337Ser
Second site mutation											?19	?Q□ ₁₉H		?L		?S		?418		?Mz337Ser
Second site mutation											?11		?QΔ ₁₁S		?P		?L		?143		?AraC11 ?8Leu
?12		?IGGΔ ₁₂		?T		?I，A		?274		?AraC24 ?9Ile ?Ara ?C249Al ?a	?11		?QΔ ₁₁S		?P		?L		?143		?AraC11 ?8Leu
?12		?IGGΔ ₁₂		?T		?I，A		?274		?AraC24 ?9Ile ?Ara ?C249Al ?a	?13		?SWXLΔ ₁₃		?S，T		?L		?330		?AraC30 ?5Leu
?14		?LΔ ₁₄Y		?N		?S		?450		?AraC42 ?5Ser	?13		?SWXLΔ ₁₃		?S，T		?L		?330		?AraC30 ?5Leu
?14		?LΔ ₁₄Y		?N		?S		?450		?AraC42 ?5Ser	?15		?GΔ ₁₅XGL		?Y，H，F，V		?C		?523		?AraC49 ?8Cys
Dual sudden change											?15		?GΔ ₁₅XGL		?Y，H，F，V		?C		?523		?AraC49 ?8Cys
Dual sudden change											?16		?TΔ ₁₆G		?L		?S		?428		?Mz347S ?er-453 ?Thr
?17		?YVΔ ₁₇G		?A，(S)		?T		?534			?16		?TΔ ₁₆G		?L		?S		?428		?Mz347S ?er-453 ?Thr

Table 2

Mouse ear mustard belongs to the comparison of (SEQ ID NO:4) and corn (SEQ ID NO:8) protox-2 aminoacid sequence.Identical residue is represented by the vertical bar between two sequences.Use Deveraux etc. carries out the sequence contrast in the GAP program described in nucleic acids research (the Nucleic Acids Res.) 12:387-395 (1984).Similarity per-cent: 75.889/ identity per-cent: 57.905.

1?............................MASGAVAD.HQIEAVSGKRVAV?21

.|??|:|:?.:??|..::.|||

1?MLALTASASSASSHPYRHASAHTRRPRLRAVLAMAGSDDPRAAPARSVAV?50

22?VGAGVSGLAAAYKLKSRGLNVTVFEADGRVGGKLRSVMQNGLIWDEGANT?71

||||||||||||:|:?.|:|||||||.:|.|||:|.??:.|::|||||||

51?VGAGVSGLAAAYRLRQSGVNVTVFEAADRAGGKIRTNSEGGFVWDEGANT?100

72?MTEAEPEVGSLLDDLGLREKQQFPISQKKRYIVRNGVPVMLPTNPIELVT?121

|||:|?|.:.|:|||||.:|||:|?||.|||||::|.|.::|.:||.|:.

101?MTEGEWEASRLIDDLGLQDKQQYPNSQHKRYIVKDGAPALIPSDPISLMK?150

122?SSVLSTQSKFQILLEPFLWKK....KSSKVSDASAEESVSEFFQRHFGQE?167

||||||.||:.:::||||:||???.|:|||:.??.|||:.|?:||||.|

151?SSVLSTKSKIALFFEPFLYKKANTRNSGKVSEEHLSESVGSFCERHFGRE?200

168?VVDYLIDPFVGGTSAADPDSLSMKHSFPDLWNVEKSFGSIIVGAIRTKFA?217

||||::||||:||||:||:|||::|.||.|||:|:.:||:|||||?.|:|

201?VVDYFVDPFVAGTSAGDPESLSIRHAFPALWNLERKYGSVIVGAILSKLA?250

218?AKGGKSRDTKSSPGTKKGSRGSFSFKGGMQILPDTLCKSLSHDEINLDSK?267

|||:.?:.?..|.|.::..|.||||.||||?|?:.|?..::.|::.|:..

251?AKGDPVKTRHDSSGKRRNRRVSFSFHGGMQSLINALHNEVGDDNVKLGTE?300268?VLSLS..YNSGSRQENWSLSCVSHNETQRQ...NPHYDAVIMTAPLCNVK?312

||||.??:::..??:.||:|.?|.:..:::??|.?:|||||||||:||:301?VLSLACTFDGVPALGRWSISVDSKDSGDKDLASNQTFDAVIMTAPLSNVR?350313?EMKVMKGGQPFQLNFLPEINYMPLSVLITTFTKEKVKRPLEGFGVLIPSK?362

||.?|||.|.?|:|||.::|:|||:::|.|.|:.||:||||||||||?|351?RMKFTKGGAPVVLDFLPKMDYLPLSLMVTAFKKDDVKKPLEGFGVLIPYK?400363?E.QKHGFKTLGTLFSSMMFPDRSPSDVHLYTTFIGGSRNQELAKASTDEL?411

|?||||:|||||||||||||||.|.|?.|||||:|||:|.:||?|.|.?|401?EQQKHGLKTLGTLFSSMMFPDRAPDDQYLYTTFVGGSHNRDLAGAPTSIL?450412?KQVVTSDLQRLLGVEGEPVSVNHYYWRKAFPLYDSSYDSVMEAIDKMEND?461

||:|||||.:||||||:|.?|.|?||?.|||||:?.|.||:|||:|||.:451?KQLVTSDLKKLLGVEGQPTFVKHVYWGNAFPLYGHDYSSVLEAIEKMEKN?500462?LPGFFYAGNHRGGLSVGKSIASGCKAADLVISYLESCSNDKKPNDSL*?509

|||||||||?::||.||.?||||:|||||.||||||?......:501?LPGFFYAGNSKDGLAVGSVIASGSKAADLAISYLESHTKHNNSH*...?545

Table 3A

Plant protox mutant is to the cross resistance of various protox inhibitor

General formula	AraC-1Val	AraC-2Cys	AraC-1Thr	AraC-3Thr	MzC-1Val
General formula	AraC-1Val	AraC-2Cys	AraC-1Thr	AraC-3Thr	MzC-1Val	XVII	+	+	+	+	+
VIIa	+	+	+	-	+	XVII	+	+	+	+	+
VIIa	+	+	+	-	+	IV	++	-	++	++	-
XV	+	+	+	+	+	IV	++	-	++	++	-
XV	+	+	+	+	+	XI	-	+	+	++	+
XVI	-	-	-	-	+	XI	-	+	+	++	+
XVI	-	-	-	-	+	XII	+	-	++	++	++
XIV	+	-	+	+	+	XII	+	-	++	++	++
XIV	+	-	+	+	+	*X

+=tolerance is higher than WT more than 10 times or 10 times ++=tolerance be higher than WT more than 100 times or 100 times-=no cross resistance *=this compound is through test but do not provide information

Table 3B

Plant protox mutant is to the cross resistance of various protox inhibitor

General formula	?AraC- ?1Leu	?AraC- ?2Ile	AraC- 1Leu + AraC- 2Met	AraC 1Leu + AraC 2Leu	AraC- 2Ile + AraC 305Leu	AraC- + AraC 425Ser	AraC- 2Leu + AraC 425Ser	AraC- 2Met + AraC 425Ser
General formula	?AraC- ?1Leu	?AraC- ?2Ile	AraC- 1Leu + AraC- 2Met	AraC 1Leu + AraC 2Leu	AraC- 2Ile + AraC 305Leu	AraC- + AraC 425Ser	AraC- 2Leu + AraC 425Ser	AraC- 2Met + AraC 425Ser	??XVII	??+	??+	??+	??+	??+	??+	??+	??+
??VIIa	??++	??++	??++	??++	??++	??++	??++	??++	??XVII	??+	??+	??+	??+	??+	??+	??+	??+
??VIIa	??++	??++	??++	??++	??++	??++	??++	??++	??IV	??++	??-	??+	??++	??+	??-	??+	??+
??IV	??++	??-	??+	??++	??+	??-	??+	??+	??IV	??++	??-	??+	??++	??+	??-	??+	??+
??IV	??++	??-	??+	??++	??+	??-	??+	??+	??XV	??++	??+++	??+++	??+++	??+++	??++	??+++	??++
??XI	??++	??++	??++	??++	??++	??++	??++	??++	??XV	??++	??+++	??+++	??+++	??+++	??++	??+++	??++
??XI	??++	??++	??++	??++	??++	??++	??++	??++	??XVI	??+++	??+++	??+++	??+++	??+++	??+	??++	??++
??XII									??XVI	??+++	??+++	??+++	??+++	??+++	??+	??++	??++
??XII									??XIV	??++	??++	??++	??++	??++	??-	??++	??++

In the sub-germination test of table 4 to the cross resistance of various protox inhibitor

General formula	Common name	Tolerance
General formula	Common name	Tolerance	II	Acifluorfen	+
III	fomasafen	+	II	Acifluorfen	+
III	fomasafen	+	IV	Fluoroglycofenethyl	±
IVb	Bifenox	+	IV	Fluoroglycofenethyl	±
IVb	Bifenox	+	IVc	Oxyfluorfen	+
IVd	Lactofen	±	IVc	Oxyfluorfen	+
IVd	Lactofen	±	VIIa	fluthiacet-methyl	++
X	Sulfentrazone	+	VIIa	fluthiacet-methyl	++
X	Sulfentrazone	+	XI	flupropazil	++
XIV	flumiclorac	+	XI	flupropazil	++
XIV	flumiclorac	+	XVI	Flumioxazin	+++
XVII		++	XVI	Flumioxazin	+++
XVII		++	XXIa	BAY?11340	+
XXII		++	XXIa	BAY?11340	+

± tolerance is less than or equal to 10 times+tolerance of wt more than or equal to 10 times of wt ++ and tolerance equals 100 times of wt greater than meeting ++ and+tolerance equals or equals 1000 times of wt

Various changes of the present invention as herein described are apparent to those skilled in the art.This class changes the scope that belongs to claims.

Sequence table＜110〉Novartis＜120〉proporphyrinogen oxidase＜130 of herbicide-resistant〉PB/5-30911A＜140〉＜141＜150〉US 09/373691＜151〉1999-08-13＜160〉44＜170〉PatentIn Ver.2.2＜210〉1＜211〉1719＜212〉DNA＜213〉mouse ear mustard＜220〉＜221〉CDS＜222, (31) .., (1644)＜223〉mouse ear mustard belongs to protox-1＜400〉1tgacaaaatt ccgaattctc tgcgatttcc atg gag tta tct ctt ctc cgt ccg 54

Met?Glu?Leu?Ser?Leu?Leu?Arg?Pro

1????????????5acg?act?caa?tcg?ctt?ctt?ccg?tcg?ttt?tcg?aag?ccc?aat?ctc?cga?tta??102Thr?Thr?Gln?Ser?Leu?Leu?Pro?Ser?Phe?Ser?Lys?Pro?Asn?Leu?Arg?Leu

10???????????????15??????????????20aat?gtt?tat?aag?cct?ctt?aga?ctc?cgt?tgt?tca?gtg?gcc?ggt?gga?cca??150Asn?Val?Tyr?Lys?Pro?Leu?Arg?Leu?Arg?Cys?Ser?Val?Ala?Gly?Gly?Pro?25??????????????30???????????????35??????????????40acc?gtc?gga?tct?tca?aaa?atc?gaa?ggc?gga?gga?ggc?acc?acc?atc?acg??198Thr?Val?Gly?Ser?Ser?Lys?Ile?Glu?Gly?Gly?Gly?Gly?Thr?Thr?Ile?Thr

45??????????????50???????????????55acg?gat?tgt?gtg?att?gtc?ggc?gga?ggt?att?agt?ggt?ctt?tgc?atc?gct??246Thr?Asp?Cys?Val?Ile?Val?Gly?Gly?Gly?Ile?Ser?Gly?Leu?Cys?Ile?Ala

60???????????????65???????????????70cag?gcg?ctt?gct?act?aag?cat?cct?gat?gct?gct?ccg?aat?tta?att?gtg??294Gln?Ala?Leu?Ala?Thr?Lys?His?Pro?Asp?Ala?Ala?Pro?Asn?Leu?Ile?Val

75???????????????80??????????????85acc?gag?gct?aag?gat?cgt?gtt?gga?ggc?aac?att?atc?act?cgt?gaa?gag??342Thr?Glu?Ala?Lys?Asp?Arg?Val?Gly?Gly?Asn?Ile?Ile?Thr?Arg?Glu?Glu

90??????????????95??????????????100aat?ggt?ttt?ctc?tgg?gaa?gaa?ggt?ccc?aat?agt?ttt?caa?ccg?tct?gat??390Asn?Gly?Phe?Leu?Trp?Glu?Glu?Gly?Pro?Asn?Ser?Phe?Gln?Pro?Ser?Asp105?????????????110???????????????115?????????????120cct?atg?ctc?act?atg?gtg?gta?gat?agt?ggt?ttg?aag?gat?gat?ttg?gtg??438Pro?Met?Leu?Thr?Met?Val?Val?Asp?Ser?Gly?Leu?Lys?Asp?Asp?Leu?Val

125??????????????130?????????????135ttg?gga?gat?cct?act?gcg?cca?agg?ttt?gtg?ttg?tgg?aat?ggg?aaa?ttg??486Leu?Gly?Asp?Pro?Thr?Ala?Pro?Arg?Phe?Val?Leu?Trp?Asn?Gly?Lys?Leu

140?????????????145??????????????150agg?ccg?gtt?cca?tcg?aag?cta?aca?gac?tta?ccg?ttc?ttt?gat?ttg?atg??534Arg?Pro?Val?Pro?Ser?Lys?Leu?Thr?Asp?Leu?Pro?Phe?Phe?Asp?Leu?Met

155??????????????160??????????????165agt?att?ggt?ggg?aag?att?aga?gct?ggt?ttt?ggt?gca?ctt?ggc?att?cga??582Ser?Ile?Gly?Gly?Lys?Ile?Arg?Ala?Gly?Phe?Gly?Ala?Leu?Gly?Ile?Arg???170??????????????175??????????????180ccg?tca?cct?cca?ggt?cgt?gaa?gaa?tct?gtg?gag?gag?ttt?gta?cgg?cgt??630Pro?Ser?Pro?Pro?Gly?Arg?Glu?Glu?Ser?Val?Glu?Glu?Phe?Val?Arg?Arg185??????????????190?????????????195??????????????200aac?ctc?ggt?gat?gag?gtt?ttt?gag?cgc?ctg?att?gaa?ccg?ttt?tgt?tca??678Asn?Leu?Gly?Asp?Glu?Val?Phe?Glu?Arg?Leu?Ile?Glu?Pro?Phe?Cys?Ser

205??????????????210?????????????215ggt?gtt?tat?gct?ggt?gat?cct?tca?aaa?ctg?agc?atg?aaa?gca?gcg?ttt??726Gly?Val?Tyr?Ala?Gly?Asp?Pro?Ser?Lys?Leu?Ser?Met?Lys?Ala?Ala?Phe

220?????????????225??????????????230ggg?aag?gtt?tgg?aaa?cta?gag?caa?aat?ggt?gga?agc?ata?ata?ggt?ggt??774Gly?Lys?Val?Trp?Lys?Leu?Glu?Gln?Asn?Gly?Gly?Ser?Ile?Ile?Gly?Gly

235??????????????240??????????????245act?ttt?aag?gca?att?cag?gag?agg?aaa?aac?gct?ccc?aag?gca?gaa?cga??822Thr?Phe?Lys?Ala?Ile?Gln?Glu?Arg?Lys?Asn?Ala?Pro?Lys?Ala?Glu?Arg???250??????????????255??????????????260gac?ccg?cgc?ctg?cca?aaa?cca?cag?ggc?caa?aca?gtt?ggt?tct?ttc?agg??870Asp?Pro?Arg?Leu?Pro?Lys?Pro?Gln?Gly?Gln?Thr?Val?Gly?Ser?Phe?Arg265??????????????270?????????????275??????????????280aag?gga?ctt?cga?atg?ttg?cca?gaa?gca?ata?tct?gca?aga?tta?ggt?agc??918Lys?Gly?Leu?Arg?Met?Leu?Pro?Glu?Ala?Ile?Ser?Ala?Arg?Leu?Gly?Ser

285??????????????290?????????????295aaa?gtt?aag?ttg?tct?tgg?aag?ctc?tca?ggt?atc?act?aag?ctg?gag?agc??966Lys?Val?Lys?Leu?Ser?Trp?Lys?Leu?Ser?Gly?Ile?Thr?Lys?Leu?Glu?Ser

300?????????????305??????????????310gga?gga?tac?aac?tta?aca?tat?gag?act?cca?gat?ggt?tta?gtt?tcc?gtg??1014Gly?Gly?Tyr?Asn?Leu?Thr?Tyr?Glu?Thr?Pro?Asp?Gly?Leu?Val?Ser?Val

315??????????????320??????????????325cag?agc?aaa?agt?gtt?gta?atg?acg?gtg?cca?tct?cat?gtt?gca?agt?ggt??1062Gln?Ser?Lys?Ser?Val?Val?Met?Thr?Val?Pro?Ser?His?Val?Ala?Ser?Gly???330??????????????335??????????????340ctc?ttg?cgc?cct?ctt?tct?gaa?tct?gct?gca?aat?gca?ctc?tca?aaa?cta??1110Leu?Leu?Arg?Pro?Leu?Ser?Glu?Ser?Ala?Ala?Asn?Ala?Leu?Ser?Lys?Leu345??????????????350??????????????355?????????????360tat?tac?cca?cca?gtt?gca?gca?gta?tct?atc?tcg?tac?ccg?aaa?gaa?gca??1158Tyr?Tyr?Pro?Pro?Val?Ala?Ala?Val?Ser?Ile?Ser?Tyr?Pro?Lys?Glu?Ala

365??????????????370?????????????375atc?cga?aca?gaa?tgt?ttg?ata?gat?ggt?gaa?cta?aag?ggt?ttt?ggg?caa??1206Ile?Arg?Thr?Glu?Cys?Leu?Ile?Asp?Gly?Glu?Leu?Lys?Gly?Phe?Gly?Gln

380?????????????385??????????????390ttg?cat?cca?cgc?acg?caa?gga?gtt?gaa?aca?tta?gga?act?atc?tac?agc??1254Leu?His?Pro?Arg?Thr?Gln?Gly?Val?Glu?Thr?Leu?Gly?Thr?Ile?Tyr?Ser

395?????????????400??????????????405tcc?tca?ctc?ttt?cca?aat?cgc?gca?ccg?ccc?gga?aga?att?ttg?ctg?ttg??1302Ser?Ser?Leu?Phe?Pro?Asn?Arg?Ala?Pro?Pro?Gly?Arg?Ile?Leu?Leu?Leu???410??????????????415??????????????420aac?tac?att?ggc?ggg?tct?aca?aac?acc?gga?att?ctg?tcc?aag?tct?gaa??1350Asn?Tyr?Ile?Gly?Gly?Ser?Thr?Asn?Thr?Gly?Ile?Leu?Ser?Lys?Ser?Glu425??????????????430?????????????435??????????????440ggt?gag?tta?gtg?gaa?gca?gtt?gac?aga?gat?ttg?agg?aaa?atg?cta?att??1398Gly?Glu?Leu?Val?Glu?Ala?Val?Asp?Arg?Asp?Leu?Arg?Lys?Met?Leu?Ile

445??????????????450??????????????455aag?cct?aat?tcg?acc?gat?cca?ctt?aaa?tta?gga?gtt?agg?gta?tgg?cct??1446Lys?Pro?Asn?Ser?Thr?Asp?Pro?Leu?Lys?Leu?Gly?Val?Arg?Val?Trp?Pro

460?????????????465??????????????470caa?gcc?att?cct?cag?ttt?cta?gtt?ggt?cac?ttt?gat?atc?ctt?gac?acg??1494Gln?Ala?Ile?Pro?Gln?Phe?Leu?Val?Gly?His?Phe?Asp?Ile?Leu?Asp?Thr

475??????????????480??????????????485gct?aaa?tca?tct?cta?acg?tct?tcg?ggc?tac?gaa?ggg?cta?ttt?ttg?ggt??1542Ala?Lys?Ser?Ser?Leu?Thr?Set?Ser?Gly?Tyr?Glu?Gly?Leu?Phe?Leu?Gly???490??????????????495??????????????500ggc?aat?tac?gtc?gct?ggt?gta?gcc?tta?ggc?cgg?tgt?gta?gaa?ggc?gca??1590Gly?Asn?Tyr?Val?Ala?Gly?Val?Ala?Leu?Gly?Arg?Cys?Val?Glu?Gly?Ala505??????????????510??????????????515?????????????520tat?gaa?acc?gcg?att?gag?gtc?aac?aac?ttc?atg?tca?cgg?tac?gct?tac??1638Tyr?Glu?Thr?Ala?Ile?Glu?Val?Asn?Asn?Phe?Met?Ser?Arg?Tyr?Ala?Tyr

525 530 535aag taa atgtaaaaca ttaaatctcc cagcttgcgt gagttttatt aaatattttg 1694Lysagatatccaa aaaaaaaaaa aaaaa, 1719＜210〉2＜211〉537＜212〉PRT＜213〉mouse ear mustard＜400〉2Met Glu Leu Ser Leu Leu Arg Pro Thr Thr Gln Ser Leu Leu Pro Ser 15 10 15Phe Ser Lys Pro Asn Leu Arg Leu Asn Val Tyr Lys Pro Leu Arg Leu

20??????????????25???????????????30Arg?Cys?Ser?Val?Ala?Gly?Gly?Pro?Thr?Val?Gly?Ser?Ser?Lys?Ile?Glu

35???????????????40???????????????45Gly?Gly?Gly?Gly?Thr?Thr?Ile?Thr?Thr?Asp?Cys?Val?Ile?Val?Gly?Gly

50???????????????55??????????????60Gly?Ile?Ser?Gly?Leu?Cys?Ile?Ala?Gln?Ala?Leu?Ala?Thr?Lys?His?Pro?65??????????????70???????????????75??????????????80Asp?Ala?Ala?Pro?Asn?Leu?Ile?Val?Thr?Glu?Ala?Lys?Asp?Arg?Val?Gly

85??????????????90???????????????95Gly?Asn?Ile?Ile?Thr?Arg?Glu?Glu?Asn?Gly?Phe?Leu?Trp?Glu?Glu?Gly

100?????????????105??????????????110Pro?Asn?Ser?Phe?Gln?Pro?Ser?Asp?Pro?Met?Leu?Thr?Met?Val?Val?Asp

115??????????????120??????????????125Ser?Gly?Leu?Lys?Asp?Asp?Leu?Val?Leu?Gly?Asp?Pro?Thr?Ala?Pro?Arg???130??????????????135??????????????140Phe?Val?Leu?Trp?Asn?Gly?Lys?Leu?Arg?Pro?Val?Pro?Ser?Lys?Leu?Thr145??????????????150?????????????155??????????????160Asp?Leu?Pro?Phe?Phe?Asp?Leu?Met?Ser?Ile?Gly?Gly?Lys?Ile?Arg?Ala

165??????????????170?????????????175Gly?Phe?Gly?Ala?Leu?Gly?Ile?Arg?Pro?Ser?Pro?Pro?Gly?Arg?Glu?Glu

180??????????????185?????????????190Ser?Val?Glu?Glu?Phe?Val?Arg?Arg?Asn?Leu?Gly?Asp?Glu?Val?Phe?Glu

195??????????????200??????????????205Arg?Leu?Ile?Glu?Pro?Phe?Cys?Ser?Gly?Val?Tyr?Ala?Gly?Asp?Pro?Ser???210??????????????215??????????????220Lys?Leu?Ser?Met?Lys?Ala?Ala?Phe?Gly?Lys?Val?Trp?Lys?Leu?Glu?Gln225??????????????230?????????????235??????????????240Asn?Gly?Gly?Ser?Ile?Ile?Gly?Gly?Thr?Phe?Lys?Ala?Ile?Gln?Glu?Arg

245??????????????250?????????????255Lys?Asn?Ala?Pro?Lys?Ala?Glu?Arg?Asp?Pro?Arg?Leu?Pro?Lys?Pro?Gln

260?????????????265??????????????270Gly?Gln?Thr?Val?Gly?Ser?Phe?Arg?Lys?Gly?Leu?Arg?Met?Leu?Pro?Glu

275??????????????280??????????????285Ala?Ile?Ser?Ala?Arg?Leu?Gly?Ser?Lys?Val?Lys?Leu?Ser?Trp?Lys?Leu

290?????????????295??????????????300Ser?Gly?Ile?Thr?Lys?Leu?Glu?Ser?Gly?Gly?Tyr?Asn?Leu?Thr?Tyr?Glu305??????????????310??????????????315?????????????320Thr?Pro?Asp?Gly?Leu?Val?Ser?Val?Gln?Ser?Lys?Ser?Val?Val?Met?Thr

325??????????????330?????????????335Val?Pro?Ser?His?Val?Ala?Ser?Gly?Leu?Leu?Arg?Pro?Leu?Ser?Glu?Ser

340?????????????345??????????????350Ala?Ala?Asn?Ala?Leu?Ser?Lys?Leu?Tyr?Tyr?Pro?Pro?Val?Ala?Ala?Val

355??????????????360??????????????365Ser?Ile?Ser?Tyr?Pro?Lys?Glu?Ala?Ile?Arg?Thr?Glu?Cys?Leu?Ile?Asp???370??????????????375??????????????380Gly?Glu?Leu?Lys?Gly?Phe?Gly?Gln?Leu?His?Pro?Arg?Thr?Gln?Gly?Val385??????????????390??????????????395?????????????400Glu?Thr?Leu?Gly?Thr?Ile?Tyr?Ser?Ser?Ser?Leu?Phe?Pro?Asn?Arg?Ala

405??????????????410?????????????415Pro?Pro?Gly?Arg?Ile?Leu?Leu?Leu?Asn?Tyr?Ile?Gly?Gly?Ser?Thr?Asn

420?????????????425??????????????430Thr?Gly?Ile?Leu?Ser?Lys?Ser?Glu?Gly?Glu?Leu?Val?Glu?Ala?Val?Asp

435??????????????440??????????????445Arg?Asp?Leu?Arg?Lys?Met?Leu?Ile?Lys?Pro?Asn?Ser?Thr?Asp?Pro?Leu???450??????????????455??????????????460Lys?Leu?Gly?Val?Arg?Val?Trp?Pro?Gln?Ala?Ile?Pro?Gln?Phe?Leu?Val465??????????????470??????????????475?????????????480Gly?His?Phe?Asp?Ile?Leu?Asp?Thr?Ala?Lys?Ser?Ser?Leu?Thr?Ser?Ser

485??????????????490??????????????495Gly?Tyr?Glu?Gly?Leu?Phe?Leu?Gly?Gly?Asn?Tyr?Val?Ala?Gly?Val?Ala

500?????????????505??????????????510Leu?Gly?Arg?Cys?Val?Glu?Gly?Ala?Tyr?Glu?Thr?Ala?Ile?Glu?Val?Asn

515 520 525Asn Phe Met Ser Arg Tyr Ala Tyr Lys, 530 535＜210〉3＜211〉1738＜212〉DNA＜213〉mouse ear mustard＜220〉＜221〉CDS＜222, (70) .., (1596)＜223〉mouse ear mustard belongs to protox-2＜400〉3ttttttactt atttccgtca ctgctttcga ctggtcagag attttgactc tgaattgttg 60cagatagca atg gcg tct gga gca gta gca gat cat caa att gaa gcg gtt 111

Met?Ala?Ser?Gly?Ala?Val?Ala?Asp?His?Gln?Ile?Glu?Ala?Val

1????????????5??????????????10tca?gga?aaa?aga?gtc?gca?gtc?gta?ggt?gca?ggt?gta?agt?gga?ctt?gcg???159Ser?Gly?Lys?Arg?Val?Ala?Val?Val?Gly?Ala?Gly?Val?Ser?Gly?Leu?Ala?15??????????????20???????????????25??????????????30gcg?gct?tac?aag?ttg?aaa?tcg?agg?ggt?ttg?aat?gtg?act?gtg?ttt?gaa???207Ala?Ala?Tyr?Lys?Leu?Lys?Ser?Arg?Gly?Leu?Asn?Val?Thr?Val?Phe?Glu

35??????????????40???????????????45gct?gat?gga?aga?gta?ggt?ggg?aag?ttg?aga?agt?gtt?atg?caa?aat?ggt???255Ala?Asp?Gly?Arg?Val?Gly?Gly?Lys?Leu?Arg?Ser?Val?Met?Gln?Asn?Gly

50??????????????55??????????????60ttg?att?tgg?gat?gaa?gga?gca?aac?acc?atg?act?gag?gct?gag?cca?gaa???303Leu?Ile?Trp?Asp?Glu?Gly?Ala?Asn?Thr?Met?Thr?Glu?Ala?Glu?Pro?Glu

65???????????????70??????????????75gtt?ggg?agt?tta?ctt?gat?gat?ctt?ggg?ctt?cgt?gag?aaa?caa?caa?ttt???351Val?Gly?Ser?Leu?Leu?Asp?Asp?Leu?Gly?Leu?Arg?Glu?Lys?Gln?Gln?Phe

80???????????????85??????????????90cca?att?tca?cag?aaa?aag?cgg?tat?att?gtg?cgg?aat?ggt?gta?cct?gtg???399Pro?Ile?Ser?Gln?Lys?Lys?Arg?Tyr?Ile?Val?Arg?Asn?Gly?Val?Pro?Val?95??????????????100?????????????105??????????????110atg?cta?cct?acc?aat?ccc?ata?gag?ctg?gtc?aca?agt?agt?gtg?ctc?tct??447Met?Leu?Pro?Thr?Asn?Pro?Ile?Glu?Leu?Val?Thr?Ser?Ser?Val?Leu?Ser

115??????????????120?????????????125acc?caa?tct?aag?ttt?caa?atc?ttg?ttg?gaa?cca?ttt?tta?tgg?aag?aaa??495Thr?Gln?Ser?Lys?Phe?Gln?Ile?Leu?Leu?Glu?Pro?Phe?Leu?Trp?Lys?Lys

130?????????????135??????????????140aag?tcc?tca?aaa?gtc?tca?gat?gca?tct?gct?gaa?gaa?agt?gta?agc?gag??543Lys?Ser?Ser?Lys?Val?Ser?Asp?Ala?Ser?Ala?Glu?Glu?Ser?Val?Ser?Glu

145??????????????150??????????????155ttc?ttt?caa?cgc?cat?ttt?gga?caa?gag?gtt?gtt?gac?tat?ctc?atc?gac??591Phe?Phe?Gln?Arg?His?Phe?Gly?Gln?Glu?Val?Val?Asp?Tyr?Leu?Ile?Asp???160??????????????165??????????????170cct?ttt?gtt?ggt?gga?aca?agt?gct?gcg?gac?cct?gat?tcc?ctt?tca?atg??639Pro?Phe?Val?Gly?Gly?Thr?Ser?Ala?Ala?Asp?Pro?Asp?Ser?Leu?Ser?Met175??????????????180??????????????185?????????????190aag?cat?tct?ttc?cca?gat?ctc?tgg?aat?gta?gag?aaa?agt?ttt?ggc?tct??687Lys?His?Ser?Phe?Pro?Asp?Leu?Trp?Asn?Val?Glu?Lys?Ser?Phe?Gly?Ser

195??????????????200?????????????205att?ata?gtc?ggt?gca?atc?aga?aca?aag?ttt?gct?gct?aaa?ggt?ggt?aaa??735Ile?Ile?Val?Gly?Ala?Ile?Arg?Thr?Lys?Phe?Ala?Ala?Lys?Gly?Gly?Lys

210?????????????215??????????????220agt?aga?gac?aca?aag?agt?tct?cct?ggc?aca?aaa?aag?ggt?tcg?cgt?ggg??783Ser?Arg?Asp?Thr?Lys?Ser?Ser?Pro?Gly?Thr?Lys?Lys?Gly?Ser?Arg?Gly

225??????????????230??????????????235tca?ttc?tct?ttt?aag?ggg?gga?atg?cag?att?ctt?cct?gat?acg?ttg?tgc??831Ser?Phe?Ser?Phe?Lys?Gly?Gly?Met?Gln?Ile?Leu?Pro?Asp?Thr?Leu?Cys???240??????????????245??????????????250aaa?agt?ctc?tca?cat?gat?gag?atc?aat?tta?gac?tcc?aag?gta?ctc?tct??879Lys?Ser?Leu?Ser?His?Asp?Glu?Ile?Asn?Leu?Asp?Ser?Lys?Val?Leu?Ser255??????????????260??????????????265?????????????270ttg?tct?tac?aat?tct?gga?tca?aga?cag?gag?aac?tgg?tca?tta?tct?tgt??927Leu?Ser?Tyr?Asn?Ser?Gly?Ser?Arg?Gln?Glu?Asn?Trp?Ser?Leu?Ser?Cys

275??????????????280?????????????285gtt?tcg?cat?aat?gaa?acg?cag?aga?caa?aac?ccc?cat?tat?gat?gct?gta??975Val?Ser?His?Asn?Glu?Thr?Gln?Arg?Gln?Asn?Pro?His?Tyr?Asp?Ala?Val

290?????????????295??????????????300att?atg?acg?gct?cct?ctg?tgc?aat?gtg?aag?gag?atg?aag?gtt?atg?aaa??1023Ile?Met?Thr?Ala?Pro?Leu?Cys?Asn?Val?Lys?Glu?Met?Lys?Val?Met?Lys

305??????????????310??????????????315gga?gga?caa?ccc?ttt?cag?cta?aac?ttt?ctc?ccc?gag?att?aat?tac?atg??1071Gly?Gly?Gln?Pro?Phe?Gln?Leu?Asn?Phe?Leu?Pro?Glu?Ile?Asn?Tyr?Met???320??????????????325??????????????330ccc?ctc?tcg?gtt?tta?atc?acc?aca?ttc?aca?aag?gag?aaa?gta?aag?aga??1119Pro?Leu?Ser?Val?Leu?Ile?Thr?Thr?Phe?Thr?Lys?Glu?Lys?Val?Lys?Arg335??????????????340?????????????345??????????????350cct?ctt?gaa?ggc?ttt?ggg?gta?ctc?att?cca?tct?aag?gag?caa?aag?cat??1167Pro?Leu?Glu?Gly?Phe?Gly?Val?Leu?Ile?Pro?Ser?Lys?Glu?Gln?Lys?His

355??????????????360?????????????365ggt?ttc?aaa?act?cta?ggt?aca?ctt?ttt?tca?tca?atg?atg?ttt?cca?gat??1215Gly?Phe?Lys?Thr?Leu?Gly?Thr?Leu?Phe?Ser?Ser?Met?Met?Phe?Pro?Asp

370??????????????375?????????????380cgt?tcc?cct?agt?gac?gtt?cat?cta?tat?aca?act?ttt?att?ggt?ggg?agt??1263Arg?Ser?Pro?Ser?Asp?Val?His?Leu?Tyr?Thr?Thr?Phe?Ile?Gly?Gly?Ser

385??????????????390??????????????395agg?aac?cag?gga?cta?gcc?aaa?gct?tcc?act?gac?gaa?tta?aaa?caa?gtt??1311Arg?Asn?Gln?Glu?Leu?Ala?Lys?Ala?Ser?Thr?Asp?Glu?Leu?Lys?Gln?Val???400??????????????405??????????????410gtg?act?tct?gac?ctt?cag?cga?ctg?ttg?ggg?gtt?gaa?ggt?gaa?ccc?gtg??1359Val?Thr?Ser?Asp?Leu?Gln?Arg?Leu?Leu?Gly?Val?Glu?Gly?Glu?Pro?Val415??????????????420??????????????425?????????????430tct?gtc?aac?cat?tac?tat?tgg?agg?aaa?gca?ttc?ccg?ttg?tat?gac?agc??1407Ser?Val?Asn?His?Tyr?Tyr?Trp?Arg?Lys?Ala?Phe?Pro?Leu?Tyr?Asp?Ser

435??????????????440??????????????445agc?tat?gac?tca?gtc?atg?gaa?gca?att?gac?aag?atg?gag?aat?gat?cta???1455Ser?Tyr?Asp?Ser?Val?Met?Glu?Ala?Ile?Asp?Lys?Met?Glu?Asn?Asp?Leu

450?????????????455??????????????460cct?ggg?ttc?ttc?tat?gca?ggt?aat?cat?cga?ggg?ggg?ctc?tct?gtt?ggg???1503Pro?Gly?Phe?Phe?Tyr?Ala?Gly?Asn?His?Arg?Gly?Gly?Leu?Ser?Val?Gly

465 470 475aaa tca ata gca tca ggt tgc aaa gca gct gac ctt gtg atc tca tac 1551Lys Ser Ile Ala Ser Gly Cys Lys Ala Ala Asp Leu Val Ile Ser Tyr 480 485 490ctg gag tct tgc tca aat gac aag aaa cca aat gac agc tta taa 1596Leu Glu Ser Cys Ser Asn Asp Lys Lys Pro Asn Asp Ser Leu495 500 505cattgtcaag gttcgtccct ttttatcact tactttgtaa acttgtaaaa tgcaacaagc 1656cgccgtgcga ttagccaaca actcagcaaa acccagattc tcataaggct cactaattcc 1716agaataaact atttatgtaa aa 1738＜210〉4＜211〉508＜212〉PRT＜213〉＜400〉4Met Ala Ser Gly Ala Val Ala Asp His Gln Ile Glu Ala Val Ser Gly 1 5 10 15Lys Arg Val Ala Val Val Gly Ala Gly Val Ser Gly Leu Ala Ala Ala

20??????????????25???????????????30Tyr?Lys?Leu?Lys?Ser?Arg?Gly?Leu?Asn?Val?Thr?Val?Phe?Glu?Ala?Asp

35???????????????40??????????????45Gly?Arg?Val?Gly?Gly?Lys?Leu?Arg?Ser?Val?Met?Gln?Asn?Gly?Leu?Ile

50??????????????55???????????????60Trp?Asp?Glu?Gly?Ala?Asn?Thr?Met?Thr?Glu?Ala?Glu?Pro?Glu?Val?Gly65???????????????70???????????????75??????????????80Ser?Leu?Leu?Asp?Asp?Leu?Gly?Leu?Arg?Glu?Lys?Gln?Gln?Phe?Pro?Ile

85??????????????90???????????????95Ser?Gln?Lys?Lys?Arg?Tyr?Ile?Val?Arg?Asn?Gly?Val?Pro?Val?Met?Leu

100?????????????105??????????????110Pro?Thr?Asn?Pro?Ile?Glu?Leu?Val?Thr?Ser?Ser?Val?Leu?Ser?Thr?Gln

115??????????????120??????????????125Ser?Lys?Phe?Gln?Ile?Leu?Leu?Glu?Pro?Phe?Leu?Trp?Lys?Lys?Lys?Ser???130??????????????135??????????????140Ser?Lys?Val?Ser?Asp?Ala?Ser?Ala?Glu?Glu?Ser?Val?Ser?Glu?Phe?Phe145??????????????150??????????????155?????????????160Gln?Arg?His?Phe?Gly?Gln?Glu?Val?Val?Asp?Tyr?Leu?Ile?Asp?Pro?Phe

165??????????????170?????????????175Val?Gly?Gly?Thr?Ser?Ala?Ala?Asp?Pro?Asp?Ser?Leu?Ser?Met?Lys?His

180?????????????185??????????????190Ser?Phe?Pro?Asp?Leu?Trp?Asn?Val?Glu?Lys?Ser?Phe?Gly?Ser?Ile?Ile

195??????????????200??????????????205Val?Gly?Ala?Ile?Arg?Thr?Lys?Phe?Ala?Ala?Lys?Gly?Gly?Lys?Ser?Arg???210??????????????2l5??????????????220Asp?Thr?Lys?Ser?Ser?Pro?Gly?Thr?Lys?Lys?Gly?Ser?Arg?Gly?Ser?Phe225??????????????230??????????????235?????????????240Ser?Phe?Lys?Gly?Gly?Met?Gln?Ile?Leu?Pro?Asp?Thr?Leu?Cys?Lys?Ser

245??????????????250?????????????255Leu?Ser?His?Asp?Glu?Ile?Asn?Leu?Asp?Ser?Lys?Val?Leu?Ser?Leu?Ser

260??????????????265?????????????270Tyr?Asn?Ser?Gly?Ser?Arg?Gln?Glu?Asn?Trp?Ser?Leu?Ser?Cys?Val?Ser

275??????????????280??????????????285His?Asn?Glu?Thr?Gln?Arg?Gln?Asn?Pro?His?Tyr?Asp?Ala?Val?Ile?Met???290??????????????295??????????????300Thr?Ala?Pro?Leu?Cys?Asn?Val?Lys?Glu?Met?Lys?Val?Met?Lys?Gly?Gly305??????????????310??????????????315?????????????320Gln?Pro?Phe?Gln?Leu?Asn?Phe?Leu?Pro?Glu?Ile?Asn?Tyr?Met?Pro?Leu

325??????????????330??????????????335Ser?Val?Leu?Ile?Thr?Thr?Phe?Thr?Lys?Glu?Lys?Val?Lys?Arg?Pro?Leu

340?????????????345??????????????350Glu?Gly?Phe?Gly?Val?Leu?Ile?Pro?Ser?Lys?Glu?Gln?Lys?His?Gly?Phe

355??????????????360??????????????365Lys?Thr?Leu?Gly?Thr?Leu?Phe?Ser?Ser?Met?Met?Phe?Pro?Asp?Arg?Ser???370??????????????375??????????????380Pro?Ser?Asp?Val?His?Leu?Tyr?Thr?Thr?Phe?Ile?Gly?Gly?Ser?Arg?Asn385??????????????390?????????????395??????????????400Gln?Glu?Leu?Ala?Lys?Ala?Ser?Thr?Asp?Glu?Leu?Lys?Gln?Val?Val?Thr

405??????????????410?????????????415Ser?Asp?Leu?Gln?Arg?Leu?Leu?Gly?Val?Glu?Gly?Glu?Pro?Val?Ser?Val

420?????????????425??????????????430Asn?His?Tyr?Tyr?Trp?Arg?Lys?Ala?Phe?Pro?Leu?Tyr?Asp?Ser?Ser?Tyr

435??????????????440??????????????445Asp?Ser?Val?Met?Glu?Ala?Ile?Asp?Lys?Met?Glu?Asn?Asp?Leu?Pro?Gly???450??????????????455??????????????460Phe?Phe?Tyr?Ala?Gly?Asn?His?Arg?Gly?Gly?Leu?Ser?Val?Gly?Lys?Ser465??????????????470??????????????475?????????????480Ile?Ala?Ser?Gly?Cys?Lys?Ala?Ala?Asp?Leu?Val?Ile?Ser?Tyr?Leu?Glu

485??????????????490?????????????495Ser?Cys?Ser?Asn?Asp?Lys?Lys?Pro?Asn?Asp?Ser?Leu

500 505＜210〉5＜211〉1691＜212〉DNA＜213〉corn＜220〉＜221〉CDS＜222〉(1) .. (1443)＜223〉corn protox-1 c-DNA (non-total length)＜400〉5gcg gac tgc gtc gtg gtg ggc gga ggc atc agt ggc ctc tgc acc gcg 48Ala Asp Cys Val Val Val Gly Gly Gly Ile Ser Gly Leu Cys Thr Ala 15 10 15cag gcg ctg gcc acg cgg cac ggc gtc ggg gac gtg ctt gtc acg gag 96Gln Ala Leu Ala Thr Arg His Gly Val Gly Asp Val Leu Val Thr Glu

20??????????????25???????????????30gcc?cgc?gcc?cgc?ccc?ggc?ggc?aac?att?acc?acc?gtc?gag?cgc?ccc?gag??144Ala?Arg?Ala?Arg?Pro?Gly?Gly?Asn?Ile?Thr?Thr?Val?Glu?Arg?Pro?Glu

35??????????????40??????????????45gaa?ggg?tac?ctc?tgg?gag?gag?ggt?ccc?aac?agc?ttc?cag?ccc?tcc?gac??192Glu?Gly?Tyr?Leu?Trp?Glu?Glu?Gly?Pro?Asn?Ser?Phe?Gln?Pro?Ser?Asp

50??????????????55???????????????60ccc?gtt?ctc?acc?atg?gcc?gtg?gac?agc?gga?ctg?aag?gat?gac?ttg?gtt??240Pro?Val?Leu?Thr?Met?Ala?Val?Asp?Ser?Gly?Leu?Lys?Asp?Asp?Leu?Val?65??????????????70???????????????75??????????????80ttt?ggg?gac?cca?aac?gcg?ccg?cgt?ttc?gtg?ctg?tgg?gag?ggg?aag?ctg??288Phe?Gly?Asp?Pro?Asn?Ala?Pro?Arg?Phe?Val?Leu?Trp?Glu?Gly?Lys?Leu

85??????????????90???????????????95agg?ccc?gtg?cca?tcc?aag?ccc?gcc?gac?ctc?ccg?ttc?ttc?gat?ctc?atg??336Arg?Pro?Val?Pro?Ser?Lys?Pro?Ala?Asp?Leu?Pro?Phe?Phe?Asp?Leu?Met

100?????????????105???????????????110agc?atc?cca?ggg?aag?ctc?agg?gcc?ggt?cta?ggc?gcg?ctt?ggc?atc?cgc??384Ser?Ile?Pro?Gly?Lys?Leu?Arg?Ala?Gly?Leu?Gly?Ala?Leu?Gly?Ile?Arg

115??????????????120??????????????125ccg?cct?cct?cca?ggc?cgc?gaa?gag?tca?gtg?gag?gag?ttc?gtg?cgc?cgc??432Pro?Pro?Pro?Pro?Gly?Arg?Glu?Glu?Ser?Val?Glu?Glu?Phe?Val?Arg?Arg???130??????????????135??????????????140aac?ctc?ggt?gct?gag?gtc?ttt?gag?cgc?ctc?att?gag?cct?ttc?tgc?tca??480Asn?Leu?Gly?Ala?Glu?Val?Phe?Glu?Arg?Leu?Ile?Glu?Pro?Phe?Cys?Ser145??????????????150?????????????155??????????????160ggt?gtc?tat?gct?ggt?gat?cct?tct?aag?ctc?agc?atg?aag?gct?gca?ttt??528Gly?Val?Tyr?Ala?Gly?Asp?Pro?Ser?Lys?Leu?Ser?Met?Lys?Ala?Ala?Phe

165??????????????170?????????????175ggg?aag?gtt?tgg?cgg?ttg?gaa?gaa?act?gga?ggt?agt?att?att?ggt?gga??576Gly?Lys?Val?Trp?Arg?Leu?Glu?Glu?Thr?Gly?Gly?Ser?Ile?Ile?Gly?Gly

180?????????????185??????????????190acc?atc?aag?aca?att?cag?gag?agg?agc?aag?aat?cca?aaa?cca?ccg?agg??624Thr?Ile?Lys?Thr?Ile?Gln?Glu?Arg?Ser?Lys?Asn?Pro?Lys?Pro?Pro?Arg

195??????????????200??????????????205gat?gcc?cgc?ctt?ccg?aag?cca?aaa?ggg?cag?aca?gtt?gca?tct?ttc?agg??672Asp?Ala?Arg?Leu?Pro?Lys?Pro?Lys?Gly?Gln?Thr?Val?Ala?Ser?Phe?Arg???210??????????????215??????????????220aag?ggt?ctt?gcc?atg?ctt?cca?aat?gcc?att?aca?tcc?agc?ttg?ggt?agt??720Lys?Gly?Leu?Ala?Met?Leu?Pro?Asn?Ala?Ile?Thr?Ser?Ser?Leu?Gly?Ser225??????????????230??????????????235?????????????240aaa?gtc?aaa?cta?tca?tgg?aaa?ctc?acg?agc?att?aca?aaa?tca?gat?gac??768Lys?Val?Lys?Leu?Ser?Trp?Lys?Leu?Thr?Ser?Ile?Thr?Lys?Ser?Asp?Asp

245??????????????250??????????????255aag?gga?tat?gtt?ttg?gag?tat?gaa?acg?cca?gaa?ggg?gtt?gtt?tcg?gtg??816Lys?Gly?Tyr?Val?Leu?Glu?Tyr?Glu?Thr?Pro?Glu?Gly?Val?Val?Ser?Val

260?????????????265??????????????270cag?gct?aaa?agt?gtt?atc?atg?act?att?cca?tca?tat?gtt?gct?agc?aac??864Gln?Ala?Lys?Ser?Val?Ile?Met?Thr?Ile?Pro?Ser?Tyr?Val?Ala?Ser?Asn

275??????????????280??????????????285att?ttg?cgt?cca?ctt?tca?agc?gat?gct?gca?gat?gct?cta?tca?aga?ttc??912Ile?Leu?Arg?Pro?Leu?Ser?Ser?Asp?Ala?Ala?Asp?Ala?Leu?Ser?Arg?Phe???290??????????????295??????????????300tat?tat?cca?ccg?gtt?gct?gct?gta?act?gtt?tcg?tat?cca?aag?gaa?gca??960Tyr?Tyr?Pro?Pro?Val?Ala?Ala?Val?Thr?Val?Ser?Tyr?Pro?Lys?Glu?Ala305??????????????310?????????????315??????????????320att?aga?aaa?gaa?tgc?tta?att?gat?ggg?gaa?ctc?cag?ggc?ttt?ggc?cag??1008Ile?Arg?Lys?Glu?Cys?Leu?Ile?Asp?Gly?Glu?Leu?Gln?Gly?Phe?Gly?Gln

325??????????????330?????????????335ttg?cat?cca?cgt?agt?caa?gga?gtt?gag?aca?tta?gga?aca?ata?tac?agt??1056Leu?His?Pro?Arg?Ser?Gln?Gly?Val?Glu?Thr?Leu?Gly?Thr?Ile?Tyr?Ser

340?????????????345??????????????350tcc?tca?ctc?ttt?cca?aat?cgt?gct?cct?gac?ggt?agg?gtg?tta?ctt?cta??1104Ser?Ser?Leu?Phe?Pro?Asn?Arg?Ala?Pro?Asp?Gly?Arg?Val?Leu?Leu?Leu

355?????????????360??????????????365aac?tac?ata?gga?ggt?gct?aca?aac?aca?gga?att?gtt?tcc?aag?act?gaa??1152Asn?Tyr?Ile?Gly?Gly?Ala?Thr?Asn?Thr?Gly?Ile?Val?Ser?Lys?Thr?Glu???370??????????????375??????????????380agt?gag?ctg?gtc?gaa?gca?gtt?gac?cgt?gac?ctc?cga?aaa?atg?ctt?ata??1200Ser?Glu?Leu?Val?Glu?Ala?Val?Asp?Arg?Asp?Leu?Arg?Lys?Met?Leu?Ile385??????????????390??????????????395?????????????400aat?tct?aca?gca?gtg?gac?cct?tta?gtc?ctt?ggt?gtt?cga?gtt?tgg?cca??1248Asn?Ser?Thr?Ala?Val?Asp?Pro?Leu?Val?Leu?Gly?Val?Arg?Val?Trp?Pro

405??????????????410?????????????415caa?gcc?ata?cct?cag?ttc?ctg?gta?gga?cat?ctt?gat?ctt?ctg?gaa?gcc??1296Gln?Ala?Ile?Pro?Gln?Phe?Leu?Val?Gly?His?Leu?Asp?Leu?Leu?Glu?Ala

420?????????????425??????????????430gca?aaa?gct?gcc?ctg?gac?cga?ggt?ggc?tac?gat?ggg?ctg?ttc?cta?gga??1344Ala?Lys?Ala?Ala?Leu?Asp?Arg?Gly?Gly?Tyr?Asp?Gly?Leu?Phe?Leu?Gly

435 440 445ggg aac tat gtt gca gga gtt gcc ctg ggc aga tgc gtt gag ggc gcg 1392Gly Asn Tyr Val Ala Gly Val Ala Leu Gly Arg Cys Val Glu Gly Ala 450 455 460tat gaa agt gcc tcg caa ata tct gac ttc ttg acc aag tat gcc tac 1440Tyr Glu Ser Ala Ser Gln Ile Ser Asp Phe Leu Thr Lys Tyr Ala Tyr465 470 475 480aag tgatgaaaga agtggagcgc tacttgttaa tcgtttatgt tgcatagatg 1493Lysaggtgcctcc ggggaaaaaa aagcttgaat agtatttttt attcttattt tgtaaattgc 1553atttctgttc ttttttctat cagtaattag ttatatttta gttctgtagg agattgttct 1613gttcactgcc cttcaaaaga aattttattt ttcattcttt tatgagagct gtgctactta 1673aaaaaaaaaa aaaaaaaa 1691＜210〉6＜211〉481＜212〉PRT＜213〉＜400〉6Ala Asp Cys Val Val Val Gly Gly Gly Ile Ser Gly Leu Cys Thr Ala 1 5 10 15Gln Ala Leu Ala Thr Arg His Gly Val Gly Asp Val Leu Val Thr Glu

20??????????????25???????????????30Ala?Arg?Ala?Arg?Pro?Gly?Gly?Asn?Ile?Thr?Thr?Val?Glu?Arg?Pro?Glu

35???????????????40??????????????45Glu?Gly?Tyr?Leu?Trp?Glu?Glu?Gly?Pro?Asn?Ser?Phe?Gln?Pro?Ser?Asp

50???????????????55??????????????60Pro?Val?Leu?Thr?Met?Ala?Val?Asp?Ser?Gly?Leu?Lys?Asp?Asp?Leu?Val?65???????????????70??????????????75??????????????80Phe?Gly?Asp?Pro?Asn?Ala?Pro?Arg?Phe?Val?Leu?Trp?Glu?Gly?Lys?Leu

85??????????????90???????????????95Arg?Pro?Val?Pro?Ser?Lys?Pro?Ala?Asp?Leu?Pro?Phe?Phe?Asp?Leu?Met

100?????????????105??????????????110Ser?Ile?Pro?Gly?Lys?Leu?Arg?Ala?Gly?Leu?Gly?Ala?Leu?Gly?Ile?Arg

115??????????????120??????????????125Pro?Pro?Pro?Pro?Gly?Arg?Glu?Glu?Ser?Val?Glu?Glu?Phe?Val?Arg?Arg???130??????????????135??????????????140Asn?Leu?Gly?Ala?Glu?Val?Phe?Glu?Arg?Leu?Ile?Glu?Pro?Phe?Cys?Ser145??????????????150??????????????155?????????????160Gly?Val?Tyr?Ala?Gly?Asp?Pro?Ser?Lys?Leu?Ser?Met?Lys?Ala?Ala?Phe

165??????????????170??????????????175Gly?Lys?Val?Trp?Arg?Leu?Glu?Glu?Thr?Gly?Gly?Ser?Ile?Ile?Gly?Gly

180?????????????185??????????????190Thr?Ile?Lys?Thr?Ile?Gln?Glu?Arg?Ser?Lys?Asn?Pro?Lys?Pro?Pro?Arg

195??????????????200??????????????205Asp?Ala?Arg?Leu?Pro?Lys?Pro?Lys?Gly?Gln?Thr?Val?Ala?Ser?Phe?Arg???210??????????????215??????????????220Lys?Gly?Leu?Ala?Met?Leu?Pro?Asn?Ala?Ile?Thr?Ser?Ser?Leu?Gly?Ser225??????????????230??????????????235?????????????240Lys?Val?Lys?Leu?Ser?Trp?Lys?Leu?Thr?Ser?Ile?Thr?Lys?Ser?Asp?Asp

245??????????????250??????????????255Lys?Gly?Tyr?Val?Leu?Glu?Tyr?Glu?Thr?Pro?Glu?Gly?Val?Val?Ser?Val

260?????????????265??????????????270Gln?Ala?Lys?Ser?Val?Ile?Met?Thr?Ile?Pro?Ser?Tyr?Val?Ala?Ser?Asn

275?????????????280??????????????285Ile?Leu?Arg?Pro?Leu?Ser?Ser?Asp?Ala?Ala?Asp?Ala?Leu?Ser?Arg?Phe???290??????????????295??????????????300Tyr?Tyr?Pro?Pro?Val?Ala?Ala?Val?Thr?Val?Ser?Tyr?Pro?Lys?Glu?Ala305??????????????310??????????????315?????????????320Ile?Ara?Lys?Glu?Cys?Leu?Ile?Asp?Gly?Glu?Leu?Gln?Gly?Phe?Gly?Gln

325??????????????330?????????????335Leu?His?Pro?Arg?Ser?Gln?Gly?Val?Glu?Thr?Leu?Gly?Thr?Ile?Tyr?Ser

340?????????????345??????????????350Ser?Ser?Leu?Phe?Pro?Asn?Arg?Ala?Pro?Asp?Gly?Arg?Val?Leu?Leu?Leu

355??????????????360??????????????365Asn?Tyr?Ile?Gly?Gly?Ala?Thr?Asn?Thr?Gly?Ile?Val?Ser?Lys?Thr?Glu???370??????????????375??????????????380Ser?Glu?Leu?Val?Glu?Ala?Val?Asp?Arg?Asp?Leu?Arg?Lys?Met?Leu?Ile385??????????????390?????????????395??????????????400Asn?Ser?Thr?Ala?Val?Asp?Pro?Leu?Val?Leu?Gly?Val?Arg?Val?Trp?Pro

405??????????????410??????????????415Gln?Ala?Ile?Pro?Gln?Phe?Leu?Val?Gly?His?Leu?Asp?Leu?Leu?Glu?Ala

420?????????????425??????????????430Ala?Lys?Ala?Ala?Leu?Asp?Arg?Gly?Gly?Tyr?Asp?Gly?Leu?Phe?Leu?Gly

435 440 445Gly Asn Tyr Val Ala Gly Val Ala Leu Gly Arg Cys Val Glu Gly Ala, 450 455 460Tyr Glu Ser Ala Ser Gln Ile Ser Asp Phe Leu Thr Lys Tyr Ala Tyr465,470 475 480Lys＜210〉7＜211〉2061＜212〉DNA＜213〉corn＜220〉＜221〉CDS＜222〉(64) .. (1698)＜223〉corn protox-2＜400〉7ctctcctacc tccacctcca cgacaacaag caaatcccca tccagttcca aaccctaact 60caa atg ctc gct ttg act gcc tca gcc tca tcc gct tcg tcc cat cct 108 Met Leu Ala Leu Thr Ala Ser Ala Ser Ser Ala Ser Ser His Pro

1????????????5??????????????10???????????????15tat?cgc?cac?gcc?tcc?gcg?cac?act?cgt?cgc?ccc?cgc?cta?cgt?gcg?gtc???156Tyr?Arg?His?Ala?Ser?Ala?His?Thr?Arg?Arg?Pro?Arg?Leu?Arg?Ala?Val

20??????????????25???????????????30ctc?gcg?atg?gcg?ggc?tcc?gac?gac?ccc?cgt?gca?gcg?ccc?gcc?aga?tcg???204Leu?Ala?Met?Ala?Gly?Ser?Asp?Asp?Pro?Arg?Ala?Ala?Pro?Ala?Arg?Ser

35???????????????40???????????????45gtc?gcc?gtc?gtc?ggc?gcc?ggg?gtc?agc?ggg?ctc?gcg?gcg?gcg?tac?agg???252Val?Ala?Val?Val?Gly?Ala?Gly?Val?Ser?Gly?Leu?Ala?Ala?Ala?Tyr?Arg

50???????????????55??????????????60ctc?aga?cag?agc?ggc?gtg?aac?gta?acg?gtg?ttc?gaa?gcg?gcc?gac?agg???300Leu?Arg?Gln?Ser?Gly?Val?Asn?Val?Thr?Val?Phe?Glu?Ala?Ala?Asp?Arg

65??????????????70???????????????75gcg?gga?gga?aag?ata?cgg?acc?aat?tcc?gag?ggc?ggg?ttt?gtc?tgg?gat???348Ala?Gly?Gly?Lys?Ile?Arg?Thr?Asn?Ser?Glu?Gly?Gly?Phe?Val?Trp?Asp?80??????????????85???????????????90??????????????95gaa?gga?gct?aac?acc?atg?aca?gaa?ggt?gaa?tgg?gag?gcc?agt?aga?ctg??396Glu?Gly?Ala?Asn?Thr?Met?Thr?Glu?Gly?Glu?Trp?Glu?Ala?Ser?Arg?Leu

100??????????????105?????????????110att?gat?gat?ctt?ggt?cta?caa?gac?aaa?cag?cag?tat?cct?aac?tcc?caa??444Ile?Asp?Asp?Leu?Gly?Leu?Gln?Asp?Lys?Gln?Gln?Tyr?Pro?Asn?Ser?Gln

115?????????????120??????????????125cac?aag?cgt?tac?att?gtc?aaa?gat?gga?gca?cca?gca?ctg?att?cct?tcg??492His?Lys?Arg?Tyr?Ile?Val?Lys?Asp?Gly?Ala?Pro?Ala?Leu?Ile?Pro?Ser

130??????????????135??????????????140gat?ccc?att?tcg?cta?atg?aaa?agc?agt?gtt?ctt?tcg?aca?aaa?tca?aag??540Asp?Pro?Ile?Ser?Leu?Met?Lys?Ser?Ser?Val?Leu?Ser?Thr?Lys?Ser?Lys???145??????????????150??????????????155att?gcg?tta?ttt?ttt?gaa?cca?ttt?ctc?tac?aag?aaa?gct?aac?aca?aga??588Ile?Ala?Leu?Phe?Phe?Glu?Pro?Phe?Leu?Tyr?Lys?Lys?Ala?Asn?Thr?Arg160??????????????165?????????????170??????????????175aac?tct?gga?aaa?gtg?tct?gag?gag?cac?ttg?agt?gag?agt?gtt?ggg?agc??636Asn?Ser?Gly?Lys?Val?Ser?Glu?Glu?His?Leu?Ser?Glu?Ser?Val?Gly?Ser

180??????????????185??????????????190ttc?tgt?gaa?cgc?cac?ttt?gga?aga?gaa?gtt?gtt?gac?tat?ttt?gtt?gat??684Phe?Cys?Glu?Arg?His?Phe?Gly?Arg?Glu?Val?Val?Asp?Tyr?Phe?Val?Asp

195??????????????200??????????????205cca?ttt?gta?gct?gga?aca?agt?gca?gga?gat?cca?gag?tca?cta?tct?att??732Pro?Phe?Val?Ala?Gly?Thr?Ser?Ala?Gly?Asp?Pro?Glu?Ser?Leu?Ser?Ile

210??????????????215??????????????220cgt?cat?gca?ttc?cca?gca?ttg?tgg?aat?ttg?gaa?aga?aag?tat?ggt?tca??780Arg?His?Ala?Phe?Pro?Ala?Leu?Trp?Asn?Leu?Glu?Arg?Lys?Tyr?Gly?Ser???225??????????????230??????????????235gtt?att?gtt?ggt?gcc?atc?ttg?tct?aag?cta?gca?gct?aaa?ggt?gat?cca??828Val?Ile?Val?Gly?Ala?Ile?Leu?Ser?Lys?Leu?Ala?Ala?Lys?Gly?Asp?Pro240??????????????245??????????????250?????????????255gta?aag?aca?aga?cat?gat?tca?tca?ggg?aaa?aga?agg?aat?aga?cga?gtg??876Val?Lys?Thr?Arg?His?Asp?Ser?Ser?Gly?Lys?Arg?Arg?Asn?Arg?Arg?Val

260??????????????265?????????????270tcg?ttt?tca?ttt?cat?ggt?gga?atg?cag?tca?cta?ata?aat?gca?ctt?cac??924Ser?Phe?Ser?Phe?His?Gly?Gly?Met?Gln?Ser?Leu?Ile?Asn?Ala?Leu?His

275?????????????280??????????????285aat?gaa?gtt?gga?gat?gat?aat?gtg?aag?ctt?ggt?aca?gaa?gtg?ttg?tca??972Asn?Glu?Val?Gly?Asp?Asp?Asn?Val?Lys?Leu?Gly?Thr?Glu?Val?Leu?Ser

290??????????????295??????????????300ttg?gca?tgt?aca?ttt?gat?gga?gtt?cct?gca?cta?ggc?agg?tgg?tca?att??1020Leu?Ala?Cys?Thr?Phe?Asp?Gly?Val?Pro?Ala?Leu?Gly?Arg?Trp?Ser?Ile???305??????????????310??????????????315tct?gtt?gat?tcg?aag?gat?agc?ggt?gac?aag?gac?ctt?gct?agt?aac?caa??1068Ser?Val?Asp?Ser?Lys?Asp?Ser?Gly?Asp?Lys?Asp?Leu?Ala?Ser?Asn?Gln320??????????????325?????????????330??????????????335acc?ttt?gat?gct?gtt?ata?atg?aca?gct?cca?ttg?tca?aat?gtc?cgg?agg??1116Thr?Phe?Asp?Ala?Val?Ile?Met?Thr?Ala?Pro?Leu?Ser?Asn?Val?Arg?Arg

340??????????????345??????????????350atg?aag?ttc?acc?aaa?ggt?gga?gct?ccg?gtt?gtt?ctt?gac?ttt?ctt?cct??1164Met?Lys?Phe?Thr?Lys?Gly?Gly?Ala?Pro?Val?Val?Leu?Asp?Phe?Leu?Pro

355??????????????360?????????????365aag?atg?gat?tat?cta?cca?cta?tct?ctc?atg?gtg?act?gct?ttt?aag?aag??1212Lys?Met?Asp?Tyr?Leu?Pro?Leu?Ser?Leu?Met?Val?Thr?Ala?Phe?Lys?Lys

370??????????????375??????????????380gat?gat?gtc?aag?aaa?cct?ctg?gaa?gga?ttt?ggg?gtc?tta?ata?cct?tac??1260Asp?Asp?Val?Lys?Lys?Pro?Leu?Glu?Gly?Phe?Gly?Val?Leu?Ile?Pro?Tyr???385??????????????390??????????????395aag?gaa?cag?caa?aaa?cat?ggt?ctg?aaa?acc?ctt?ggg?act?ctc?ttt?tcc??1308Lys?Glu?Gln?Gln?Lys?His?Gly?Leu?Lys?Thr?Leu?Gly?Thr?Leu?Phe?Ser400??????????????405?????????????410??????????????415tca?atg?atg?ttc?cca?gat?cga?gct?cct?gat?gac?caa?tat?tta?tat?aca??1356Ser?Met?Met?Phe?Pro?Asp?Arg?Ala?Pro?Asp?Asp?Gln?Tyr?Leu?Tyr?Thr

420??????????????425?????????????430aca?ttt?gtt?ggg?ggt?agc?cac?aat?aga?gat?ctt?gct?gga?gct?cca?acg??1404Thr?Phe?Val?Gly?Gly?Ser?His?Asn?Arg?Asp?Leu?Ala?Gly?Ala?Pro?Thr

435?????????????440??????????????445tct?att?ctg?aaa?caa?ctt?gtg?acc?tct?gac?ctt?aaa?aaa?ctc?ttg?ggc??1452Ser?Ile?Leu?Lys?Gln?Leu?Val?Thr?Ser?Asp?Leu?Lys?Lys?Leu?Leu?Gly

450??????????????455??????????????460gta?gag?ggg?caa?cca?act?ttt?gtc?aag?cat?gta?tac?tgg?gga?aat?gct??1500Val?Glu?Gly?Gln?Pro?Thr?Phe?Val?Lys?His?Val?Tyr?Trp?Gly?Asn?Ala???465??????????????470??????????????475ttt?cct?ttg?tat?ggc?cat?gat?tat?agt?tct?gta?ttg?gaa?gct?ata?gaa??1548Phe?Pro?Leu?Tyr?Gly?His?Asp?Tyr?Ser?Ser?Val?Leu?Glu?Ala?Ile?Glu480??????????????485?????????????490??????????????495aag?atg?gag?aaa?aac?ctt?cca?ggg?ttc?ttc?tac?gca?gga?aat?agc?aag??1596Lys?Met?Glu?Lys?Asn?Leu?Pro?Gly?Phe?Phe?Tyr?Ala?Gly?Asn?Ser?Lys

500??????????????505?????????????510gat?ggg?ctt?gct?gtt?gga?agt?gtt?ata?gct?tca?gga?agc?aag?gct?gct??1644Asp?Gly?Leu?Ala?Val?Gly?Ser?Val?Ile?Ala?Ser?Gly?Ser?Lys?Ala?Ala

515?????????????520??????????????525gac?ctt?gca?atc?tca?tat?ctt?gaa?tct?cac?acc?aag?cat?aat?aat?tca??1692Asp?Leu?Ala?Ile?Ser?Tyr?Leu?Glu?Ser?His?Thr?Lys?His?Asn?Asn?Ser

530 535 540cat tga aagtgtctga cctatcctct agcagttgtc gacaaatttc tccagttcat 1748His 545gtacagtaga aaccgatgcg ttgcagtttc agaacatctt cacttcttca gatattaacc 1808cttcgttgaa catccaccag aaaggtagtc acatgtgtaa gtgggaaaat gaggttaaaa 1868actattatgg cggccgaaat gttccttttt gttttcctca caagtggcct acgacacttg 1928atgttggaaa tacatttaaa tttgttgaat tgtttgagaa cacatgcgtg acgtgtaata 1988tttgcctatt gtgattttag cagtagtctt ggccagatta tgctttacgc ctttaaaaaa 2048aaaaaaaaaa aaa 2061＜210〉8＜211〉544＜212〉PRT＜213〉＜400〉8Met Leu Ala Leu Thr Ala Ser Ala Ser Ser Ala Ser Ser His Pro Tyr 1 5 10 15Arg His Ala Ser Ala His Thr Arg Arg Pro Arg Leu Arg Ala Val Leu

20???????????????25???????????????30Ala?Met?Ala?Gly?Ser?Asp?Asp?Pro?Arg?Ala?Ala?Pro?Ala?Arg?Ser?Val

35???????????????40??????????????45Ala?Val?Val?Gly?Ala?Gly?Val?Ser?Gly?Leu?Ala?Ala?Ala?Tyr?Arg?Leu

50??????????????55???????????????60Arg?Gln?Ser?Gly?Val?Asn?Val?Thr?Val?Phe?Glu?Ala?Ala?Asp?Arg?Ala?65??????????????70???????????????75??????????????80Gly?Gly?Lys?Ile?Arg?Thr?Asn?Ser?Glu?Gly?Gly?Phe?Val?Trp?Asp?Glu

85??????????????90???????????????95Gly?Ala?Asn?Thr?Met?Thr?Glu?Gly?Glu?Trp?Glu?Ala?Ser?Arg?Leu?Ile

100?????????????105??????????????110Asp?Asp?Leu?Gly?Leu?Gln?Asp?Lys?Gln?Gln?Tyr?Pro?Asn?Ser?Gln?His

115??????????????120??????????????125Lys?Arg?Tyr?Ile?Val?Lys?Asp?Gly?Ala?Pro?Ala?Leu?Ile?Pro?Ser?Asp???130??????????????135??????????????140Pro?Ile?Ser?Leu?Met?Lys?Ser?Ser?Val?Leu?Ser?Thr?Lys?Ser?Lys?Ile145??????????????150?????????????155??????????????160Ala?Leu?Phe?Phe?Glu?Pro?Phe?Leu?Tyr?Lys?Lys?Ala?Asn?Thr?Arg?Asn

165??????????????170?????????????175Ser?Gly?Lys?Val?Ser?Glu?Glu?His?Leu?Ser?Glu?Ser?Val?Gly?Ser?Phe

180?????????????185??????????????190Cys?Glu?Arg?His?Phe?Gly?Arg?Glu?Val?Val?Asp?Tyr?Phe?Val?Asp?Pro

195??????????????200??????????????205Phe?Val?Ala?Gly?Thr?Ser?Ala?Gly?Asp?Pro?Glu?Ser?Leu?Ser?Ile?Arg???210??????????????215??????????????220His?Ala?Phe?Pro?Ala?Leu?Trp?Asn?Leu?Glu?Arg?Lys?Tyr?Gly?Ser?Val225??????????????230??????????????235?????????????240Ile?Val?Gly?Ala?Ile?Leu?Ser?Lys?Leu?Ala?Ala?Lys?Gly?Asp?Pro?Val

245??????????????250?????????????255Lys?Thr?Arg?His?Asp?Ser?Ser?Gly?Lys?Arg?Arg?Asn?Arg?Arg?Val?Ser

260?????????????265??????????????270Phe?Ser?Phe?His?Gly?Gly?Met?Gln?Ser?Leu?Ile?Asn?Ala?Leu?His?Asn

275??????????????280??????????????285Glu?Val?Gly?Asp?Asp?Asn?Val?Lys?Leu?Gly?Thr?Glu?Val?Leu?Ser?Leu???290??????????????295??????????????300Ala?Cys?Thr?Phe?Asp?Gly?Val?Pro?Ala?Leu?Gly?Arg?Trp?Ser?Ile?Ser305??????????????310??????????????315?????????????320Val?Asp?Ser?Lys?Asp?Ser?Gly?Asp?Lys?Asp?Leu?Ala?Ser?Asn?Gln?Thr

325??????????????330?????????????335Phe?Asp?Ala?Val?Ile?Met?Thr?Ala?Pro?Leu?Ser?Asn?Val?Arg?Ara?Met

340?????????????345??????????????350Lys?Phe?Thr?Lys?Gly?Gly?Ala?Pro?Val?Val?Leu?Asp?Phe?Leu?Pro?Lys

355??????????????360??????????????365Met?Asp?Tyr?Leu?Pro?Leu?Ser?Leu?Met?Val?Thr?Ala?Phe?Lys?Lys?Asp???370??????????????375??????????????380Asp?Val?Lys?Lys?Pro?Leu?Glu?Gly?Phe?Gly?Val?Leu?Ile?Pro?Tyr?Lys385??????????????390??????????????395?????????????400Glu?Gln?Gln?Lys?His?Gly?Leu?Lys?Thr?Leu?Gly?Thr?Leu?Phe?Ser?Ser

405??????????????410?????????????415Met?Met?Phe?Pro?Asp?Arg?Ala?Pro?Asp?Asp?Gln?Tyr?Leu?Tyr?Thr?Thr

420?????????????425??????????????430Phe?Val?Gly?Gly?Ser?His?Asn?Arg?Asp?Leu?Ala?Gly?Ala?Pro?Thr?Ser

435??????????????440??????????????445Ile?Leu?Lys?Gln?Leu?Val?Thr?Ser?Asp?Leu?Lys?Lys?Leu?Leu?Gly?Val???450??????????????455??????????????460Glu?Gly?Gln?Pro?Thr?Phe?Val?Lys?His?Val?Tyr?Trp?Gly?Asn?Ala?Phe465??????????????470?????????????475??????????????480Pro?Leu?Tyr?Gly?His?Asp?Tyr?Ser?Ser?Val?Leu?Glu?Ala?Ile?Glu?Lys

485??????????????490?????????????495Met?Glu?Lys?Asn?Leu?Pro?Gly?Phe?Phe?Tyr?Ala?Gly?Asn?Ser?Lys?Asp

500??????????????505?????????????510Gly?Leu?Ala?Val?Gly?Ser?Val?Ile?Ala?Ser?Gly?Ser?Lys?Ala?Ala?Asp

515 520 525Leu Ala Ile Ser Tyr Leu Glu Ser His Thr Lys His Asn Asn Ser His 530 535 540＜210〉9＜211〉1811＜212〉DNA＜213〉common wheat＜220〉＜221〉CDS＜222, (3) .., (1589)＜223〉wheat protox-1＜400〉9gc gca aca atg gcc acc gcc acc gtc gcg gcc gcg tcg ccg ctc cgc 47 Ala Thr Met Ala Thr Ala Thr Val Ala Ala Ala Ser Pro Leu Arg

1????????????5??????????????10???????????????15ggc?agg?gtc?acc?ggg?cgc?cca?cac?cgc?gtc?cgc?ccg?cgt?tgc?gct?acc??95Gly?Arg?Val?Thr?Gly?Arg?Pro?His?Arg?Val?Arg?Pro?Arg?Cys?Ala?Thr

20??????????????25???????????????30gcg?agc?agc?gcg?acc?gag?act?ccg?gcg?gcg?ccc?ggc?gtg?cgg?ctg?tcc??143Ala?Ser?Ser?Ala?Thr?Glu?Thr?Pro?Ala?Ala?Pro?Gly?Val?Ara?Leu?Ser

35??????????????40??????????????45gcg?gaa?tgc?gtc?att?gtg?ggc?gcc?ggc?atc?agc?ggc?ctc?tgc?acc?gcg??191Ala?Glu?Cys?Val?Ile?Val?Gly?Ala?Gly?Ile?Ser?Gly?Leu?Cys?Thr?Ala

50???????????????55??????????????60cag?gcg?ctg?gcc?acc?cga?tac?ggc?gtc?agc?gac?ctg?ctc?gtc?acg?gag??239Gln?Ala?Leu?Ala?Thr?Arg?Tyr?Gly?Val?Ser?Asp?Leu?Leu?Val?Thr?Glu

65???????????????70??????????????75gcc?cgc?gac?cgc?ccg?ggc?ggc?aac?atc?acc?acc?gtc?gag?cgt?ccc?gac??287Ala?Arg?Asp?Arg?Pro?Gly?Gly?Asn?Ile?Thr?Thr?Val?Glu?Arg?Pro?Asp?80??????????????85???????????????90??????????????95gag?ggg?tac?ctg?tgg?gag?gag?gga?ccc?aac?agc?ttc?cag?ccc?tcc?gac??335Glu?Gly?Tyr?Leu?Trp?Glu?Glu?Gly?Pro?Asn?Ser?Phe?Gln?Pro?Ser?Asp

100??????????????105?????????????110ccg?gtc?ctc?acc?atg?gcc?gtg?gac?agc?ggg?ctc?aag?gat?gac?ttg?gtg??383Pro?Val?Leu?Thr?Met?Ala?Val?Asp?Ser?Gly?Leu?Lys?Asp?Asp?Leu?Val

115??????????????120??????????????125ttc?ggg?gac?ccc?aac?gcg?ccc?cgg?ttc?gtg?ctg?tgg?gag?ggg?aag?ctg??431Phe?Gly?Asp?Pro?Asn?Ala?Pro?Arg?Phe?Val?Leu?Trp?Glu?Gly?Lys?Leu

130??????????????135??????????????140agg?ccg?gtg?ccg?tcg?aag?cca?ggc?gac?ctg?cct?ttc?ttc?agc?ctc?atg??479Arg?Pro?Val?Pro?Ser?Lys?Pro?Gly?Asp?Leu?Pro?Phe?Phe?Ser?Leu?Met???145??????????????150??????????????155agt?atc?cct?ggg?aag?ctc?agg?gcc?ggc?ctt?ggc?gcg?ctc?ggc?att?cgc??527Ser?Ile?Pro?Gly?Lys?Leu?Arg?Ala?Gly?Leu?Gly?Ala?Leu?Gly?Ile?Arg160??????????????165??????????????170?????????????175cca?cct?cct?cca?ggg?cgc?gag?gag?tcg?gtg?gag?gag?ttt?gtg?cgc?cgc??575Pro?Pro?Pro?Pro?Gly?Arg?Glu?Glu?Ser?Val?Glu?Glu?Phe?Val?Arg?Arg

180??????????????185?????????????190aac?ctc?ggt?gcc?gag?gtc?ttt?gag?cgc?ctc?atc?gag?cct?ttc?tgc?tca??623Asn?Leu?Gly?Ala?Glu?Val?Phe?Glu?Arg?Leu?Ile?Glu?Pro?Phe?Cys?Ser

195?????????????200??????????????205ggt?gta?tat?gct?ggt?gat?cct?tcg?aag?ctt?agt?atg?aag?gct?gca?ttt??671Gly?Val?Tyr?Ala?Gly?Asp?Pro?Ser?Lys?Leu?Ser?Met?Lys?Ala?Ala?Phe

210?????????????215??????????????220ggg?aag?gtc?tgg?agg?ttg?gag?gag?att?gga?ggt?agt?att?att?ggt?gga??719Gly?Lys?Val?Trp?Arg?Leu?Glu?Glu?Ile?Gly?Gly?Ser?Ile?Ile?Gly?Gly???225??????????????230??????????????235acc?atc?aag?gcg?att?cag?gat?aaa?ggg?aag?aac?ccc?aaa?ccg?cca?agg??767Thr?Ile?Lys?Ala?Ile?Gln?Asp?Lys?Gly?Lys?Asn?Pro?Lys?Pro?Pro?Arg240??????????????245??????????????250?????????????255gat?ccc?cga?ctt?ccg?gca?cca?aag?gga?cag?acg?gtg?gca?tct?ttc?agg??815Asp?Pro?Arg?Leu?Pro?Ala?Pro?Lys?Gly?Gln?Thr?Val?Ala?Ser?Phe?Arg

260??????????????265??????????????270aag?ggt?cta?gcc?atg?ctc?ccg?aat?gcc?atc?gca?tct?agg?ctg?ggt?agt??863Lys?Gly?Leu?Ala?Met?Leu?Pro?Asn?Ala?Ile?Ala?Ser?Arg?Leu?Gly?Ser

275?????????????280??????????????285aaa?gtc?aag?ctg?tca?tgg?aag?ctt?acg?agc?att?aca?aag?gcg?gac?aac??911Lys?Val?Lys?Leu?Ser?Trp?Lys?Leu?Thr?Ser?Ile?Thr?Lys?Ala?Asp?Asn

290?????????????295??????????????300caa?gga?tat?gta?tta?ggt?tat?gaa?aca?cca?gaa?gga?ctt?gtt?tca?gtg??959Gln?Gly?Tyr?Val?Leu?Gly?Tyr?Glu?Thr?Pro?Glu?Gly?Leu?Val?Ser?Val???305??????????????310?????????????315cag?gct?aaa?agt?gtt?atc?atg?acc?atc?ccg?tca?tat?gtt?gct?agt?gat??1007Gln?Ala?Lys?Ser?Val?Ile?Met?Thr?Ile?Pro?Ser?Tyr?Val?Ala?Ser?Asp320??????????????325??????????????330?????????????335atc?ttg?cgc?cca?ctt?tca?att?gat?gca?gca?gat?gca?ctc?tca?aaa?ttc??1055Ile?Leu?Arg?Pro?Leu?Ser?Ile?Asp?Ala?Ala?Asp?Ala?Leu?Ser?Lys?Phe

340??????????????345?????????????350tat?tat?ccg?cca?gtt?gct?gct?gta?act?gtt?tca?tat?cca?aaa?gaa?gct??1103Tyr?Tyr?Pro?Pro?Val?Ala?Ala?Val?Thr?Val?Ser?Tyr?Pro?Lys?Glu?Ala

355?????????????360??????????????365att?aga?aaa?gaa?tgc?tta?att?gat?ggg?gag?ctc?cag?ggt?ttc?ggc?cag??1151Ile?Arg?Lys?Glu?Cys?Leu?Ile?Asp?Gly?Glu?Leu?Gln?Gly?Phe?Gly?Gln

370??????????????375??????????????380ttg?cat?cca?cgt?agc?caa?gga?gtc?gag?act?tta?ggg?aca?ata?tat?agc??1199Leu?His?Pro?Arg?Ser?Gln?Gly?Val?Glu?Thr?Leu?Gly?Thr?Ile?Tyr?Ser???385??????????????390??????????????395tct?tct?ctc?ttt?cct?aat?cgt?gct?cct?gct?gga?aga?gtg?tta?ctt?ctg??1247Ser?Ser?Leu?Phe?Pro?Asn?Arg?Ala?Pro?Ala?Gly?Arg?Val?Leu?Leu?Leu400??????????????405??????????????410?????????????415aac?tat?atc?ggg?ggt?tct?aca?aat?aca?ggg?atc?gtc?tcc?aag?act?gag??1295Asn?Tyr?Ile?Gly?Gly?Ser?Thr?Asn?Thr?Gly?Ile?Val?Ser?Lys?Thr?Glu

420??????????????425?????????????430agt?gac?tta?gta?gga?gcc?gtt?gac?cgt?gac?ctc?aga?aaa?atg?ttg?ata??1343Ser?Asp?Leu?Val?Gly?Ala?Val?Asp?Arg?Asp?Leu?Arg?Lys?Met?Leu?Ile

435?????????????440??????????????445aac?cct?aga?gca?gca?gac?cct?tta?gca?tta?ggg?gtt?cga?gtg?tgg?cca??1391Asn?Pro?Arg?Ala?Ala?Asp?Pro?Leu?Ala?Leu?Gly?Val?Arg?Val?Trp?Pro

450?????????????455??????????????460caa?gca?ata?cca?cag?ttt?ttg?att?ggg?cac?ctt?gat?cgc?ctt?gct?gct??1439Gln?Ala?Ile?Pro?Gln?Phe?Leu?Ile?Gly?His?Leu?Asp?Arg?Leu?Ala?Ala???465??????????????470??????????????475gca?aaa?tct?gca?ctg?ggc?caa?ggc?ggc?tac?gac?ggg?ttg?ttc?cta?gga??1487Ala?Lys?Ser?Ala?Leu?Gly?Gln?Gly?Gly?Tyr?Asp?Gly?Leu?Phe?Leu?Gly480??????????????485??????????????490?????????????495gga?aac?tac?gtc?gca?gga?gtt?gcc?ttg?ggc?cga?tgc?atc?gag?ggt?gcg??1535Gly?Asn?Tyr?Val?Ala?Gly?Val?Ala?Leu?Gly?Arg?Cys?Ile?Glu?Gly?Ala

500??????????????505?????????????510tac?gag?agt?gcc?tca?caa?gta?tct?gac?ttc?ttg?acc?aag?tat?gcc?tac???1583Tyr?Glu?Ser?Ala?Ser?Gln?Val?Ser?Asp?Phe?Leu?Thr?Lys?Tyr?Ala?Tyr

515 520 525aag tga tggaagtagt gcatctcttc attttgttgc atatacgagg tgaggctagg 1639Lysatcggtaaaa catcatgaga ttctgtagtg tttctttaat tgaaaaaaca aattttagtg 1699atgcaatatg tgctctttcc tgtagttcga gcatgtacat cggtatggga taaagtagaa 1759taagctattc tgcaaaagca gtgatttttt ttgaaaaaaa aaaaaaaaaa aa, 1811＜210〉10＜211〉528＜212〉PRT＜213〉common wheat＜400〉10Ala Thr Met Ala Thr Ala Thr Val Ala Ala Ala Ser Pro Leu Arg Gly, 15 10 15Arg Val Thr Gly Arg Pro His Arg Val Arg Pro Arg Cys Ala Thr Ala

20??????????????25???????????????30Ser?Ser?Ala?Thr?Glu?Thr?Pro?Ala?Ala?Pro?Gly?Val?Arg?Leu?Ser?Ala

35???????????????40??????????????45Glu?Cys?Val?Ile?Val?Gly?Ala?Gly?Ile?Ser?Gly?Leu?Cys?Thr?Ala?Gln

50???????????????55??????????????60Ala?Leu?Ala?Thr?Arg?Tyr?Gly?Val?Ser?Asp?Leu?Leu?Val?Thr?Glu?Ala?65???????????????70??????????????75??????????????80Arg?Asp?Arg?Pro?Gly?Gly?Asn?Ile?Thr?Thr?Val?Glu?Arg?Pro?Asp?Glu

85??????????????90???????????????95Gly?Tyr?Leu?Trp?Glu?Glu?Gly?Pro?Asn?Ser?Phe?Gln?Pro?Ser?Asp?Pro

100?????????????105??????????????110Val?Leu?Thr?Met?Ala?Val?Asp?Ser?Gly?Leu?Lys?Asp?Asp?Leu?Val?Phe

115??????????????120??????????????125Gly?Asp?Pro?Asn?Ala?Pro?Arg?Phe?Val?Leu?Trp?Glu?Gly?Lys?Leu?Arg???130??????????????135??????????????140Pro?Val?Pro?Ser?Lys?Pro?Gly?Asp?Leu?Pro?Phe?Phe?Ser?Leu?Met?Ser145??????????????150??????????????155?????????????160Ile?Pro?Gly?Lys?Leu?Arg?Ala?Gly?Leu?Gly?Ala?Leu?Gly?Ile?Arg?Pro

165??????????????170?????????????175Pro?Pro?Pro?Gly?Arg?Glu?Glu?Ser?Val?Glu?Glu?Phe?Val?Arg?Arg?Asn

180?????????????185??????????????190Leu?Gly?Ala?Glu?Val?Phe?Glu?Arg?Leu?Ile?Glu?Pro?Phe?Cys?Ser?Gly

195??????????????200??????????????205Val?Tyr?Ala?Gly?Asp?Pro?Ser?Lys?Leu?Ser?Met?Lys?Ala?Ala?Phe?Gly???210??????????????215??????????????220Lys?Val?Trp?Arg?Leu?Glu?Glu?Ile?Gly?Gly?Ser?Ile?Ile?Gly?Gly?Thr225??????????????230?????????????235??????????????240Ile?Lys?Ala?Ile?Gln?Asp?Lys?Gly?Lys?Asn?Pro?Lys?Pro?Pro?Arg?Asp

245??????????????250??????????????255Pro?Arg?Leu?Pro?Ala?Pro?Lys?Gly?Gln?Thr?Val?Ala?Ser?Phe?Arg?Lys

260??????????????265??????????????270Gly?Leu?Ala?Met?Leu?Pro?Asn?Ala?Ile?Ala?Ser?Arg?Leu?Gly?Ser?Lys

275??????????????280??????????????285Val?Lys?Leu?Ser?Trp?Lys?Leu?Thr?Ser?Ile?Thr?Lys?Ala?Asp?Asn?Gln???290??????????????295??????????????300Gly?Tyr?Val?Leu?Gly?Tyr?Glu?Thr?Pro?Glu?Gly?Leu?Val?Ser?Val?Gln305??????????????310??????????????315?????????????320Ala?Lys?Ser?Val?Ile?Met?Thr?Ile?Pro?Ser?Tyr?Val?Ala?Ser?Asp?Ile

325??????????????330?????????????335Leu?Arg?Pro?Leu?Ser?Ile?Asp?Ala?Ala?Asp?Ala?Leu?Ser?Lys?Phe?Tyr

340?????????????345??????????????350Tyr?Pro?Pro?Val?Ala?Ala?Val?Thr?Val?Ser?Tyr?Pro?Lys?Glu?Ala?Ile

355??????????????360??????????????365Arg?Lys?Glu?Cys?Leu?Ile?Asp?Gly?Glu?Leu?Gln?Gly?Phe?Gly?Gln?Leu???370??????????????375??????????????380His?Pro?Arg?Ser?Gln?Gly?Val?Glu?Thr?Leu?Gly?Thr?Ile?Tyr?Ser?Ser385??????????????390??????????????395?????????????400Ser?Leu?Phe?Pro?Asn?Arg?Ala?Pro?Ala?Gly?Arg?Val?Leu?Leu?Leu?Asn

405??????????????410?????????????415Tyr?Ile?Gly?Gly?Ser?Thr?Asn?Thr?Gly?Ile?Val?Ser?Lys?Thr?Glu?Ser

420?????????????425??????????????430Asp?Leu?Val?Gly?Ala?Val?Asp?Arg?Asp?Leu?Arg?Lys?Met?Leu?Ile?Asn

435??????????????440??????????????445Pro?Arg?Ala?Ala?Asp?Pro?Leu?Ala?Leu?Gly?Val?Arg?Val?Trp?Pro?Gln???450??????????????455??????????????460Ala?Ile?Pro?Gln?Phe?Leu?Ile?Gly?His?Leu?Asp?Arg?Leu?Ala?Ala?Ala465??????????????470?????????????475??????????????480Lys?Ser?Ala?Leu?Gly?Gln?Gly?Gly?Tyr?Asp?Gly?Leu?Phe?Leu?Gly?Gly

485??????????????490?????????????495Asn?Tyr?Val?Ala?Gly?Val?Ala?Leu?Gly?Arg?Cys?Ile?Glu?Gly?Ala?Tyr

500?????????????505??????????????510Glu?Ser?Ala?Ser?Gln?Val?Ser?Asp?Phe?Leu?Thr?Lys?Tyr?Ala?Tyr?Lys

515 520 525＜210〉11＜211〉1847＜212〉DNA＜213〉soybean＜220〉＜221〉CDS＜222〉(55) .. (1683)＜223〉soybean protox-1＜400〉11ctttagcaca gtgttgaaga taacgaacga atagtgccat tactgtaacc aacc atg 57

Met

1gtt?tcc?gtc?ttc?aac?gag?atc?cta?ttc?ccg?ccg?aac?caa?acc?ctt?ctt??105Val?Ser?Val?Phe?Asn?Glu?Ile?Leu?Phe?Pro?Pro?Asn?Gln?Thr?Leu?Leu

5???????????????10??????????????15cgc?ccc?tcc?ctc?cat?tcc?cca?acc?tct?ttc?ttc?acc?tct?ccc?act?cga??153Arg?Pro?Ser?Leu?His?Ser?Pro?Thr?Ser?Phe?Phe?Thr?Ser?Pro?Thr?Arg

20???????????????25??????????????30aaa?ttc?cct?cgc?tct?cgc?cct?aac?cct?att?cta?cgc?tgc?tcc?att?gcg??201Lys?Phe?Pro?Arg?Ser?Arg?Pro?Asn?Pro?Ile?Leu?Arg?Cys?Ser?Ile?Ala

35??????????????40???????????????45gag?gaa?tcc?acc?gcg?tct?ccg?ccc?aaa?acc?aga?gac?tcc?gcc?ccc?gtg??249Glu?Glu?Ser?Thr?Ala?Ser?Pro?Pro?Lys?Thr?Arg?Asp?Ser?Ala?Pro?Val?50??????????????55???????????????60??????????????65gac?tgc?gtc?gtc?gtc?ggc?gga?ggc?gtc?agc?ggc?ctc?tgc?atc?gcc?cag??297Asp?Cys?Val?Val?Val?Gly?Gly?Gly?Val?Ser?Gly?Leu?Cys?Ile?Ala?Gln

70??????????????75???????????????80gcc?ctc?gcc?acc?aaa?cac?gcc?aat?gcc?aac?gtc?gtc?gtc?acg?gag?gcc??345Ala?Leu?Ala?Thr?Lys?His?Ala?Asn?Ala?Asn?Val?Val?Val?Thr?Glu?Ala

85??????????????90??????????????95cga?gac?cgc?gtc?ggc?ggc?aac?atc?acc?acg?atg?gag?agg?gac?gga?tac??393Arg?Asp?Arg?Val?Gly?Gly?Asn?Ile?Thr?Thr?Met?Glu?Arg?Asp?Gly?Tyr

100??????????????105?????????????110ctc?tgg?gaa?gaa?ggc?ccc?aac?agc?ttc?cag?cct?tct?gat?cca?atg?ctc??441Leu?Trp?Glu?Glu?Gly?Pro?Asn?Ser?Phe?Gln?Pro?Ser?Asp?Pro?Met?Leu???115??????????????120??????????????125acc?atg?gtg?gtg?gac?agt?ggt?tta?aag?gat?gag?ctt?gtt?ttg?ggg?gat??489Thr?Met?Val?Val?Asp?Ser?Gly?Leu?Lys?Asp?Glu?Leu?Val?Leu?Gly?Asp130??????????????135??????????????140?????????????145cct?gat?gca?cct?cgg?ttt?gtg?ttg?tgg?aac?agg?aag?ttg?agg?ccg?gtg??537Pro?Asp?Ala?Pro?Arg?Phe?Val?Leu?Trp?Asn?Arg?Lys?Leu?Arg?Pro?Val

150??????????????155?????????????160ccc?ggg?aag?ctg?act?gat?ttg?cct?ttc?ttt?gac?ttg?atg?agc?att?ggt??585Pro?Gly?Lys?Leu?Thr?Asp?Leu?Pro?Phe?Phe?Asp?Leu?Met?Ser?Ile?Gly

165??????????????170?????????????175ggc?aaa?atc?agg?gct?ggc?ttt?ggt?gcg?ctt?gga?att?cgg?cct?cct?cct??633Gly?Lys?Ile?Arg?Ala?Gly?Phe?Gly?Ala?Leu?Gly?Ile?Arg?Pro?Pro?Pro

180??????????????185??????????????190cca?ggt?cat?gag?gaa?tcg?gtt?gaa?gag?ttt?gtt?cgt?cgg?aac?ctt?ggt??681Pro?Gly?His?Glu?Glu?Ser?Val?Glu?Glu?Phe?Val?Arg?Arg?Asn?Leu?Gly???195??????????????200??????????????205gat?gag?gtt?ttt?gaa?cgg?ttg?ata?gag?cct?ttt?tgt?tca?ggg?gtc?tat??729Asp?Glu?Val?Phe?Glu?Ara?Leu?Ile?Glu?Pro?Phe?Cys?Ser?Gly?Val?Tyr210??????????????215?????????????220??????????????225gca?ggc?gat?cct?tca?aaa?tta?agt?atg?aaa?gca?gca?ttc?ggg?aaa?gtt??777Ala?Gly?Asp?Pro?Ser?Lys?Leu?Ser?Met?Lys?Ala?Ala?Phe?Gly?Lys?Val

230??????????????235?????????????240tgg?aag?ctg?gaa?aaa?aat?ggt?ggt?agc?att?att?ggt?gga?act?ttc?aaa??825Trp?Lys?Leu?Glu?Lys?Asn?Gly?Gly?Ser?Ile?Ile?Gly?Gly?Thr?Phe?Lys

245?????????????250??????????????255gca?ata?caa?gag?aga?aat?gga?gct?tca?aaa?cca?cct?cga?gat?ccg?cgt??873Ala?Ile?Gln?Glu?Arg?Asn?Gly?Ala?Ser?Lys?Pro?Pro?Arg?Asp?Pro?Arg

260??????????????265??????????????270ctg?cca?aaa?cca?aaa?ggt?cag?act?gtt?gga?tct?ttc?cgg?aag?gga?ctt??921Leu?Pro?Lys?Pro?Lys?Gly?Gln?Thr?Val?Gly?Ser?Phe?Arg?Lys?Gly?Leu???275??????????????280??????????????285acc?atg?ttg?cct?gat?gca?att?tct?gcc?aga?cta?ggc?aac?aaa?gta?aag??969Thr?Met?Leu?Pro?Asp?Ala?Ile?Ser?Ala?Arg?Leu?Gly?Asn?Lys?Val?Lys290??????????????295?????????????300??????????????305tta?tct?tgg?aag?ctt?tca?agt?att?agt?aaa?ctg?gat?agt?gga?gag?tac??1017Leu?Ser?Trp?Lys?Leu?Ser?Ser?Ile?Ser?Lys?Leu?Asp?Ser?Gly?Glu?Tyr

310??????????????315?????????????320agt?ttg?aca?tat?gaa?aca?cca?gaa?gga?gtg?gtt?tct?ttg?cag?tgc?aaa??1065Ser?Leu?Thr?Tyr?Glu?Thr?Pro?Glu?Gly?Val?Val?Ser?Leu?Gln?Cys?Lys

325?????????????330??????????????335act?gtt?gtc?ctg?acc?att?cct?tcc?tat?gtt?gct?agt?aca?ttg?ctg?cgt??1113Thr?Val?Val?Leu?Thr?Ile?Pro?Ser?Tyr?Val?Ala?Ser?Thr?Leu?Leu?Arg

340??????????????345??????????????350cct?ctg?tct?gct?gct?gct?gca?gat?gca?ctt?tca?aag?ttt?tat?tac?cct??1161Pro?Leu?Ser?Ala?Ala?Ala?Ala?Asp?Ala?Leu?Ser?Lys?Phe?Tyr?Tyr?Pro???355??????????????360??????????????365cca?gtt?gct?gca?gtt?tcc?ata?tcc?tat?cca?aaa?gaa?gct?att?aga?tca??1209Pro?Val?Ala?Ala?Val?Ser?Ile?Ser?Tyr?Pro?Lys?Glu?Ala?Ile?Arg?Ser370??????????????375??????????????380?????????????385gaa?tgc?ttg?ata?gat?ggt?gag?ttg?aag?ggg?ttt?ggt?caa?ttg?cat?cca??1257Glu?Cys?Leu?Ile?Asp?Gly?Glu?Leu?Lys?Gly?Phe?Gly?Gln?Leu?His?Pro

390??????????????395??????????????400cgt?agc?caa?gga?gtg?gaa?aca?tta?gga?act?ata?tac?agc?tca?tca?cta??1305Arg?Ser?Gln?Gly?Val?Glu?Thr?Leu?Gly?Thr?Ile?Tyr?Ser?Ser?Ser?Leu

405?????????????410??????????????415ttc?ccc?aac?cga?gca?cca?cct?gga?agg?gtt?cta?ctc?ttg?aat?tac?att??1353Phe?Pro?Asn?Arg?Ala?Pro?Pro?Gly?Arg?Val?Leu?Leu?Leu?Asn?Tyr?Ile

420?????????????425??????????????430gga?gga?gca?act?aat?act?gga?att?tta?tcg?aag?acg?gac?agt?gaa?ctt??1401Gly?Gly?Ala?Thr?Asn?Thr?Gly?Ile?Leu?Ser?Lys?Thr?Asp?Ser?Glu?Leu???435??????????????440??????????????445gtg?gaa?aca?gtt?gat?cga?gat?ttg?agg?aaa?atc?ctt?ata?aac?cca?aat??1449Val?Glu?Thr?Val?Asp?Arg?Asp?Leu?Arg?Lys?Ile?Leu?Ile?Asn?Pro?Asn450??????????????455??????????????460?????????????465gcc?cag?gat?cca?ttt?gta?gtg?ggg?gtg?aga?ctg?tgg?cct?caa?gct?att??1497Ala?Gln?Asp?Pro?Phe?Val?Val?Gly?Val?Arg?Leu?Trp?Pro?Gln?Ala?Ile

470??????????????475?????????????480cca?cag?ttc?tta?gtt?ggc?cat?ctt?gat?ctt?cta?gat?gtt?gct?aaa?gct??1545Pro?Gln?Phe?Leu?Val?Gly?His?Leu?Asp?Leu?Leu?Asp?Val?Ala?Lys?Ala

485?????????????490??????????????495tct?atc?aga?aat?act?ggg?ttt?gaa?ggg?ctc?ttc?ctt?ggg?ggt?aat?tat???1593Ser?Ile?Arg?Asn?Thr?Gly?Phe?Glu?Gly?Leu?Phe?Leu?Gly?Gly?Asn?Tyr

500 505 510gtg tct ggt gtt gcc ttg gga cga tgc gtt gag gga gcc tat gag gta 1641Val Ser Gly Val Ala Leu Gly Arg Cys Val Glu Gly Ala Tyr Glu Val 515 520 525gca gct gaa gta aac gat ttt ctc aca aat aga gtg tac aaa 1683Ala Ala Glu Val Asn Asp Phe Leu Thr Asn Arg Val Tyr Lys530 535 540tagtagcagt ttttgttttt gtggtggaat gggtgatggg actctcgtgt tccattgaat 1743tataataatg tgaaagtttc tcaaattcgt tcgataggtt tttggcggct tctattgctg 1803ataatgtaaa atcctcttta agtttgaaaa aaaaaaaaaa aaaa 1847＜210〉12＜211〉543＜212〉PRT＜213〉＜400〉12Met Val Ser Val Phe Asn Glu Ile Leu Phe Pro Pro Asn Gln Thr Leu 1 5 10 15Leu Arg Pro Ser Leu His Ser Pro Thr Ser Phe Phe Thr Ser Pro Thr

20???????????????25???????????????30Arg?Lys?Phe?Pro?Arg?Ser?Arg?Pro?Asn?Pro?Ile?Leu?Arg?Cys?Ser?Ile

35???????????????40??????????????45Ala?Glu?Glu?Ser?Thr?Ala?Ser?Pro?Pro?Lys?Thr?Arg?Asp?Ser?Ala?Pro

50??????????????55???????????????60Val?Asp?Cys?Val?Val?Val?Gly?Gly?Gly?Val?Ser?Gly?Leu?Cys?Ile?Ala?65??????????????70???????????????75???????????????80Gln?Ala?Leu?Ala?Thr?Lys?His?Ala?Asn?Ala?Asn?Val?Val?Val?Thr?Glu

85??????????????90???????????????95Ala?Arg?Asp?Arg?Val?Gly?Gly?Asn?Ile?Thr?Thr?Met?Glu?Arg?Asp?Gly

100?????????????105??????????????110Tyr?Leu?Trp?Glu?Glu?Gly?Pro?Asn?Ser?Phe?Gln?Pro?Ser?Asp?Pro?Met

115??????????????120??????????????125Leu?Thr?Met?Val?Val?Asp?Ser?Gly?Leu?Lys?Asp?Glu?Leu?Val?Leu?Gly???130??????????????135??????????????140Asp?Pro?Asp?Ala?Pro?Arg?Phe?Val?Leu?Trp?Asn?Arg?Lys?Leu?Arg?Pro145??????????????150?????????????155??????????????160Val?Pro?Gly?Lys?Leu?Thr?Asp?Leu?Pro?Phe?Phe?Asp?Leu?Met?Ser?Ile

165??????????????170?????????????175Gly?Gly?Lys?Ile?Arg?Ala?Gly?Phe?Gly?Ala?Leu?Gly?Ile?Arg?Pro?Pro

180?????????????185??????????????190Pro?Pro?Gly?His?Glu?Glu?Ser?Val?Glu?Glu?Phe?Val?Arg?Arg?Asn?Leu

195??????????????200??????????????205Gly?Asp?Glu?Val?Phe?Glu?Arg?Leu?Ile?Glu?Pro?Phe?Cys?Ser?Gly?Val???210??????????????215??????????????220Tyr?Ala?Gly?Asp?Pro?Ser?Lys?Leu?Ser?Met?Lys?Ala?Ala?Phe?Gly?Lys225??????????????230??????????????235?????????????240Val?Trp?Lys?Leu?Glu?Lys?Asn?Gly?Gly?Ser?Ile?Ile?Gly?Gly?Thr?Phe

245??????????????250?????????????255Lys?Ala?Ile?Gln?Glu?Arg?Asn?Gly?Ala?Ser?Lys?Pro?Pro?Arg?Asp?Pro

260?????????????265??????????????270Arg?Leu?Pro?Lys?Pro?Lys?Gly?Gln?Thr?Val?Gly?Ser?Phe?Arg?Lys?Gly

275??????????????280??????????????285Leu?Thr?Met?Leu?Pro?Asp?Ala?Ile?Ser?Ala?Arg?Leu?Gly?Asn?Lys?Val???290??????????????295??????????????300Lys?Leu?Ser?Trp?Lys?Leu?Ser?Ser?Ile?Ser?Lys?Leu?Asp?Ser?Gly?Glu305??????????????310??????????????315?????????????320Tyr?Ser?Leu?Thr?Tyr?Glu?Thr?Pro?Glu?Gly?Val?Val?Ser?Leu?Gln?Cys

325?????????????330?????????????335Lys?Thr?Val?Val?Leu?Thr?Ile?Pro?Ser?Tyr?Val?Ala?Ser?Thr?Leu?Leu

340?????????????345??????????????350Ara?Pro?Leu?Ser?Ala?Ala?Ala?Ala?Asp?Ala?Leu?Ser?Lys?Phe?Tyr?Tyr

355??????????????360??????????????365Pro?Pro?Val?Ala?Ala?Val?Ser?Ile?Ser?Tyr?Pro?Lys?Glu?Ala?Ile?Arg???370??????????????375??????????????380Ser?Glu?Cys?Leu?Ile?Asp?Gly?Glu?Leu?Lys?Gly?Phe?Gly?Gln?Leu?His385??????????????390??????????????395?????????????400Pro?Arg?Ser?Gln?Gly?Val?Glu?Thr?Leu?Gly?Thr?Ile?Tyr?Ser?Ser?Ser

405??????????????410?????????????415Leu?Phe?Pro?Asn?Arg?Ala?Pro?Pro?Gly?Arg?Val?Leu?Leu?Leu?Asn?Tyr

420?????????????425??????????????430Ile?Gly?Gly?Ala?Thr?Asn?Thr?Gly?Ile?Leu?Ser?Lys?Thr?Asp?Ser?Glu

435??????????????440??????????????445Leu?Val?Glu?Thr?Val?Asp?Arg?Asp?Leu?Arg?Lys?Ile?Leu?Ile?Asn?Pro???450??????????????455??????????????460Asn?Ala?Gln?Asp?Pro?Phe?Val?Val?Gly?Val?Arg?Leu?Trp?Pro?Gln?Ala465??????????????470??????????????475?????????????480Ile?Pro?Gln?Phe?Leu?Val?Gly?His?Leu?Asp?Leu?Leu?Asp?Val?Ala?Lys

485??????????????490?????????????495Ala?Ser?Ile?Arg?Asn?Thr?Gly?Phe?Glu?Gly?Leu?Phe?Leu?Gly?Gly?Asn

500?????????????505??????????????510Tyr?Val?Ser?Gly?Val?Ala?Leu?Gly?Arg?Cys?Val?Glu?Gly?Ala?Tyr?Glu

515 520 525Val Ala Ala Glu Val Asn Asp Phe Leu Thr Asn Ara Val Tyr Lys 530 535 540＜210〉13＜211〉583＜212〉DNA＜213〉＜220〉＜221〉misc_＜222〉 (1).. (583)＜223〉protox-1＜400〉13gaattccgat cgaattatat aattatcata aatttgaata agcatgttgc cttttattaa 60agaggtttaa taaagtttgg taataatgga ctttgacttc aaactcgatt ctcatgtaat 120taattaatat ttacatcaaa atttggtcac taatattacc aaattaatat actaaaatgt 180taattcgcaa ataaaacact aattccaaat aaagggtcat tatgataaac acgtattgaa 240cttgataaag caaagcaaaa ataatgggtt tcaaggtttg ggttatatat gacaaaaaaa 300aaaaaaggtt tggttatata tctattgggc ctataaccat gttatacaaa tttgggccta 360actaaaataa taaaataaac gtaatggtcc tttttatatt tgggtcaaac ccaactctaa 420acccaaacca aagaaaaagt atacggtacg gtacacagac ttatggtgtg tgtgattgca 480ggtgaatatt tctcgtcgtc ttctcctttc ttctgaagaa gattacccaa tctgaaaaaa 540accaagaagc tgacaaaatt ccgaattctc tgcgatttcc atg 583＜210〉14＜211〉3848＜212〉DNA＜213〉＜220〉＜221〉misc_＜222〉 (1).. (3848)＜223〉protox-1＜400〉14tcgatctttc taggctgatc cccaaatctt cctccgaagc ccctggcgcc tctgcccctt 60ggagctggtg gcctgaaaga gctttgctgt tgccccgaag attgtgaggt atattgtgac 120ctctgagact gacttccttt gtcgtcactt tgagtggagt tatggattga cctgacgtgc 180ctcagatgga ttcttcctcc gaagcccctg gtcatttcgg agaatctgta atcttattcc 240cttctttggc gaaaatctgt cagcttggat gtactcatcc atcttctgaa gcagcttctc 300cagagtttgt ggaggcttcc tggcgaaata ttgggctgta ggtcctggac gaagaccctt 360gatcatggcc tcaatgacaa tctcattggg caccgtaggc gcttgtgccc tcaatcgcaa 420gaaccttcgt acatatgcct gaaggtattc ttcgtgatct tgtgtgcatt ggaacagagc 480ctgagctgtg accgacttcg tttgaaagcc ttggaagcta gtaaccaaca tgtgcttaag 540cttctgccac gacgtgatag tccctggccg aagagaagaa taccatgttt gggctacatt 600ccggactgcc atgacgaagg acttcgccat gactacagtg ttgaccccat acgaagatat 660agttgcttcg tagctcatca gaaactgctt tggatctgag tgcccatcat acatggggag 720ctgaggtggc ttgtatgatg ggggccatgg ggtagcctgc agttctgctg ccaagggaga 780agcatcatca aaagtaaagg catcatgatt aaaatcatca taccatccat cctcgttgaa 840taagccttct tgacgaagct ccctgtgttg gggccttcga tcttgttcat cttgaacaag 900atgacgcact tcttcagtgg cttcgtcgat ctttctttgg agatcagcca gtcgcaccat 960cttctccttc tttctttgta cttgttgatg gatgatctcc atgtccctga tctcttggtc 1020caactcctcc tcttggagtg tcagactggt ggctttcctc ttctggcttc gagcctctcg 1080aagagaaaga gtttcttgat ttgggtccag cggctgcagt gcagtggtcc ctggtgctga 1140agctttcttc ggtggcatga caaaggtcag tgcttgccga aggtggtcga aaagggttca 1200ctagaggtgg gagccaatgt tggggacttc tcaagtgcta tgagttaaga acaaggcaac 1260acaaaatgtt aaatattaat agctttcatc tttcgaagca ttatttccct ttgggtataa 1320tgatcttcag acgaaagagt ccttcatcat tgcgatatat gttaatagaa ggaggagcat 1380atgaaatgta agagacaaca tgaacaatcg tgtagcattg ttaattcatc atcattttat 1440tattatggaa aaatagaaac aatattgaat tacaaatgta cctttggctt gacagaagat 1500aaaagtacaa gcttgacgca cgagcaagta caagtcagtg tgaacagtac gggggtactg 1560ttcatctatt tataggcaca ggacacagcc tgtgagaaat tacagtcatg ccctttacat 1620ttactattga cttatagaaa aatctatgag gactggatag ccttttcccc tttaagtcgg 1680tgcctttttc cgcgattaag ccgaatctcc cttgcgcata gcttcggagc atcggcaacc 1740ttcgtcacga tcatgccctt ctcattgtgt atgcttttaa tcctgaattc gaaggtacct 1800gtccataaac catacttgga agacattgtt aaattatgtt tttgaggacc ttcggaggac 1860gaaggccccc aacagtcgtg tttttgagga ccttcggaag atgaaggccc ccaacaagac 1920ctatccataa aaccaaccta tccacaaaac cgaccccatt cacccttcat ttgcctcacc 1980aacaacccga attaggttgt tggtttaaat tttttagggt caatttggtc atcaccatcc 2040actgtcactc cacaaactca atatcaataa acagactcaa tcacccaaac tgaccatacc 2100cataaaaccg ccccaccctt ctagcgcctc gccagaaacc agaaaccctg attcagagtt 2160caaacttaaa acgaccataa ctttcacctt ggaactcgaa tcaggtccat ttttttccaa 2220atcacacaaa attaaatttc gcatccgata atcaagccat ctcttcacta tggttttaag 2280tgttgctcac actagtgtat ttatggacta atcacctgtg tatctcatac aataacatat 2340cagtacatct aagttgttac tcaattacca aaaccgaatt atagccttcg aaaaaggtta 2400tcgactagtc actcaattac caaaactaaa ctttagactt tcatgtatga catccaacat 2460gacactgtac tggactaaac cacctttcaa gctacacaag gagcaaaaat aactaatttt 2520cgtagttgta ggagctaaag tatatgtcca caacaatagt taagggaagc ccccaaggac 2580ttaaaagtcc ttttacctct tgaaactttt gtcgtggtct actttttcac tttaaacttc 2640aaaatttgac attttatcac cccttaactc ttaaaaccat ttaaattaca ttcttactag 2700attatagatg attttgttgt gaaaagtttt taagacatgt ttacacattg attaaaatca 2760tttgttcaat ttcctagagt taaatctaat cttattaaaa ctattagaga tactttcacg 2820agctctaaat atttttattt tttcattatg gaattttgtt agaattctta tagacctttt 2880tttgtggttt aaaagccttg ccatgttttt aacaagtttt ttttctattt tttgaaattt 2940tcttggaaac cacttctaac ccggtagaag atttattttg ctacacttat atctacaaca 3000aaatcaactt atgaaattgt cttggaaact acctctaacc cggtagaatg aatttgaatg 3060aaaattaaac caacttacgg aatcgcccaa catatgtcga ttaaagtgga tatggataca 3120tatgaagaag ccctagagat aatctaaatg gtttcagaat tgagggttat tttttgaagt 3180ttgatgggaa gataagacca taacggtagt tcacagagat aaaagggtta tttttttcag 3240aaatatttgt gctgcaattg atcctgtgcc tcaaattcag cctgcaacca aggccaggtt 3300ctagagcgaa caaggcccac gtcacccgtg gcccgtcagg cgaagcaggt cttgtgcaga 3360ctttgagagg gattggatat caacggaacc aatcacgcac ggcaatgcga ttcccagccc 3420acctgtaacg ttccagtggg ccatccttaa ctccaagccc aacggcccta ccccatctcg 3480tcgtgtcatc cactccgccg cacaggcgct cagctccgca acgccgccgg aaatggtcgc 3540cgccacagcc accgccatgg ccaccgctgc atcgccgcta ctcaacggga cccgaatacc 3600tgcgcggctc cgccatcgag gactcagcgt gcgctgcgct gctgtggcgg gcggcgcggc 3660cgaggcaccg gcatccaccg gcgcgcggct gtccgcggac tgcgttgtgg tgggcggagg 3720catcagtggc ctctgcaccg cgcaggcgct ggccacgcgg cacggcgtcg gggacgtgct 3780tgtcacggag gcccgcgccc gccccggcgg caacattacc accgtcgagc gccccgagga 3840agggtacc 3848＜210〉15＜211〉1826＜212〉DNA＜213〉＜220〉＜221〉CDS＜222〉 (31).. (1647)＜223〉protox-1＜400〉15cctctcgctc gcctggcccc accaccaatc atg acg gct cta atc gac ctt tct 54

Met?Thr?Ala?Leu?Ile?Asp?Leu?Ser

1????????????5ctt?ctc?cgt?tcc?tcg?ccc?tcc?gtt?tcc?cct?ttc?tcc?ata?ccc?cac?cac??102Leu?Leu?Arg?Ser?Ser?Pro?Ser?Val?Ser?Pro?Phe?Ser?Ile?Pro?His?His

10???????????????15??????????????20cag?cat?ccg?ccc?cgc?ttt?cgt?aaa?cct?ttc?aag?ctc?cga?tgc?tcc?ctc??150Gln?His?Pro?Pro?Arg?Phe?Arg?Lys?Pro?Phe?Lys?Leu?Arg?Cys?Ser?Leu?25??????????????30???????????????35??????????????40gcc?gag?ggt?ccc?acg?att?tcc?tca?tct?aaa?atc?gac?ggg?gga?gaa?tca??198Ala?Glu?Gly?Pro?Thr?Ile?Ser?Ser?Ser?Lys?Ile?Asp?Gly?Gly?Glu?Ser

45??????????????50???????????????55tcc?atc?gcg?gat?tgc?gtc?atc?gtt?gga?ggt?ggt?atc?agt?gga?ctt?tgc??246Ser?Ile?Ala?Asp?Cys?Val?Ile?Val?Gly?Gly?Gly?Ile?Ser?Gly?Leu?Cys

60??????????????65???????????????70att?gct?caa?gct?ctc?gcc?acc?aag?cac?cgt?gac?gtc?gct?tcc?aat?gtg??294Ile?Ala?Gln?Ala?Leu?Ala?Thr?Lys?His?Arg?Asp?Val?Ala?Ser?Asn?Val

75???????????????80??????????????85att?gtg?acg?gag?gcc?aga?gac?cgt?gtt?ggt?ggc?aac?atc?act?acc?gtt??342Ile?Val?Thr?Glu?Ala?Arg?Asp?Arg?Val?Gly?Gly?Asn?Ile?Thr?Thr?Val

90??????????????95??????????????100gag?aga?gat?gga?tat?ctg?tgg?gaa?gaa?ggc?ccc?aac?agt?ttt?cag?ccc??390Glu?Arg?Asp?Gly?Tyr?Leu?Trp?Glu?Glu?Gly?Pro?Asn?Ser?Phe?Gln?Pro105??????????????110??????????????115?????????????120tcc?gat?cct?att?cta?acc?atg?gcc?gtg?gat?agt?gga?ttg?aag?gac?gat??438Ser?Asp?Pro?Ile?Leu?Thr?Met?Ala?Val?Asp?Ser?Gly?Leu?Lys?Asp?Asp

125?????????????130??????????????135ttg?gtt?tta?ggt?gac?cct?aat?gca?ccg?cga?ttt?gta?cta?tgg?gag?gga??486Leu?Val?Leu?Gly?Asp?Pro?Asn?Ala?Pro?Arg?Phe?Val?Leu?Trp?Glu?Gly

140?????????????145??????????????150aaa?cta?agg?cct?gtg?ccc?tcc?aag?cca?acc?gac?ttg?ccg?ttt?ttt?gat??534Lys?Leu?Arg?Pro?Val?Pro?Ser?Lys?Pro?Thr?Asp?Leu?Pro?Phe?Phe?Asp

155??????????????160??????????????165ttg?atg?agc?att?gct?gga?aaa?ctt?agg?gct?ggg?ttc?ggg?gct?att?ggc??582Leu?Met?Ser?Ile?Ala?Gly?Lys?Leu?Arg?Ala?Gly?Phe?Gly?Ala?Ile?Gly???170??????????????175?????????????180att?cgg?cct?ccc?cct?ccg?ggt?tat?gaa?gaa?tcg?gtg?gag?gag?ttt?gtg??630Ile?Arg?Pro?Pro?Pro?Pro?Gly?Tyr?Glu?Glu?Ser?Val?Glu?Glu?Phe?Val185??????????????190??????????????195?????????????200cgc?cgt?aat?ctt?ggt?gct?gag?gtt?ttt?gaa?cgc?ttt?att?gaa?cca?ttt??678Arg?Arg?Asn?Leu?Gly?Ala?Glu?Val?Phe?Glu?Arg?Phe?Ile?Glu?Pro?Phe

205??????????????210?????????????215tgt?tca?ggt?gtt?tat?gca?ggg?gat?cct?tca?aaa?tta?agc?atg?aaa?gca??726Cys?Ser?Gly?Val?Tyr?Ala?Gly?Asp?Pro?Ser?Lys?Leu?Ser?Met?Lys?Ala

220?????????????225??????????????230gca?ttt?gga?aga?gta?tgg?aag?cta?gaa?gag?att?ggt?ggc?agc?atc?att??774Ala?Phe?Gly?Arg?Val?Trp?Lys?Leu?Glu?Glu?Ile?Gly?Gly?Ser?Ile?Ile

235?????????????240??????????????245ggt?ggc?act?ttc?aag?aca?atc?cag?gag?aga?aat?aag?aca?cct?aag?cca??822Gly?Gly?Thr?Phe?Lys?Thr?Ile?Gln?Glu?Arg?Asn?Lys?Thr?Pro?Lys?Pro???250??????????????255??????????????260ccc?aga?gac?ccg?cgt?ctg?cca?aaa?ccg?aag?ggc?caa?aca?gtt?gga?tct??870Pro?Arg?Asp?Pro?Arg?Leu?Pro?Lys?Pro?Lys?Gly?Gln?Thr?Val?Gly?Ser265??????????????270??????????????275?????????????280ttt?agg?aag?gga?ctt?acc?atg?ctg?cct?gag?gca?att?gct?aac?agt?ttg??918Phe?Arg?Lys?Gly?Leu?Thr?Met?Leu?Pro?Glu?Ala?Ile?Ala?Asn?Ser?Leu

285??????????????290?????????????295ggt?agc?aat?gta?aaa?tta?tct?tgg?aag?ctt?tcc?agt?att?acc?aaa?ttg??966Gly?Ser?Asn?Val?Lys?Leu?Ser?Trp?Lys?Leu?Ser?Ser?Ile?Thr?Lys?Leu

300?????????????305??????????????310ggc?aat?gga?ggg?tat?aac?ttg?aca?ttt?gaa?aca?cct?gaa?gga?atg?gta??1014Gly?Asn?Gly?Gly?Tyr?Asn?Leu?Thr?Phe?Glu?Thr?Pro?Glu?Gly?Met?Val

315??????????????320??????????????325tct?ctt?cag?agt?aga?agt?gtt?gta?atg?acc?att?cca?tcc?cat?gtt?gcc??1062Ser?Leu?Gln?Ser?Arg?Ser?Val?Val?Met?Thr?Ile?Pro?Ser?His?Val?Ala???330??????????????335??????????????340agt?aac?ttg?ttg?cat?cct?ctc?tcg?gct?gct?gct?gca?gat?gca?tta?tcc??1110Ser?Asn?Leu?Leu?His?Pro?Leu?Ser?Ala?Ala?Ala?Ala?Asp?Ala?Leu?Ser345??????????????350??????????????355?????????????360caa?ttt?tat?tat?cct?cca?gtt?gca?tca?gtc?aca?gtc?tcc?tat?cca?aaa??1158Gln?Phe?Tyr?Tyr?Pro?Pro?Val?Ala?Ser?Val?Thr?Val?Ser?Tyr?Pro?Lys

365??????????????370?????????????375gaa?gcc?att?cga?aaa?gaa?tgt?ttg?att?gat?ggt?gaa?ctt?aag?ggg?ttt??1206Glu?Ala?Ile?Arg?Lys?Glu?Cys?Leu?Ile?Asp?Gly?Glu?Leu?Lys?Gly?Phe

380?????????????385??????????????390ggc?cag?ttg?cac?cca?cgc?agc?caa?gga?att?gaa?act?tta?ggg?acg?ata??1254Gly?Gln?Leu?His?Pro?Arg?Ser?Gln?Gly?Ile?Glu?Thr?Leu?Gly?Thr?Ile

395??????????????400??????????????405tac?agt?tca?tca?ctt?ttc?ccc?aat?cga?gct?cca?tct?ggc?agg?gtg?ttg??1302Tyr?Ser?Ser?Ser?Leu?Phe?Pro?Asn?Arg?Ala?Pro?Ser?Gly?Arg?Val?Leu???410??????????????415??????????????420ctc?ttg?aac?tac?ata?gga?gga?gct?acc?aac?act?gga?att?ttg?tcc?aag??1350Leu?Leu?Asn?Tyr?Ile?Gly?Gly?Ala?Thr?Asn?Thr?Gly?Ile?Leu?Ser?Lys425??????????????430??????????????435?????????????440act?gaa?ggg?gaa?ctt?gta?gaa?gca?gtt?gat?cgt?gat?ttg?aga?aaa?atg??1398Thr?Glu?Gly?Glu?Leu?Val?Glu?Ala?Val?Asp?Arg?Asp?Leu?Arg?Lys?Met

445??????????????450?????????????455ctt?ata?aat?cct?aat?gca?aag?gat?cct?ctt?gtt?ttg?ggt?gta?aga?gta??1446Leu?Ile?Asn?Pro?Asn?Ala?Lys?Asp?Pro?Leu?Val?Leu?Gly?Val?Arg?Val

460?????????????465??????????????470tgg?cca?aaa?gcc?att?cca?cag?ttc?ttg?gtt?ggt?cat?ttg?gat?ctc?ctt???1494Trp?Pro?Lys?Ala?Ile?Pro?Gln?Phe?Leu?Val?Gly?His?Leu?Asp?Leu?Leu

475?????????????480??????????????485gat?agt?gca?aaa?atg?gct?ctc?agg?gat?tct?ggg?ttt?cat?gga?ctg?ttt???1542Asp?Ser?Ala?Lys?Met?Ala?Leu?Arg?Asp?Ser?Gly?Phe?His?Gly?Leu?Phe???490??????????????495??????????????500ctt?ggg?ggc?aac?tat?gta?tct?ggt?gtg?gca?tta?gga?cgg?tgt?gtg?gaa???1590Leu?Gly?Gly?Asn?Tyr?Val?Ser?Gly?Val?Ala?Leu?Gly?Arg?Cys?Val?Glu505??????????????510??????????????515?????????????520ggt?gct?tac?gag?gtt?gca?gct?gaa?gtg?aag?gaa?ttc?ctg?tca?caa?tat???1638Gly?Ala?Tyr?Glu?Val?Ala?Ala?Glu?Val?Lys?Glu?Phe?Leu?Ser?Gln?Tyr

525 530 535gca tac aaa taatattgaa attcttgtca ggctgcaaat gtagaagtca 1687Ala Tyr Lysgttattggat agtatctctt tagctaaaaa attgggtagg gttttttttg ttagttcctt 1747gaccactttt tggggttttc attagaactt catatttgta tatcatgttg caatatcaaa 1807aaaaaaaaaa aaaaaaaaa, 1826＜210〉16＜211〉539＜212〉PRT＜213〉upland cotton＜400〉16Met Thr Ala Leu Ile Asp Leu Ser Leu Leu Arg Ser Ser Pro Ser Val, 15 10 15Ser Pro Phe Ser Ile Pro His His Gln His Pro Pro Arg Phe Arg Lys

20??????????????25???????????????30Pro?Phe?Lys?Leu?Arg?Cys?Ser?Leu?Ala?Glu?Gly?Pro?Thr?Ile?Ser?Ser

35??????????????40??????????????45Ser?Lys?Ile?Asp?Gly?Gly?Glu?Ser?Ser?Ile?Ala?Asp?Cys?Val?Ile?Val

50???????????????55??????????????60Gly?Gly?Gly?Ile?Ser?Gly?Leu?Cys?Ile?Ala?Gln?Ala?Leu?Ala?Thr?Lys?65???????????????70??????????????75??????????????80His?Arg?Asp?Val?Ala?Ser?Asn?Val?Ile?Val?Thr?Glu?Ala?Arg?Asp?Arg

85??????????????90???????????????95Val?Gly?Gly?Asn?Ile?Thr?Thr?Val?Glu?Arg?Asp?Gly?Tyr?Leu?Trp?Glu

100?????????????105??????????????110Glu?Gly?Pro?Asn?Ser?Phe?Gln?Pro?Ser?Asp?Pro?Ile?Leu?Thr?Met?Ala

115?????????????120??????????????125Val?Asp?Ser?Gly?Leu?Lys?Asp?Asp?Leu?Val?Leu?Gly?Asp?Pro?Asn?Ala

130?????????????135??????????????140Pro?Arg?Phe?Val?Leu?Trp?Glu?Gly?Lys?Leu?Arg?Pro?Val?Pro?Ser?Lys145??????????????150??????????????155?????????????160Pro?Thr?Asp?Leu?Pro?Phe?Phe?Asp?Leu?Met?Ser?Ile?Ala?Gly?Lys?Leu

165??????????????170?????????????175Arg?Ala?Gly?Phe?Gly?Ala?Ile?Gly?Ile?Arg?Pro?Pro?Pro?Pro?Gly?Tyr

180?????????????185??????????????190Glu?Glu?Ser?Val?Glu?Glu?Phe?Val?Arg?Arg?Asn?Leu?Gly?Ala?Glu?Val

195??????????????200??????????????205Phe?Glu?Arg?Phe?Ile?Glu?Pro?Phe?Cys?Ser?Gly?Val?Tyr?Ala?Gly?Asp???210??????????????215??????????????220Pro?Ser?Lys?Leu?Ser?Met?Lys?Ala?Ala?Phe?Gly?Arg?Val?Trp?Lys?Leu225??????????????230?????????????235??????????????240Glu?Glu?Ile?Gly?Gly?Ser?Ile?Ile?Gly?Gly?Thr?Phe?Lys?Thr?Ile?Gln

245??????????????250??????????????255Glu?Arg?Asn?Lys?Thr?Pro?Lys?Pro?Pro?Arg?Asp?Pro?Arg?Leu?Pro?Lys

260?????????????265??????????????270Pro?Lys?Gly?Gln?Thr?Val?Gly?Ser?Phe?Arg?Lys?Gly?Leu?Thr?Met?Leu

275??????????????280??????????????285Pro?Glu?Ala?Ile?Ala?Asn?Ser?Leu?Gly?Ser?Asn?Val?Lys?Leu?Ser?Trp

290?????????????295??????????????300Lys?Leu?Ser?Ser?Ile?Thr?Lys?Leu?Gly?Asn?Gly?Gly?Tyr?Asn?Leu?Thr305??????????????310?????????????315??????????????320Phe?Glu?Thr?Pro?Glu?Gly?Met?Val?Ser?Leu?Gln?Ser?Arg?Ser?Val?Val

325??????????????330?????????????335Met?Thr?Ile?Pro?Ser?His?Val?Ala?Ser?Asn?Leu?Leu?His?Pro?Leu?Ser

340?????????????345??????????????350Ala?Ala?Ala?Ala?Asp?Ala?Leu?Ser?Gln?Phe?Tyr?Tyr?Pro?Pro?Val?Ala

355??????????????360??????????????365Ser?Val?Thr?Val?Ser?Tyr?Pro?Lys?Glu?Ala?Ile?Arg?Lys?Glu?Cys?Leu???370??????????????375??????????????380Ile?Asp?Gly?Glu?Leu?Lys?Gly?Phe?Gly?Gln?Leu?His?Pro?Arg?Ser?Gln385?????????????390??????????????395??????????????400Gly?Ile?Glu?Thr?Leu?Gly?Thr?Ile?Tyr?Ser?Ser?Ser?Leu?Phe?Pro?Asn

405??????????????410?????????????415Arg?Ala?Pro?Ser?Gly?Arg?Val?Leu?Leu?Leu?Asn?Tyr?Ile?Gly?Gly?Ala

420?????????????425??????????????430Thr?Asn?Thr?Gly?Ile?Leu?Ser?Lys?Thr?Glu?Gly?Glu?Leu?Val?Glu?Ala

435??????????????440??????????????445Val?Asp?Arg?Asp?Leu?Arg?Lys?Met?Leu?Ile?Asn?Pro?Asn?Ala?Lys?Asp???450??????????????455??????????????460Pro?Leu?Val?Leu?Gly?Val?Arg?Val?Trp?Pro?Lys?Ala?Ile?Pro?Gln?Phe465??????????????470?????????????475??????????????480Leu?Val?Gly?His?Leu?Asp?Leu?Leu?Asp?Ser?Ala?Lys?Met?Ala?Leu?Arg

485??????????????490?????????????495Asp?Ser?Gly?Phe?His?Gly?Leu?Phe?Leu?Gly?Gly?Asn?Tyr?Val?Ser?Gly

500?????????????505??????????????510Val?Ala?Leu?Gly?Arg?Cys?Val?Glu?Gly?Ala?Tyr?Glu?Val?Ala?Ala?Glu

515 520 525Val Lys Glu Phe Leu Ser Gln Tyr Ala Tyr Lys 530 535＜210〉17＜211〉1910＜212〉DNA＜213〉beet＜220〉＜221〉CDS＜222〉(1) .. (1680)＜223〉beet protox-1 coded sequence＜400〉17atg aaa tca atg gcg tta tca aac tgc att cca cag aca cag tgc atg 48Met Lys Ser Met Ala Leu Ser Asn Cys Ile Pro Gln Thr Gln Cys Met, 15 10 15cca ttg cgc agc agc ggg cat tac agg ggt aat tgt atc atg ttg tca 96Pro Leu Arg Ser Ser Gly His Tyr Arg Gly Asn Cys Ile Met Leu Ser

20??????????????25???????????????30att?cca?tgt?agt?tta?att?gga?aga?cga?ggt?tat?tat?tca?cat?aag?aag??144Ile?Pro?Cys?Ser?Leu?Ile?Gly?Arg?Arg?Gly?Tyr?Tyr?Ser?His?Lys?Lys

35???????????????40??????????????45agg?agg?atg?agc?atg?agt?tgc?agc?aca?agc?tca?ggc?tca?aag?tca?gcg??192Arg?Arg?Met?Ser?Met?Ser?Cys?Ser?Thr?Ser?Ser?Gly?Ser?Lys?Ser?Ala

50??????????????55???????????????60gtt?aaa?gaa?gca?gga?tca?gga?tca?ggt?gca?gga?gga?ttg?cta?gac?tgc??240Val?Lys?Glu?Ala?Gly?Ser?Gly?Ser?Gly?Ala?Gly?Gly?Leu?Leu?Asp?Cys?65??????????????70???????????????75??????????????80gta?atc?gtt?gga?ggt?gga?att?agc?ggg?ctt?tgc?atc?gcg?cag?gct?ctt??288Val?Ile?Val?Gly?Gly?Gly?Ile?Ser?Gly?Leu?Cys?Ile?Ala?Gln?Ala?Leu

85???????????????90??????????????95tgt?aca?aaa?cac?tcc?tct?tcc?tct?tta?tcc?cca?aat?ttt?ata?gtt?aca??336Cys?Thr?Lys?His?Ser?Ser?Ser?Ser?Leu?Ser?Pro?Asn?Phe?Ile?Val?Thr

100?????????????105??????????????110gag?gcc?aaa?gac?aga?gtt?ggc?ggc?aac?atc?gtc?act?gtg?gag?gcc?gat??384Glu?Ala?Lys?Asp?Arg?Val?Gly?Gly?Asn?Ile?Val?Thr?Val?Glu?Ala?Asp

115??????????????120??????????????125ggc?tat?atc?tgg?gag?gag?gga?ccc?aat?agc?ttc?cag?cct?tcc?gac?gcg??432Gly?Tyr?Ile?Trp?Glu?Glu?Gly?Pro?Asn?Ser?Phe?Gln?Pro?Ser?Asp?Ala???130??????????????135??????????????140gtg?ctc?acc?atg?gcg?gtc?gac?agt?ggc?ttg?aaa?gat?gag?ttg?gtg?ctc??480Val?Leu?Thr?Met?Ala?Val?Asp?Ser?Gly?Leu?Lys?Asp?Glu?Leu?Val?Leu145??????????????150??????????????155?????????????160gga?gat?ccc?aat?gct?cct?cgc?ttt?gtg?cta?tgg?aat?gac?aaa?tta?agg??528Gly?Asp?Pro?Asn?Ala?Pro?Arg?Phe?Val?Leu?Trp?Asn?Asp?Lys?Leu?Arg

165??????????????170?????????????175ccc?gta?cct?tcc?agt?ctc?acc?gac?ctc?cct?ttc?ttc?gac?ctc?atg?acc??576Pro?Val?Pro?Ser?Ser?Leu?Thr?Asp?Leu?Pro?Phe?Phe?Asp?Leu?Met?Thr

180?????????????185??????????????190att?ccg?ggc?aag?att?agg?gct?gct?ctt?ggt?gct?ctc?gga?ttt?cgc?cct??624Ile?Pro?Gly?Lys?Ile?Arg?Ala?Ala?Leu?Gly?Ala?Leu?Gly?Phe?Arg?Pro

195??????????????200??????????????205tct?cct?cca?cct?cat?gag?gaa?tct?gtt?gaa?cac?ttt?gtg?cgt?cgt?aat??672Ser?Pro?Pro?Pro?His?Glu?Glu?Ser?Val?Glu?His?Phe?Val?Arg?Arg?Asn???210??????????????215??????????????220ctc?gga?gat?gag?gtc?ttt?gaa?cgc?ttg?att?gaa?ccc?ttt?tgt?tca?ggt??720Leu?Gly?Asp?Glu?Val?Phe?Glu?Arg?Leu?Ile?Glu?Pro?Phe?Cys?Ser?Gly225??????????????230?????????????235??????????????240gtg?tat?gcc?ggt?gat?cct?gcc?aag?ctg?agt?atg?aaa?gct?gct?ttt?ggg??768Val?Tyr?Ala?Gly?Asp?Pro?Ala?Lys?Leu?Ser?Met?Lys?Ala?Ala?Phe?Gly

245??????????????250?????????????255aag?gtc?tgg?aag?ttg?gag?caa?aag?ggt?ggc?agc?ata?att?ggt?ggc?act??816Lys?Val?Trp?Lys?Leu?Glu?Gln?Lys?Gly?Gly?Ser?Ile?Ile?Gly?Gly?Thr

260?????????????265??????????????270ctc?aaa?gct?ata?cag?gaa?aga?ggg?agt?aat?cct?aag?ccg?ccc?cgt?gac??864Leu?Lys?Ala?Ile?Gln?Glu?Arg?Gly?Ser?Asn?Pro?Lys?Pro?Pro?Arg?Asp

275??????????????280??????????????285cag?cgc?ctc?cct?aaa?cca?aag?ggt?cag?act?gtt?gga?tcc?ttt?aga?aag??912Gln?Arg?Leu?Pro?Lys?Pro?Lys?Gly?Gln?Thr?Val?Gly?Ser?Phe?Arg?Lys

290?????????????295??????????????300gga?ctc?gtt?atg?ttg?cct?acc?gcc?att?tct?gct?cga?ctt?ggc?agt?aga??960Gly?Leu?Val?Met?Leu?Pro?Thr?Ala?Ile?Ser?Ala?Arg?Leu?Gly?Ser?Arg305??????????????310?????????????315??????????????320gtg?aaa?cta?tct?tgg?acc?ctt?tct?agt?atc?gta?aag?tca?ctc?aat?gga??1008Val?Lys?Leu?Ser?Trp?Thr?Leu?Ser?Ser?Ile?Val?Lys?Ser?Leu?Asn?Gly

325??????????????330?????????????335gaa?tat?agt?ctg?act?tat?gat?acc?cca?gat?ggc?ttg?gtt?tct?gta?aga??1056Glu?Tyr?Ser?Leu?Thr?Tyr?Asp?Thr?Pro?Asp?Gly?Leu?Val?Ser?Val?Arg

340?????????????345??????????????350acc?aaa?agt?gtt?gtg?atg?act?gtt?cca?tca?tat?gtt?gca?agt?agg?ctt??1104Thr?Lys?Ser?Val?Val?Met?Thr?Val?Pro?Ser?Tyr?Val?Ala?Ser?Arg?Leu

355??????????????360??????????????365ctt?cgt?cca?ctt?tca?gac?tct?gct?gca?gat?tct?ctt?tca?aaa?ttt?tac??1152Leu?Arg?Pro?Leu?Ser?Asp?Ser?Ala?Ala?Asp?Ser?Leu?Ser?Lys?Phe?Tyr???370??????????????375??????????????380tat?cca?cca?gtt?gca?gca?gtg?tca?ctt?tcc?tat?cct?aaa?gaa?gcg?atc??1200Tyr?Pro?Pro?Val?Ala?Ala?Val?Ser?Leu?Ser?Tyr?Pro?Lys?Glu?Ala?Ile385??????????????390??????????????395?????????????400aga?tca?gaa?tgc?ttg?att?aat?ggt?gaa?ctt?caa?ggt?ttc?ggg?caa?cta??1248Arg?Ser?Glu?Cys?Leu?Ile?Asn?Gly?Glu?Leu?Gln?Gly?Phe?Gly?Gln?Leu

405??????????????410?????????????415cat?ccc?cgc?agt?cag?ggt?gta?gaa?acc?ttg?gaa?aca?att?tat?agt?tcg??1296His?Pro?Ara?Ser?Gln?Gly?Val?Glu?Thr?Leu?Gly?Thr?Ile?Tyr?Ser?Ser

420?????????????425??????????????430tct?ctt?ttc?cct?ggt?cga?gca?cca?cct?ggt?agg?atc?ttg?atc?ttg?agc??1344Ser?Leu?Phe?Pro?Gly?Arg?Ala?Pro?Pro?Gly?Arg?Ile?Leu?Ile?Leu?Ser

435??????????????440??????????????445tac?atc?gga?ggt?gct?aaa?aat?cct?ggc?ata?tta?aac?aag?tcg?aaa?gat??1392Tyr?Ile?Gly?Gly?Ala?Lys?Asn?Pro?Gly?Ile?Leu?Asn?Lys?Ser?Lys?Asp

450?????????????455??????????????460gaa?ctt?gcc?aag?aca?gtt?gac?aag?gac?ctg?aga?aga?atg?ctt?ata?aat???1440Glu?Leu?Ala?Lys?Thr?Val?Asp?Lys?Asp?Leu?Arg?Arg?Met?Leu?Ile?Asn465?????????????470???????????????475?????????????480cct?gat?gca?aaa?ctt?cct?cgt?gta?ctg?ggt?gtg?aga?gta?tgg?cct?caa???1488Pro?Asp?Ala?Lys?Leu?Pro?Arg?Val?Leu?Gly?Val?Arg?Val?Trp?Pro?Gln

485??????????????490?????????????495gca?ata?ccc?cag?ttt?tct?att?ggg?cac?ttt?gat?ctg?ctc?gat?gct?gca???1536Ala?Ile?Pro?Gln?Phe?Ser?Ile?Gly?His?Phe?Asp?Leu?Leu?Asp?Ala?Ala

500?????????????505??????????????510aaa?gct?gct?ctg?aca?gat?aca?ggg?gtc?aaa?gga?ctg?ttt?ctt?ggt?ggc???1584Lys?Ala?Ala?Leu?Thr?Asp?Thr?Gly?Val?Lys?Gly?Leu?Phe?Leu?Gly?Gly

515 520 525aac tat gtt tca ggt gtt gcc ttg ggg cgg tgt ata gag ggt gct tat 1632Asn Tyr Val Ser Gly Val Ala Leu Gly Arg Cys Ile Glu Gly Ala Tyr 530 535 540gag tct gca gct gag gta gta gat ttc ctc tca cag tac tca gac aaa 1680Glu Ser Ala Ala Glu Val Val Asp Phe Leu Ser Gln Tyr Ser Asp Lys545 550 555 560tagagcttca gcatcctgtg taattcaaca caggcctttt tgtatctgtt gtgcgcgcat 1740gtagtctggt cgtggtgcta ggattgatta gttgctctgc tgtgtgatcc acaagaattt 1800tgatggaatt tttccagatg tgggcattat atgttgctgt cttataaatc cttaatttgt 1860acgtttagtg aattacaccg catttgatga ctaaaaaaaa aaaaaaaaaa 1910＜210〉18＜211〉560＜212〉PRT＜213〉＜400〉18Met Lys Ser Met Ala Leu Ser Asn Cys Ile Pro Gln Thr Gln Cys Met 1 5 10 15Pro Leu Arg Ser Ser Gly His Tyr Arg Gly Asn Cys Ile Met Leu Ser

20???????????????25???????????????30Ile?Pro?Cys?Ser?Leu?Ile?Gly?Arg?Arg?Gly?Tyr?Tyr?Ser?His?Lys?Lys

35???????????????40???????????????45Arg?Arg?Met?Ser?Met?Ser?Cys?Ser?Thr?Ser?Ser?Gly?Ser?Lys?Ser?Ala

50??????????????55???????????????60Val?Lys?Glu?Ala?Gly?Ser?Gly?Ser?Gly?Ala?Gly?Gly?Leu?Leu?Asp?Cys?65??????????????70???????????????75???????????????80Val?Ile?Val?Gly?Gly?Gly?Ile?Ser?Gly?Leu?Cys?Ile?Ala?Gln?Ala?Leu

85??????????????90???????????????95Cys?Thr?Lys?His?Ser?Ser?Ser?Ser?Leu?Ser?Pro?Asn?Phe?Ile?Val?Thr

100?????????????105??????????????110Glu?Ala?Lys?Asp?Arg?Val?Gly?Gly?Asn?Ile?Val?Thr?Val?Glu?Ala?Asp

115??????????????120??????????????125Gly?Tyr?Ile?Trp?Glu?Glu?Gly?Pro?ASn?Ser?Phe?Gln?Pro?Ser?Asp?Ala???130??????????????135??????????????140Val?Leu?Thr?Met?Ala?Val?Asp?Ser?Gly?Leu?Lys?Asp?Glu?Leu?Val?Leu145??????????????150??????????????155?????????????160Gly?Asp?Pro?Asn?Ala?Pro?Arg?Phe?Val?Leu?Trp?Asn?Asp?Lys?Leu?Arg

165??????????????170?????????????175Pro?Val?Pro?Ser?Ser?Leu?Thr?Asp?Leu?Pro?Phe?Phe?Asp?Leu?Met?Thr

180?????????????185??????????????190Ile?Pro?Gly?Lys?Ile?Arg?Ala?Ala?Leu?Gly?Ala?Leu?Gly?Phe?Arg?Pro

195??????????????200??????????????205Ser?Pro?Pro?Pro?His?Glu?Glu?Ser?Val?Glu?His?Phe?Val?Arg?Arg?Asn???210??????????????215??????????????220Leu?Gly?Asp?Glu?Val?Phe?Glu?Arg?Leu?Ile?Glu?Pro?Phe?Cys?Ser?Gly225??????????????230??????????????235?????????????240Val?Tyr?Ala?Gly?Asp?Pro?Ala?Lys?Leu?Ser?Met?Lys?Ala?Ala?Phe?Gly

245??????????????250?????????????255Lys?Val?Trp?Lys?Leu?Glu?Gln?Lys?Gly?Gly?Ser?Ile?Ile?Gly?Gly?Thr

260?????????????265??????????????270Leu?Lys?Ala?Ile?Gln?Glu?Arg?Gly?Ser?Asn?Pro?Lys?Pro?Pro?Arg?Asp

275??????????????280??????????????285Gln?Arg?Leu?Pro?Lys?Pro?Lys?Gly?Gln?Thr?Val?Gly?Ser?Phe?Arg?Lys???290??????????????295??????????????300Gly?Leu?Val?Met?Leu?Pro?Thr?Ala?Ile?Ser?Ala?Arg?Leu?Gly?Ser?Arg305?????????????310??????????????315??????????????320Val?Lys?Leu?Ser?Trp?Thr?Leu?Ser?Ser?Ile?Val?Lys?Ser?Leu?Asn?Gly

325??????????????330?????????????335Glu?Tyr?Ser?Leu?Thr?Tyr?Asp?Thr?Pro?Asp?Gly?Leu?Val?Ser?Val?Arg

340?????????????345??????????????350Thr?Lys?Ser?Val?Val?Met?Thr?Val?Pro?Ser?Tyr?Val?Ala?Ser?Arg?Leu

355??????????????360??????????????365Leu?Arg?Pro?Leu?Ser?Asp?Ser?Ala?Ala?Asp?Ser?Leu?Ser?Lys?Phe?Tyr???370??????????????375??????????????380Tyr?Pro?Pro?Val?Ala?Ala?Val?Ser?Leu?Ser?Tyr?Pro?Lys?Glu?Ala?Ile385??????????????390?????????????395??????????????400Arg?Ser?Glu?Cys?Leu?Ile?Asn?Gly?Glu?Leu?Gln?Gly?Phe?Gly?Gln?Leu

405?????????????410?????????????415His?Pro?Arg?Ser?Gln?Gly?Val?Glu?Thr?Leu?Gly?Thr?Ile?Tyr?Ser?Ser

420?????????????425?????????????430Ser?Leu?Phe?Pro?Gly?Arg?Ala?Pro?Pro?Gly?Arg?Ile?Leu?Ile?Leu?Ser

435?????????????440???????????????445Tyr?Ile?Gly?Gly?Ala?Lys?Asn?Pro?Gly?Ile?Leu?Asn?Lys?Ser?Lys?Asp???450??????????????455?????????????460Glu?Leu?Ala?Lys?Thr?Val?Asp?Lys?Asp?Leu?Arg?Arg?Met?Leu?Ile?Asn465??????????????470??????????????475?????????????480Pro?Asp?Ala?Lys?Leu?Pro?Arg?Val?Leu?Gly?Val?Arg?Val?Trp?Pro?Gln

485??????????????490?????????????495Ala?Ile?Pro?Gln?Phe?Ser?Ile?Gly?His?Phe?Asp?Leu?Leu?Asp?Ala?Ala

500?????????????505??????????????510Lys?Ala?Ala?Leu?Thr?Asp?Thr?Gly?Val?Lys?Gly?Leu?Phe?Leu?Gly?Gly

515 520 525Asn Tyr Val Ser Gly Val Ala Leu Gly Arg Cys Ile Glu Gly Ala Tyr, 530 535 540Glu Ser Ala Ala Glu Val Val Asp Phe Leu Ser Gln Tyr Ser Asp Lys545 550 555 560＜210〉19＜211〉1784＜212〉DNA＜213〉colea＜220〉＜221〉CDS＜222〉(47) .. (1654)＜223〉rape protox-1 coded sequence＜400〉19gggccccccc caaaattgag gattctcctt ctcgcgggcg atcgcc atg gat tta 55

Met?Asp?Leu

1tct?ctt?ctc?cgt?ccg?cag?cca?ttc?cta?tcg?cca?ttc?tca?aat?cca?ttt???103Ser?Leu?Leu?Arg?Pro?Gln?Pro?Phe?Leu?Ser?Pro?Phe?Ser?Asn?Pro?Phe

5??????????????10???????????????15cct?cgg?tcg?cgt?ccc?tac?aag?cct?ctc?aac?ctc?cgt?tgc?tcc?gta?tcc???151Pro?Arg?Ser?Arg?Pro?Tyr?Lys?Pro?Leu?Asn?Leu?Arg?Cys?Ser?Val?Ser?20??????????????25???????????????30??????????????35ggt?gga?tcc?gtc?gtc?ggc?tct?tct?aca?atc?gaa?ggc?gga?gga?gga?ggt???199Gly?Gly?Ser?Val?Val?Gly?Ser?Ser?Thr?Ile?Glu?Gly?Gly?Gly?Gly?Gly

40??????????????45???????????????50aaa?acc?gtc?acg?gcg?gac?tgc?gtg?atc?gtc?ggc?gga?gga?atc?agc?ggc??247Lys?Thr?Val?Thr?Ala?Asp?Cys?Val?Ile?Val?Gly?Gly?Gly?Ile?Ser?Gly

55???????????????60??????????????65ctg?tgc?att?gcg?caa?gcg?ctc?gtg?acg?aag?cac?cca?gac?gct?gca?aag??295Leu?Cys?Ile?Ala?Gln?Ala?Leu?Val?Thr?Lys?His?Pro?Asp?Ala?Ala?Lys

70???????????????75??????????????80aat?gtg?atg?gtg?acg?gag?gcg?aag?gac?cgt?gtg?gga?ggg?aat?atc?atc??343Asn?Val?Met?Val?Thr?Glu?Ala?Lys?Asp?Arg?Val?Gly?Gly?Asn?Ile?Ile

85???????????????90??????????????95acg?cga?gag?gag?caa?ggg?ttt?cta?tgg?gaa?gaa?ggt?ccc?aat?agc?ttt??391Thr?Arg?Glu?Glu?Gln?Gly?Phe?Leu?Trp?Glu?Glu?Gly?Pro?Asn?Ser?Phe100??????????????105?????????????110??????????????115cag?ccg?tct?gat?cct?atg?ctc?act?atg?gtg?gta?gat?agt?ggt?ttg?aaa??439Gln?Pro?Ser?Asp?Pro?Met?Leu?Thr?Met?Val?Val?Asp?Ser?Gly?Leu?Lys

120??????????????125?????????????130gat?gat?cta?gtc?ttg?gga?gat?cct?act?gct?ccg?agg?ttt?gtg?ttg?tgg??487Asp?Asp?Leu?Val?Leu?Gly?Asp?Pro?Thr?Ala?Pro?Arg?Phe?Val?Leu?Trp

135??????????????140??????????????145aat?ggg?aag?ctg?agg?ccg?gtt?ccg?tcg?aag?cta?act?gac?ttg?cct?ttc??535Asn?Gly?Lys?Leu?Arg?Pro?Val?Pro?Ser?Lys?Leu?Thr?Asp?Leu?Pro?Phe

150?????????????155??????????????160ttt?gac?ttg?atg?agt?att?gga?ggg?aag?att?aga?gct?ggg?ttt?ggt?gcc??583Phe?Asp?Leu?Met?Ser?Ile?Gly?Gly?Lys?Ile?Arg?Ala?Gly?Phe?Gly?Ala

165?????????????170?????????????175att?ggt?att?cga?cct?tca?cct?ccg?ggt?cgt?gag?gaa?tca?gtg?gaa?gag??631Ile?Gly?Ile?Arg?Pro?Ser?Pro?Pro?Gly?Arg?Glu?Glu?Ser?Val?Glu?Glu180??????????????185?????????????190??????????????195ttt?gta?agg?cgt?aat?ctt?ggt?gat?gag?gtt?ttt?gag?cgc?ttg?att?gaa??679Phe?Val?Arg?Arg?Asn?Leu?Gly?Asp?Glu?Val?Phe?Glu?Arg?Leu?Ile?Glu

200?????????????205??????????????210ccc?ttt?tgc?tca?ggt?gtt?tat?gcg?gga?gat?cct?gcg?aaa?ctg?agt?atg??727Pro?Phe?Cys?Ser?Gly?Val?Tyr?Ala?Gly?Asp?Pro?Ala?Lys?Leu?Ser?Met

215??????????????220?????????????225aaa?gca?gct?ttt?ggg?aag?gtt?tgg?aag?cta?gag?gag?aat?ggt?ggg?agc??775Lys?Ala?Ala?Phe?Gly?Lys?Val?Trp?Lys?Leu?Glu?Glu?Asn?Gly?Gly?Ser

230??????????????235??????????????240atc?att?ggt?ggt?gct?ttt?aag?gca?att?caa?gcg?aaa?aat?aaa?gct?ccc??823Ile?Ile?Gly?Gly?Ala?Phe?Lys?Ala?Ile?Gln?Ala?Lys?Asn?Lys?Ala?Pro???245??????????????250??????????????255aag?aca?acc?cga?gat?ccg?cgt?ctg?cca?aag?cca?aag?ggc?caa?act?gtt??871Lys?Thr?Thr?Arg?Asp?Pro?Arg?Leu?Pro?Lys?Pro?Lys?Gly?Gln?Thr?Val260??????????????265??????????????270?????????????275ggt?tct?ttc?agg?aaa?gga?ctc?aca?atg?ctg?cca?gag?gca?atc?tcc?gca??919Gly?Ser?Phe?Arg?Lys?Gly?Leu?Thr?Met?Leu?Pro?Glu?Ala?Ile?Ser?Ala

280??????????????285?????????????290agg?ttg?ggt?gac?aag?gtg?aaa?gtt?tct?tgg?aag?ctc?tca?agt?atc?act??967Arg?Leu?Gly?Asp?Lys?Val?Lys?Val?Ser?Trp?Lys?Leu?Ser?Ser?Ile?Thr

295?????????????300??????????????305aag?ctg?gcc?agc?gga?gaa?tat?agc?tta?act?tac?gaa?act?ccg?gag?ggt??1015Lys?Leu?Ala?Ser?Gly?Glu?Tyr?Ser?Leu?Thr?Tyr?Glu?Thr?Pro?Glu?Gly

310??????????????315??????????????320ata?gtc?act?gta?cag?agc?aaa?agt?gta?gtg?atg?act?gtg?cca?tct?cat??1063Ile?Val?Thr?Val?Gln?Ser?Lys?Ser?Val?Val?Met?Thr?Val?Pro?Ser?His???325??????????????330??????????????335gtt?gct?agt?agt?ctc?ttg?cgc?cct?ctc?tct?gat?tct?gca?gct?gaa?gcg??1111Val?Ala?Ser?Ser?Leu?Leu?Arg?Pro?Leu?Ser?Asp?Ser?Ala?Ala?Glu?Ala340??????????????345??????????????350?????????????355ctc?tca?aaa?ctc?tac?tat?ccg?cca?gtt?gca?gcc?gta?tcc?atc?tca?tac??1159Leu?Ser?Lys?Leu?Tyr?Tyr?Pro?Pro?Val?Ala?Ala?Val?Ser?Ile?Ser?Tyr

360?????????????365??????????????370gcg?aaa?gaa?gca?atc?cga?agc?gaa?tgc?tta?ata?gat?ggt?gaa?cta?aaa??1207Ala?Lys?Glu?Ala?Ile?Arg?Ser?Glu?Cys?Leu?Ile?Asp?Gly?Glu?Leu?Lys

375?????????????380??????????????385ggg?ttc?ggc?cag?ttg?cat?cca?cgc?acg?caa?aaa?gtg?gaa?act?ctt?gga??1255Gly?Phe?Gly?Gln?Leu?His?Pro?Arg?Thr?Gln?Lys?Val?Glu?Thr?Leu?Gly

390?????????????395??????????????400aca?ata?tac?agt?tca?tcg?ctc?ttt?ccc?aac?cga?gca?ccg?cct?gga?aga??1303Thr?Ile?Tyr?Ser?Ser?Ser?Leu?Phe?Pro?Asn?Arg?Ala?Pro?Pro?Gly?Arg???405??????????????410??????????????415gta?ttg?cta?ttg?aac?tac?atc?ggt?gga?gct?acc?aac?act?ggg?atc?tta??1351Val?Leu?Leu?Leu?Asn?Tyr?Ile?Gly?Gly?Ala?Thr?Asn?Thr?Gly?Ile?Leu420??????????????425?????????????430??????????????435tca?aag?tcg?gaa?ggt?gag?tta?gtg?gaa?gca?gta?gat?aga?gac?ttg?agg??1399Ser?Lys?Ser?Glu?Gly?Glu?Leu?Val?Glu?Ala?Val?Asp?Arg?Asp?Leu?Arg

440?????????????445???????????????450aag?atg?ctg?ata?aag?cca?agc?tcg?acc?gat?cca?ctt?gta?ctt?gga?gta??1447Lys?Met?Leu?Ile?Lys?Pro?Ser?Ser?Thr?Asp?Pro?Leu?Val?Leu?Gly?Val

455?????????????460??????????????465aaa?tta?tgg?cct?caa?gcc?att?cct?cag?ttt?ctg?ata?ggt?cac?att?gat??1495Lys?Leu?Trp?Pro?Gln?Ala?Ile?Pro?Gln?Phe?Leu?Ile?Gly?His?Ile?Asp

470?????????????475??????????????480ttg?gta?gac?gca?gcg?aaa?gca?tcg?ctc?tcg?tca?tct?ggt?cat?gag?ggc??1543Leu?Val?Asp?Ala?Ala?Lys?Ala?Ser?Leu?Ser?Ser?Ser?Gly?His?Glu?Gly???485??????????????490??????????????495tta?ttc?ttg?ggt?gga?aat?tac?gtt?gcc?ggt?gta?gca?ttg?ggt?cgg?tgt??1591Leu?Phe?Leu?Gly?Gly?Asn?Tyr?Val?Ala?Gly?Val?Ala?Leu?Gly?Arg?Cys500??????????????505?????????????510??????????????515gtg?gaa?ggt?gct?tat?gaa?act?gca?acc?caa?gtg?aat?gat?ttc?atg?tca??1639Val?Glu?Gly?Ala?Tyr?Glu?Thr?Ala?Thr?Gln?Val?Asn?Asp?Phe?Met?Ser

520?????????????525??????????????530agg?tat?gct?tac?aag?taatgtaacg?cagcaacgat?ttgatactaa?gtagtagatt??1694Arg?Tyr?Ala?Tyr?Lys

535ttgcagtttt gactttaaga acactctgtt tgtgaaaaat tcaagtctgt gattgagtaa 1754atttatgtat tattactaaa aaaaaaaaaa 1784＜210〉20＜211〉536＜212〉PRT＜213〉colea＜400〉20Met Asp Leu Ser Leu Leu Arg Pro Gln Pro Phe Leu Ser Pro Phe Ser 15 10 15Asn Pro Phe Pro Arg Ser Arg Pro Tyr Lys Pro Leu Asn Leu Arg Cys

20??????????????25???????????????30Ser?Val?Ser?Gly?Gly?Ser?Val?Val?Gly?Ser?Ser?Thr?Ile?Glu?Gly?Gly

35???????????????40??????????????45Gly?Gly?Gly?Lys?Thr?Val?Thr?Ala?Asp?Cys?Val?Ile?Val?Gly?Gly?Gly

50??????????????55???????????????60Ile?Ser?Gly?Leu?Cys?Ile?Ala?Gln?Ala?Leu?Val?Thr?Lys?His?Pro?Asp?65??????????????70???????????????75??????????????80Ala?Ala?Lys?Asn?Val?Met?Val?Thr?Glu?Ala?Lys?Asp?Arg?Val?Gly?Gly

85???????????????90???????????????95Asn?Ile?Ile?Thr?Arg?Glu?Glu?Gln?Gly?Phe?Leu?Trp?Glu?Glu?Gly?Pro

100?????????????105??????????????110Asn?Ser?Phe?Gln?Pro?Ser?Asp?Pro?Met?Leu?Thr?Met?Val?Val?Asp?Ser

115??????????????120??????????????125Gly?Leu?Lys?Asp?Asp?Leu?Val?Leu?Gly?Asp?Pro?Thr?Ala?Pro?Arg?Phe???130??????????????135?????????????140Val?Leu?Trp?Asn?Gly?Lys?Leu?Arg?Pro?Val?Pro?Ser?Lys?Leu?Thr?Asp145?????????????150??????????????155??????????????160Leu?Pro?Phe?Phe?Asp?Leu?Met?Ser?Ile?Gly?Gly?Lys?Ile?Arg?Ala?Gly

165??????????????170?????????????175Phe?Gly?Ala?Ile?Gly?Ile?Arg?Pro?Ser?Pro?Pro?Gly?Arg?Glu?Glu?Ser

180?????????????185??????????????190Val?Glu?Glu?Phe?Val?Arg?Arg?Asn?Leu?Gly?Asp?Glu?Val?Phe?Glu?Arg

195??????????????200??????????????205Leu?Ile?Glu?Pro?Phe?Cys?Ser?Gly?Val?Tyr?Ala?Gly?Asp?Pro?Ala?Lys???210??????????????215??????????????220Leu?Ser?Met?Lys?Ala?Ala?Phe?Gly?Lys?Val?Trp?Lys?Leu?Glu?Glu?Asn225??????????????230?????????????235??????????????240Gly?Gly?Ser?Ile?Ile?Gly?Gly?Ala?Phe?Lys?Ala?Ile?Gln?Ala?Lys?Asn

245??????????????250?????????????255Lys?Ala?Pro?Lys?Thr?Thr?Arg?Asp?Pro?Arg?Leu?Pro?Lys?Pro?Lys?Gly

260?????????????265??????????????270Gln?Thr?Val?Gly?Ser?Phe?Arg?Lys?Gly?Leu?Thr?Met?Leu?Pro?Glu?Ala

275??????????????280??????????????285Ile?Ser?Ala?Arg?Leu?Gly?Asp?Lys?Val?Lys?Val?Ser?Trp?Lys?Leu?Ser

290?????????????295?????????????300Ser?Ile?Thr?Lys?Leu?Ala?Ser?Gly?Glu?Tyr?Ser?Leu?Thr?Tyr?Glu?Thr305??????????????310??????????????315?????????????320Pro?Glu?Gly?Ile?Val?Thr?Val?Gln?Ser?Lys?Ser?Val?Val?Met?Thr?Val

325??????????????330?????????????335Pro?Ser?His?Val?Ala?Ser?Ser?Leu?Leu?Arg?Pro?Leu?Ser?Asp?Ser?Ala

340?????????????345??????????????350Ala?Glu?Ala?Leu?Ser?Lys?Leu?Tyr?Tyr?Pro?Pro?Val?Ala?Ala?Val?Ser

355?????????????360??????????????365Ile?Ser?Tyr?Ala?Lys?Glu?Ala?Ile?Arg?Ser?Glu?Cys?Leu?Ile?Asp?Gly???370??????????????375??????????????380Glu?Leu?Lys?Gly?Phe?Gly?Gln?Leu?His?Pro?Arg?Thr?Gln?Lys?Val?Glu385??????????????390?????????????395??????????????400Thr?Leu?Gly?Thr?Ile?Tyr?Ser?Ser?Ser?Leu?Phe?Pro?Asn?Arg?Ala?Pro

405?????????????410??????????????415Pro?Gly?Arg?Val?Leu?Leu?Leu?Asn?Tyr?Ile?Gly?Gly?Ala?Thr?Asn?Thr

420?????????????425??????????????430Gly?Ile?Leu?Ser?Lys?Ser?Glu?Gly?Glu?Leu?Val?Glu?Ala?Val?Asp?Arg

435??????????????440??????????????445Asp?Leu?Arg?Lys?Met?Leu?Ile?Lys?Pro?Ser?Ser?Thr?Asp?Pro?Leu?Val???450??????????????455?????????????460Leu?Gly?Val?Lys?Leu?Trp?Pro?Gln?Ala?Ile?Pro?Gln?Phe?Leu?Ile?Gly465??????????????470?????????????475??????????????480His?Ile?Asp?Leu?Val?Asp?Ala?Ala?Lys?Ala?Ser?Leu?Ser?Ser?Ser?Gly

485??????????????490?????????????495His?Glu?Gly?Leu?Phe?Leu?Gly?Gly?Asn?Tyr?Val?Ala?Gly?Val?Ala?Leu

500?????????????505??????????????510Gly?Arg?Cys?Val?Glu?Gly?Ala?Tyr?Glu?Thr?Ala?Thr?Gln?Val?Asn?Asp

515 520 525Phe Met Ser Arg Tyr Ala Tyr Lys 530 535＜210〉21＜211〉1224＜212〉DNA＜213〉rice＜220〉＜221〉CDS＜222〉(1) .. (936)＜223〉rice protox-1 part coded sequence＜400〉21cgg gct ttg aag gct gca ttt ggg aag gtg tgg agg ctg gag gat act 48Arg Ala Leu Lys Ala Ala Phe Gly Lys Val Trp Arg Leu Glu Asp Thr, 15 10 15gga ggt agc att att ggt gga acc atc aag aca atc cag gag agg ggg 96Gly Gly Ser Ile Ile Gly Gly Thr Ile Lys Thr Ile Gln Glu Arg Gly

20???????????????25??????????????30aaa?aac?ccc?aaa?ccg?ccg?agg?gat?ccc?cgc?ctt?cca?acg?cca?aag?ggg?144Lys?Asn?Pro?Lys?Pro?Pro?Arg?Asp?Pro?Arg?Leu?Pro?Thr?Pro?Lys?Gly

35???????????????40??????????????45cag?aca?gtt?gca?tct?ttc?agg?aag?ggt?ctg?act?atg?ctc?ccg?gat?gct??192Gln?Thr?Val?Ala?Ser?Phe?Arg?Lys?Gly?Leu?Thr?Met?Leu?Pro?Asp?Ala

50???????????????55??????????????60att?aca?tct?agg?ttg?ggt?agc?aaa?gtc?aaa?ctt?tca?tgg?aag?ttg?aca??240Ile?Thr?Ser?Arg?Leu?Gly?Ser?Lys?Val?Lys?Leu?Ser?Trp?Lys?Leu?Thr?65??????????????70???????????????75??????????????80agc?att?aca?aag?tca?gac?aac?aaa?gga?tat?gca?tta?gtg?tat?gaa?aca??288Ser?Ile?Thr?Lys?Ser?Asp?Asn?Lys?Gly?Tyr?Ala?Leu?Val?Tyr?Glu?Thr

85???????????????90??????????????95cca?gaa?ggg?gtg?gtc?tcg?gtg?caa?gct?aaa?act?gtt?gtc?atg?acc?atc??336Pro?Glu?Gly?Val?Val?Ser?Val?Gln?Ala?Lys?Thr?Val?Val?Met?Thr?Ile

100??????????????105??????????????110cca?tca?tat?gtt?gct?agt?gat?atc?ttg?cgg?cca?ctt?tca?agt?gat?gca??384Pro?Ser?Tyr?Val?Ala?Ser?Asp?Ile?Leu?Arg?Pro?Leu?Ser?Ser?Asp?Ala

115?????????????120??????????????125gca?gat?gct?ctg?tca?ata?ttc?tat?tat?cca?cca?gtt?gct?gct?gta?act??432Ala?Asp?Ala?Leu?Ser?Ile?Phe?Tyr?Tyr?Pro?Pro?Val?Ala?Ala?Val?Thr???130??????????????135?????????????140gtt?tca?tat?cca?aaa?gaa?gca?att?aga?aaa?gaa?tgc?tta?att?gac?gga??480Val?Ser?Tyr?Pro?Lys?Glu?Ala?Ile?Arg?Lys?Glu?Cys?Leu?Ile?Asp?Gly145??????????????150?????????????155??????????????160gag?ctc?cag?ggt?ttc?ggc?cag?ctg?cat?ccg?cgt?agt?cag?gga?gtt?gag??528Glu?Leu?Gln?Gly?Phe?Gly?Gln?Leu?His?Pro?Arg?Ser?Gln?Gly?Val?Glu

165?????????????170??????????????175act?tta?gga?aca?ata?tat?agc?tca?tca?ctc?ttt?cca?aat?cgt?gct?cca??576Thr?Leu?Gly?Thr?Ile?Tyr?Ser?Ser?Ser?Leu?Phe?Pro?Asn?Arg?Ala?Pro

180??????????????185??????????????190gct?gga?agg?gtg?tta?ctt?ctg?aac?tac?ata?gga?ggt?tct?aca?aat?aca??624Ala?Gly?Arg?Val?Leu?Leu?Leu?Asn?Tyr?Ile?Gly?Gly?Ser?Thr?Asn?Thr

195??????????????200??????????????205ggg?att?gtt?tcc?aag?act?gaa?agt?gag?ctg?gta?gaa?gca?gtt?gac?cgt??672Gly?Ile?Val?Ser?Lys?Thr?Glu?Ser?Glu?Leu?Val?Glu?Ala?Val?Asp?Arg???210??????????????215??????????????220gac?ctc?agg?aag?atg?ctg?ata?aat?cct?aga?gca?gtg?gac?cct?ttg?gtc??720Asp?Leu?Arg?Lys?Met?Leu?Ile?Asn?Pro?Arg?Ala?Val?Asp?Pro?Leu?Val225??????????????230??????????????235?????????????240ctt?ggc?gtc?cgg?gta?tgg?cca?caa?gcc?ata?cca?cag?ttc?ctc?att?ggc??768Leu?Gly?Val?Arg?Val?Trp?Pro?Gln?Ala?Ile?Pro?Gln?Phe?Leu?Ile?Gly

245??????????????250?????????????255cat?ctt?gat?cat?ctt?gag?gct?gca?aaa?tct?gcc?ctg?ggc?aaa?ggt?ggg??816His?Leu?Asp?His?Leu?Glu?Ala?Ala?Lys?Ser?Ala?Leu?Gly?Lys?Gly?Gly

260?????????????265??????????????270tat?gat?gga?ttg?ttc?ctc?gga?ggg?aac?tat?gtt?gca?gga?gtt?gcc?ctg??864Tyr?Asp?Gly?Leu?Phe?Leu?Gly?Gly?Asn?Tyr?Val?Ala?Gly?Val?Ala?Leu

275 280 285ggc cga tgc gtt gaa ggt gca tat gag agt gcc tca caa ata tct gac 912Gly Arg Cys Val Glu Gly Ala Tyr Glu Ser Ala Ser Gln Ile Ser Asp 290 295 300tac ttg acc aag tac gcc tac aag tgatcaaagt tggcctgctc cttttggcac 966Tyr Leu Thr Lys Tyr Ala Tyr Lys305 310atagatgtga ggcttctagc agcaaaaatt tcatgggcat ctttttatcc tgattctaat 1026tagttagaat ttagaattgt agaggaatgt tccatttgca gttcataata gttgttcaga 1086tttcagccat tcaatttgtg cagccattta ctatatgtag tatgatcttg taagtactac 1146taagaacaaa tcaattatat tttcctgcaa gtgacatctt aatcgtcagc aaatccagtt 1206actagtaaaa aaaaaaaa 1224＜210〉22＜211〉312＜212〉PRT＜213〉＜400〉22Arg Ala Leu Lys Ala Ala Phe Gly Lys Val Trp Arg Leu Glu Asp Thr 1 5 10 15Gly Gly Ser Ile Ile Gly Gly Thr Ile Lys Thr Ile Gln Glu Arg Gly

20???????????????25???????????????30Lys?Asn?Pro?LysPro?Pro?Arg?Asp?Pro?Arg?Leu?Pro?Thr?Pro?Lys?Gly

35???????????????40??????????????45Gln?Thr?Val?Ala?Ser?Phe?Ara?Lys?Gly?Leu?Thr?Met?Leu?Pro?Asp?Ala

50???????????????55??????????????60Ile?Thr?Ser?Arg?Leu?Gly?Ser?Lys?Val?Lys?Leu?Ser?Trp?Lys?Leu?Thr?65??????????????70???????????????75???????????????80Ser?Ile?Thr?Lys?Ser?Asp?Asn?Lys?Gly?Tyr?Ala?Leu?Val?Tyr?Glu?Thr

85???????????????90???????????????95Pro?Glu?Gly?Val?Val?Ser?Val?Gln?Ala?Lys?Thr?Val?Val?Met?Thr?Ile

100?????????????105??????????????110Pro?Ser?Tyr?Val?Ala?Ser?Asp?Ile?Leu?Arg?Pro?Leu?Ser?Ser?Asp?Ala

115??????????????120??????????????125Ala?Asp?Ala?Leu?Ser?Ile?Phe?Tyr?Tyr?Pro?Pro?Val?Ala?Ala?Val?Thr???130??????????????135??????????????140Val?Ser?Tyr?Pro?Lys?Glu?Ala?Ile?Arg?Lys?Glu?Cys?Leu?Ile?Asp?Gly145??????????????150?????????????155??????????????160Glu?Leu?Gln?Gly?Phe?Gly?Gln?Leu?His?Pro?Arg?Ser?Gln?Gly?Val?Glu

165?????????????170??????????????175Thr?Leu?Gly?Thr?Ile?Tyr?Ser?Ser?Ser?Leu?Phe?Pro?Asn?Arg?Ala?Pro

180?????????????185??????????????190Ala?Gly?Arg?Val?Leu?Leu?Leu?Asn?Tyr?Ile?Gly?Gly?Ser?Thr?Asn?Thr

195??????????????200??????????????205Gly?Ile?Val?Ser?Lys?Thr?Glu?Ser?Glu?Leu?Val?Glu?Ala?Val?Asp?Arg???210??????????????215??????????????220Asp?Leu?Arg?Lys?Met?Leu?Ile?Asn?Pro?Arg?Ala?Val?Asp?Pro?Leu?Val225??????????????230?????????????235??????????????240Leu?Gly?Val?Arg?Val?Trp?Pro?Gln?Ala?Ile?Pro?Gln?Phe?Leu?Ile?Gly

245?????????????250??????????????255His?Leu?Asp?His?Leu?Glu?Ala?Ala?Lys?Ser?Ala?Leu?Gly?Lys?Gly?Gly

260?????????????265??????????????270Tyr?Asp?Gly?Leu?Phe?Leu?Gly?Gly?Asn?Tyr?Val?Ala?Gly?Val?Ala?Leu

275 280 285Gly Arg Cys Val Glu Gly Ala Tyr Glu Ser Ala Ser Gln Ile Ser Asp, 290 295 300Tyr Leu Thr Lys Tyr Ala Tyr Lys305,310＜210〉23＜211〉1590＜212〉DNA＜213〉dichromatism chinese sorghum＜220〉＜221〉CDS＜222〉(1) .. (1320)＜223〉Chinese sorghum protox-1 part coded sequence＜400〉23tcc acc gtc gag cgc ccc gag gaa ggg tac ctc tgg gag gag ggt ccc 48Ser Thr Val Glu Arg Pro Glu Glu Gly Tyr Leu Trp Glu Glu Gly Pro, 15 10 15aac agc ttc cag cca tcc gac ccc gtt ctc tcc atg gcc gtg gac agc 96Asn Ser Phe Gln Pro Ser Asp Pro Val Leu Ser Met Ala Val Asp Ser

20???????????????25???????????????30ggg?ctg?aag?gat?gac?ctg?gtt?ttt?ggg?gac?ccc?aac?gcg?cca?cgg?ttc?144Gly?Leu?Lys?Asp?Asp?Leu?Val?Phe?Gly?Asp?Pro?Asn?Ala?Pro?Arg?Phe

35???????????????40??????????????45gtg?ctg?tgg?gag?ggg?aag?ctg?agg?ccc?gtg?cca?tcc?aag?ccc?gcc?gac?192Val?Leu?Trp?Glu?Gly?Lys?Leu?Arg?Pro?Val?Pro?Ser?Lys?Pro?Ala?Asp

50??????????????55???????????????60ctc?ccg?ttc?ttc?gat?ctc?atg?agc?atc?cct?ggc?aag?ctc?agg?gcc?ggt??240Leu?Pro?Phe?Phe?Asp?Leu?Met?Ser?Ile?Pro?Gly?Lys?Leu?Arg?Ala?Gly?65??????????????70??????????????75???????????????80ctc?ggc?gcg?ctt?ggc?atc?cgc?ccg?cct?gct?cca?ggc?cgc?gag?gag?tca??288Leu?Gly?Ala?Leu?Gly?Ile?Arg?Pro?Pro?Ala?Pro?Gly?Arg?Glu?Glu?Ser

85??????????????90???????????????95gtg?gag?gag?ttt?gtg?cgc?cgc?aac?ctc?ggt?gct?gag?gtc?ttt?gag?cgc??336Val?Glu?Glu?Phe?Val?Arg?Arg?Asn?Leu?Gly?Ala?Glu?Val?Phe?Glu?Arg

100?????????????105??????????????110cta?att?gag?cct?ttc?tgc?tca?ggt?gtc?tat?gct?ggc?gat?cct?tcc?aag??384Leu?Ile?Glu?Pro?Phe?Cys?Ser?Gly?Val?Tyr?Ala?Gly?Asp?Pro?Ser?Lys

115??????????????120??????????????125ctc?agt?atg?aag?gct?gca?ttt?ggg?aag?gtg?tgg?cgg?tta?gaa?gaa?gct??432Leu?Ser?Met?Lys?Ala?Ala?Phe?Gly?Lys?Val?Trp?Arg?Leu?Glu?Glu?Ala???130??????????????135??????????????140gga?ggt?agt?att?att?ggt?gga?acc?atc?aag?acg?att?cag?gag?agg?ggc??480Gly?Gly?Ser?Ile?Ile?Gly?Gly?Thr?Ile?Lys?Thr?Ile?Gln?Glu?Arg?Gly145??????????????150??????????????155?????????????160aag?aat?cca?aaa?cca?ccg?agg?gat?ccc?cgc?ctt?ccg?aag?cca?aaa?ggg??528Lys?Asn?Pro?Lys?Pro?Pro?Arg?Asp?Pro?Arg?Leu?Pro?Lys?Pro?Lys?Gly

165??????????????170?????????????175cag?aca?gtt?gca?tct?ttc?agg?aag?ggt?ctt?gcc?atg?ctt?cca?aat?gcc??576Gln?Thr?Val?Ala?Ser?Phe?Arg?Lys?Gly?Leu?Ala?Met?Leu?Pro?Asn?Ala

180?????????????185??????????????190atc?aca?tcc?agc?ttg?ggt?agt?aaa?gtc?aaa?cta?tca?tgg?aaa?ctc?acg??624Ile?Thr?Ser?Ser?Leu?Gly?Ser?Lys?Val?Lys?Leu?Ser?Trp?Lys?Leu?Thr

195??????????????200??????????????205agc?atg?aca?aaa?tca?gat?ggc?aag?ggg?tat?gtt?ttg?gag?tat?gaa?aca??672Ser?Met?Thr?Lys?Ser?Asp?Gly?Lys?Gly?Tyr?Val?Leu?Glu?Tyr?Glu?Thr???210??????????????215??????????????220cca?gaa?ggg?gtt?gtt?ttg?gtg?cag?gct?aaa?agt?gtt?atc?atg?acc?att??720Pro?Glu?Gly?Val?Val?Leu?Val?Gln?Ala?Lys?Ser?Val?Ile?Met?Thr?Ile225?????????????230??????????????235??????????????240cca?tca?tat?gtt?gct?agc?gac?att?ttg?cgt?cca?ctt?tca?ggt?gat?gct??768Pro?Ser?Tyr?Val?Ala?Ser?Asp?Ile?Leu?Arg?Pro?Leu?Ser?Gly?Asp?Ala

245??????????????250?????????????255gca?gat?gtt?cta?tca?aga?ttc?tat?tat?cca?cca?gtt?gct?gct?gta?acg??816Ala?Asp?Val?Leu?Ser?Arg?Phe?Tyr?Tyr?Pro?Pro?Val?Ala?Ala?Val?Thr

260?????????????265??????????????270gtt?tcg?tat?cca?aag?gaa?gca?att?aga?aaa?gaa?tgc?tta?att?gat?ggg??864Val?Ser?Tyr?Pro?Lys?Glu?Ala?Ile?Arg?Lys?Glu?Cys?Leu?Ile?Asp?Gly

275?????????????280??????????????285gaa?ctc?cag?ggt?ttt?ggc?cag?ttg?cat?cca?cgt?agt?caa?gga?gtt?gag??912Glu?Leu?Gln?Gly?Phe?Gly?Gln?Leu?His?Pro?Arg?Ser?Gln?Gly?Val?Glu???290??????????????295??????????????300aca?tta?gga?aca?ata?tac?agc?tca?tca?ctc?ttt?cca?aat?cgt?gct?cct??960Thr?Leu?Gly?Thr?Ile?Tyr?Ser?Ser?Ser?Leu?Phe?Pro?Asn?Arg?Ala?Pro305??????????????310?????????????315??????????????320gct?ggt?agg?gtg?tta?ctt?cta?aac?tac?ata?gga?ggt?gct?aca?aac?aca??1008Ala?Gly?Arg?Val?Leu?Leu?Leu?Asn?Tyr?Ile?Gly?Gly?Ala?Thr?Asn?Thr

325??????????????330??????????????335gga?att?gtt?tcc?aag?act?gaa?agt?gag?ctg?gta?gaa?gca?gtt?gac?cgt??1056Gly?Ile?Val?Ser?Lys?Thr?Glu?Ser?Glu?Leu?Val?Glu?Ala?Val?Asp?Arg

340??????????????345?????????????350gac?ctc?cga?aaa?atg?ctt?ata?aat?cct?aca?gca?gtg?gac?cct?tta?gtc??1104Asp?Leu?Arg?Lys?Met?Leu?Ile?Asn?Pro?Thr?Ala?Val?Asp?Pro?Leu?Val

355??????????????360??????????????365ctt?ggt?gtc?cga?gtt?tgg?cca?caa?gcc?ata?cct?cag?ttc?ctg?gta?gga??1152Leu?Gly?Val?Arg?Val?Trp?Pro?Gln?Ala?Ile?Pro?Gln?Phe?Leu?Val?Gly???370??????????????375??????????????380cat?ctt?gat?ctt?ctg?gag?gcc?gca?aaa?tct?gcc?ctg?gac?caa?ggt?ggc??1200His?Leu?Asp?Leu?Leu?Glu?Ala?Ala?Lys?Ser?Ala?Leu?Asp?Gln?Gly?Gly385??????????????390?????????????395??????????????400tat?aat?ggg?ctg?ttc?cta?gga?ggg?aac?tat?gtt?gca?gga?gtt?gcc?ctg??1248Tyr?Asn?Gly?Leu?Phe?Leu?Gly?Gly?Asn?Tyr?Val?Ala?Gly?Val?Ala?Leu

405??????????????410?????????????415ggc?aga?tgc?att?gag?ggc?gca?tat?gag?agt?gcc?gcg?caa?ata?tat?gac???1296Gly?Arg?Cys?Ile?Glu?Gly?Ala?Tyr?Glu?Ser?Ala?Ala?Gln?Ile?Tyr?Asp

420?????????????425??????????????430ttc?ttg?acc?aag?tac?gcc?tac?aag?tgatggaaga?agtggagcgc?tgcttgttaa??1350Phe?Leu?Thr?Lys?Tyr?Ala?Tyr?Lys

435 440ttgttatgtt gcatagatga ggtgagacca ggagtagtaa aaggcgtcac gagtattttt 1410cattcttatt ttgtaaattg cacttctgtt tttttttcct gtcagtaatt agttagattt 1470tagttatgta ggagattgtt gtgttcactg ccctacaaaa gaatttttat tttgcattcg 1530tttatgagag ctgtgcagac ttatgtaacg ttttactgta agtatcaaca aaatcaaata, 1590＜210〉24＜211〉440＜212〉PRT＜213〉dichromatism chinese sorghum＜400〉24Ser Thr Val Glu Arg Pro Glu Glu Gly Tyr Leu Trp Glu Glu Gly Pro 15 10 15Asn Ser Phe Gln Pro Ser Asp Pro Val Leu Ser Met Ala Val Asp Ser

20???????????????25???????????????30Gly?Leu?Lys?Asp?Asp?Leu?Val?Phe?Gly?Asp?Pro?Asn?Ala?Pro?Arg?Phe

35???????????????40??????????????45Val?Leu?Trp?Glu?Gly?Lys?Leu?Arg?Pro?Val?Pro?Ser?Lys?Pro?Ala?Asp

50???????????????55??????????????60Leu?Pro?Phe?Phe?Asp?Leu?Met?Ser?Ile?Pro?Gly?Lys?Leu?Arg?Ala?Gly?65??????????????70??????????????75???????????????80Leu?Gly?Ala?Leu?Gly?Ile?Arg?Pro?Pro?Ala?Pro?Gly?Arg?Glu?Glu?Ser

85??????????????90???????????????95Val?Glu?Glu?Phe?Val?Arg?Arg?Asn?Leu?Gly?Ala?Glu?Val?Phe?Glu?Arg

100?????????????105??????????????110Leu?Ile?Glu?Pro?Phe?Cys?Ser?Gly?Val?Tyr?Ala?Gly?Asp?Pro?Ser?Lys

115??????????????120??????????????125Leu?Ser?Met?Lys?Ala?Ala?Phe?Gly?Lys?Val?Trp?Arg?Leu?Glu?Glu?Ala???130??????????????135?????????????140Gly?Gly?Ser?Ile?Ile?Gly?Gly?Thr?Ile?Lys?Thr?Ile?Gln?Glu?Arg?Gly145??????????????150?????????????155??????????????160Lys?Asn?Pro?Lys?Pro?Pro?Arg?Asp?Pro?Arg?Leu?Pro?Lys?Pro?Lys?Gly

165??????????????170?????????????175Gln?Thr?Val?Ala?Ser?Phe?Arg?Lys?Gly?Leu?Ala?Met?Leu?Pro?Asn?Ala

180?????????????185??????????????190Ile?Thr?Ser?Ser?Leu?Gly?Ser?Lys?Val?Lys?Leu?Ser?Trp?Lys?Leu?Thr

195?????????????200??????????????205Ser?Met?Thr?Lys?Ser?Asp?Gly?Lys?Gly?Tyr?Val?Leu?Glu?Tyr?Glu?Thr

210????????????215??????????????220Pro?Glu?Gly?Val?Val?Leu?Val?Gln?Ala?Lys?Ser?Val?Ile?Met?Thr?Ile225??????????????230?????????????235??????????????240Pro?Ser?Tyr?Val?Ala?Ser?Asp?Ile?Leu?Arg?Pro?Leu?Ser?Gly?Asp?Ala

245??????????????250?????????????255Ala?Asp?Val?Leu?Ser?Arg?Phe?Tyr?Tyr?Pro?Pro?Val?Ala?Ala?Val?Thr

260?????????????265??????????????270Val?Ser?Tyr?Pro?Lys?Glu?Ala?Ile?Arg?Lys?Glu?Cys?Leu?Ile?Asp?Gly

275??????????????280??????????????285Glu?Leu?Gln?Gly?Phe?Gly?Gln?Leu?His?Pro?Arg?Ser?Gln?Gly?Val?Glu???290??????????????295??????????????300Thr?Leu?Gly?Thr?Ile?Tyr?Ser?Ser?Ser?Leu?Phe?Pro?Asn?Arg?Ala?Pro305??????????????310??????????????315?????????????320Ala?Gly?Arg?Val?Leu?Leu?Leu?Asn?Tyr?Ile?Gly?Gly?Ala?Thr?Asn?Thr

325??????????????330?????????????335Gly?Ile?Val?Ser?Lys?Thr?Glu?Ser?Glu?Leu?Val?Glu?Ala?Val?Asp?Arg

340?????????????345??????????????350Asp?Leu?Arg?Lys?Met?Leu?Ile?Asn?Pro?Thr?Ala?Val?Asp?Pro?Leu?Val

355??????????????360??????????????365Leu?Gly?Val?Arg?Val?Trp?Pro?Gln?Ala?Ile?Pro?Gln?Phe?Leu?Val?Gly???370??????????????375?????????????380His?Leu?Asp?Leu?Leu?Glu?Ala?Ala?Lys?Ser?Ala?Leu?Asp?Gln?Gly?Gly385??????????????390?????????????395??????????????400Tyr?Asn?Gly?Leu?Phe?Leu?Gly?Gly?Asn?Tyr?Val?Ala?Gly?Val?Ala?Leu

405??????????????410?????????????415Gly?Arg?Cys?Ile?Glu?Gly?Ala?Tyr?Glu?Ser?Ala?Ala?Gln?Ile?Tyr?Asp

420?????????????425??????????????430Phe?Leu?Thr?Lys?Tyr?Ala?Tyr?Lys

435 440＜210〉25＜211〉93＜212〉DNA＜213〉＜220〉＜221〉misc_＜222〉 ( 1 ) .. ( 93 )＜223〉protox-1＜400〉25gtacgctcct cgctggcgcc gcagcgtctt cttctcagac tcatgcgcag ccatggaatt 60gagatgctga atggatttta tacgcgcgcg cag 93＜210〉26＜211〉2606＜212〉DNA＜213〉＜220〉＜221〉misc_＜222〉 ( 2601 ) .. ( 2606 )＜223〉SalI＜220〉＜221〉misc_＜222〉 ( 1 ) .. ( 538 )＜223〉protox-1c-DNA＜220〉＜221〉misc_＜222〉 ( 539 ) .. ( 2606 )＜223〉protox-1 ( pWDC-20～3kb PstI-SalI )＜400〉26ctgcaggggg agggaaagag agaccgcgac ggtgagggag gggagaccgc gacggtgagg 60gaggggagaa cgcgacggtg agggagggga gaacgcgatg gtgagggagg ggagaacgcg 120acgcgcaggg gagggggata actcgacggt gcagggaggt gagggggacg acgtgacggc 180gcaggggagg ggggaaccgt cgcgggaagg ggaagaccgg ggggccgaca aggtggtgtt 240actggggtag ggagaggcgg cgtggagaat agtaacagag ggaggagtgg tggtgctagg 300gtggaagaag ggtaagaaag aggaagaaag agaattaaca ttatcttaac caaacaccac 360tctaaatcta agggttttct tttcctttcc tctcctctcc ctttcttgat tccattccct 420ttaccccgtt gcaaccaaac gcccccttat tatggaccgg aggaagtatg tagagatggt 480cacaaaacta cttaagctgg taacttataa atatactggg tattaaatga attaagtggc 540cacaaaatga ctataaatta cttcgtaatc tttaggaact atgttggtca cgaaataaca 600taaaactggt tatttaatgg ctttatgtag gtactgcatt cataaatata tttctaacat 660aatcgtggta tgtaggtgtt ttataacaca aggattaggt ttacaccaat gtcattttca 720ttagaatgta gttagaatca ctttggaact ttgaagagtg atgacacatt tttattatgc 780ttttatgaaa tgtctttgtg gtttttatga tagtattgag tttaaggcaa gttggaagta 840tatgatggag aagtacagta tataggtgac aattggtttg cttgtttcta tgagttgaaa 900gataagtagt acacgacact gagcaatgac ctcttcttag ttgtaatttt gtcttctcga 960cgtagtgaaa gtacaaacaa gattatggct ttcaagcttc caagataacg agattgtatg 1020aattttgtgg tgtatttcac atcattgttt tacgttggag acaaactaaa accaatgatg 1080agtttgtgga ttcgagattt gcccctaagt cttatttacc catggcaagc atgctgaaac 1140atgttagtca aacttacaca gctacaatgt ttagggattt tgagcaaaaa atttgggtat 1200tctttgggta ccattatgtg agttgttgac tatggattaa acaaaatcac tatataaagt 1260ctggaatgag aagcatccgc aattgacaca ccatgttact ttgattgttt caacaagttt 1320attagatgta tttgtaggaa ttttgaagag gcggagatgt tgtgttataa ttgctttggg 1380ggtgcttcac atgcactctg ttagtgagac atcttcagct tatattttaa ggcggttagt 1440gagtatgatt tttttttttc aaacttttcg atttccatgt aattaaaaaa ggtgtttgat 1500aaatacatgt taagatagcc aagaaaaggc aactttcaaa caaataaaaa aaattaagtc 1560gcttaatcat ttttccaagt actttttact tttaacacca cttattactg aatctatagc 1620cgttaagaat gcattttcac gctcatacat gcaaatcaag aacctcctca ttgaaggaga 1680taatttagtc ctcataaacc ccgttaaaga catttttagc atccagagaa atttcgattc 1740agttaaaatt gcatatataa ccagagaaac aaattcagat gttagtcagt ccagctacat 1800aggtcaatgc ctgagagttt aaaagaatcc gtatccttaa gcataagtag gtattgaggt 1860gagttacaaa ggtaagttac cggttacgca ccacctccac caaacaagta tggttagaag 1920atacatgtaa tcgtttattt agagtactat ttataaaaaa ctttttaact agaaacagtt 1980gtttcatttt gatataaggt taattagaat tcccgagcaa gcaagaaggg gatatagagg 2040ataaggaggg cgagagagcg agagagagat gaaatcaatg gcgttatcaa actgcattcc 2100acagacacag tgcatgccat tgcacagcag cgggcattac aggggcaatt gtatcatgtt 2160gtcaattcca tgtagtttaa ttggaagacg aggttattat tcacataaga agaggaggat 2220gagcatgagt tgcagcacaa gctcaggctc aaagtcagcg gttaaagaag caggatcagg 2280atcaggatca ggagcaggag gattgctaga ctgcgtaatc gttggaggtg gaattagcgg 2340gctttgcatc gcgcaggctc tttgtacaaa acagtcctct ttatccccaa attttatagt 2400gacagaggcc aaagacagag ttggcggcaa catcgtcact gtggaggccg atggctatat 2460ctgggaggag ggacccaata gcttccagcc ttccgacgcg gtgctcacca tggcggtaat 2520tctgtctctt cattattcat aatcataatt caattcaatt caattcctaa cgtggaatgt 2580ggaatgtggc atgtgcgtag gtcgac 2606＜210〉27＜211〉31＜212〉DNA＜213〉＜220〉＜223〉：

Oligonucleotide＜220〉＜221〉misc_ feature＜222〉(4) .. (9)＜223〉EcoRI restriction site＜400〉27gcggaattca tacttattta tcattagaaa g 31＜210〉28＜211〉32＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description:

Oligonucleotide＜220〉＜221〉misc_ feature＜222〉(4) .. (9)＜223〉XbaI restriction site＜400〉28gcgtctagaa agaactaaat actatatttc ac 32＜210〉29＜211〉30＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description:

Oligonucleotide＜220〉＜221〉misc_ feature＜222〉(4) .. (9)＜223〉NcoI restriction site＜400〉29gcgccatggt aaatgaaaga aagaactaaa 30＜210〉30＜211〉30＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description:

Oligonucleotide＜220〉＜221〉misc_ feature＜222〉(4) .. (9)＜223〉XbaI restriction site＜400〉30gcgtctagat caaccgaaat tcaattaagg 30＜210〉31＜211〉27＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description:

Oligonucleotide＜220〉＜221〉misc_ feature＜222〉(4) .. (9)＜223〉HindIII restriction site＜400〉31cgcaagcttc aatggaagca atgataa 27＜210〉32＜211〉36＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description:

Oligonucleotide＜400〉32gggagtccct gatgattaaa taaaccaaga ttttac 36＜210〉33＜211〉40＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description:

Oligonucleotide＜400〉33catggtaaaa tcttggttta tttaatcatc agggactccc 40＜210〉34＜211〉32＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description:

Oligonucleotide＜220〉＜221〉misc_ feature＜222〉(5) .. (10)＜223〉NcoI restriction site/ATG initiator codon＜400〉34gggaccatgg attgtgtgat tgtcggcgga gg 32＜210〉35＜211〉24＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description:

Oligonucleotide＜400〉35ctccgctctc cagcttagtg atac 24＜210〉36＜211〉633＜212〉DNA＜212〉DNA＜213〉sugarcane＜220〉＜221〉CDS＜222, (3) .., (305)＜223〉sugarcane protox-1 part encoding sequence＜400〉36tt tcc aag act gaa agt gag ctg gta gaa gca gtt gac cgt gac ctc, 47 Ser Lys Thr Glu Ser Glu Leu Val Glu Ala Val Asp Arg Asp Leu

1????????????5??????????????10???????????????15cgg?aaa?atg?ctt?ata?aat?cct?aca?gca?gtg?gac?cct?tta?gtc?ctt?ggt??95Arg?Lys?Met?Leu?Ile?Asn?Pro?Thr?Ala?Val?Asp?Pro?Leu?Val?Leu?Gly

20??????????????25???????????????30gtc?cga?gtt?tgg?cca?caa?gcc?ata?cct?cag?ttc?ctg?gta?gga?cat?ctt??143Val?Arg?Val?Trp?Pro?Gln?Ala?Ile?Pro?Gln?Phe?Leu?Val?Gly?His?Leu

Claims

1. nucleic acid molecule, it comprises that coding has the nucleotide sequence of the active modifying enzyme of proporphyrinogen oxidase (protox), wherein said modifying enzyme can tolerate the inhibitor of the natural existence form of described enzyme, and wherein said modifying enzyme comprises at least a in the following amino acid subsequence:

(a) KA Δ ₁₈F, wherein Δ ₁₈Be the amino acid of non-L-Ala;

(b) Q Δ ₁₉H, wherein Δ ₁₉The leucic amino acid of right and wrong;

(c) AP Δ ₁F, wherein Δ ₁The arginic amino acid of right and wrong;

(d) F Δ ₂S, wherein Δ ₂It is leucine;

(e) Y Δ ₃G, wherein Δ ₃It is Isoleucine;

(f) Δ ₇IG, wherein Δ ₇Be Histidine or L-Ala;

2. nucleic acid molecule according to claim 1, it comprises that coding has the nucleotide sequence of the active modifying enzyme of proporphyrinogen oxidase (protox), wherein said modifying enzyme can tolerate the inhibitor of the natural existence form of described enzyme, and wherein said modifying enzyme comprises at least a amino acid subsequence that is selected from following one group of amino acid subsequence:

(a) KA Δ ₁₈F, wherein Δ ₁₈Be the amino acid of non-L-Ala;

(b) Q Δ ₁₉H, wherein Δ ₁₉The leucic amino acid of right and wrong;

(c) AP Δ ₁F, wherein Δ ₁The arginic amino acid of right and wrong;

(d) F Δ ₂S, wherein Δ ₂It is leucine;

(e) Y Δ ₃G, wherein Δ ₃It is Isoleucine;

(f) Δ ₇IG, wherein Δ ₇Be Histidine or L-Ala;

(ii) wash among 2X SSC, the 1%SDS under 50 ℃.

3. one kind according to nucleic acid molecule any in claim 1 or 2, it comprises that coding has the nucleotide sequence of the active modifying enzyme of proporphyrinogen oxidase (protox), wherein said modifying enzyme can tolerate the inhibitor of the natural existence form of described enzyme, and wherein said modifying enzyme comprises at least a in the following amino acid subsequence:

(a) KA Δ ₁₈F, wherein Δ ₁₈Be the amino acid of non-L-Ala;

(b) Q Δ ₁₉H, wherein Δ ₁₉The leucic amino acid of right and wrong;

(c) AP Δ ₁F, wherein Δ ₁The arginic amino acid of right and wrong;

(d) Δ ₇IG, wherein Δ ₇It is Histidine;

4. nucleic acid molecule according to claim 1, wherein said modifying enzyme comprise amino acid subsequence KA Δ ₁₈F, wherein Δ ₁₈Be the amino acid of non-L-Ala, preferably Threonine or Xie Ansuan and Xie Ansuan especially.

5. nucleic acid molecule according to claim 1, wherein said modifying enzyme comprise amino acid subsequence Q Δ ₁₉H, wherein Δ ₁₉The leucic amino acid of right and wrong, Serine preferably.

6. nucleic acid molecule according to claim 1, wherein said modifying enzyme comprise amino acid subsequence AP Δ ₁F, wherein Δ ₁Be leucine or halfcystine.

7. nucleic acid molecule according to claim 1, wherein said modifying enzyme comprise amino acid subsequence Δ ₇IG, wherein Δ ₇Be Histidine or L-Ala, Histidine preferably.

8. nucleic acid molecule, it comprises that coding has the nucleotide sequence of the active modifying enzyme of proporphyrinogen oxidase (protox), wherein said modifying enzyme can tolerate the inhibitor of the natural existence form of described enzyme, and wherein said modifying enzyme comprises the first seed amino acid subsequence and further comprises at least a additional amino acid subsequence that is selected from following one group of amino acid subsequence:

(a) Q Δ ₁₁S, wherein Δ ₁₁Be the amino acid of non-proline(Pro);

(b) IGG Δ ₁₂, Δ wherein ₁₂Be the amino acid of non-Threonine;

(c) SWXL Δ ₁₃, Δ wherein ₁₃Be the amino acid of non-Serine;

(d) L Δ ₁₄Y, wherein Δ ₁₄Be the amino acid of non-l-asparagine;

(e) G Δ ₁₅XGL, wherein Δ ₁₅Be the amino acid of non-tyrosine.

9. nucleic acid molecule according to Claim 8, wherein said first kind of subsequence is sequence any among the claim 1-7.

According to Claim 8 with 9 in any described nucleic acid molecule, wherein said additional subsequence is the Q Δ ₁₁S, wherein Δ ₁₁Be the amino acid of non-proline(Pro), leucine preferably.

11. according to Claim 8 with 9 in any described nucleic acid molecule, wherein said additional subsequence is the IGG Δ ₁₂, Δ wherein ₁₂Be the amino acid of non-Threonine, preferably Isoleucine or L-Ala.

12. according to Claim 8 with 9 in any described nucleic acid molecule, wherein said additional subsequence is the SWXL Δ ₁₃, Δ wherein ₁₃Be the amino acid of non-Serine, leucine preferably.

13. according to Claim 8 with 9 in any described nucleic acid molecule, wherein said additional subsequence is the L Δ ₁₄Y, wherein Δ ₁₄Be the amino acid of non-l-asparagine, Serine preferably.

14. according to Claim 8 with 9 in any described nucleic acid molecule, wherein said additional subsequence is the G Δ ₁₅XGL, wherein Δ ₁₅Be the amino acid of non-tyrosine, halfcystine preferably.

15. according to any described nucleic acid molecule among the claim 1-9, wherein said modifying enzyme comprises: amino acid subsequence Δ ₇IG, wherein Δ ₇Be the amino acid of non-tyrosine, Isoleucine preferably; Amino acid subsequence IGG Δ ₁₂, Δ wherein ₁₂Be the amino acid of non-Threonine, Isoleucine preferably; With amino acid subsequence SWXL Δ ₁₃, Δ wherein ₁₃Be the amino acid of non-Serine, leucine preferably.

16. one kind according to any described nucleic acid molecule among claim 1 and the 8-9, it comprises that separation has the nucleotide sequence of the active modifying enzyme of proporphyrinogen oxidase (protox) from the coding of a kind of plant, wherein said modifying enzyme can tolerate the inhibitor of naturally occurring protox enzyme, and wherein said nucleotide sequence is further characterized in that and satisfies one of following condition at least:

(e) described nucleotide sequence has coded amino acid subsequence Y Δ ₃The sequence of G, wherein Δ ₃Be the amino acid of non-L-Ala, and preferably halfcystine or Isoleucine, more preferably be Isoleucine; And described nucleotide sequence also has the sequence that coding one of is selected from the following subsequence group:

(f) described nucleotide sequence has coded amino acid subsequence Δ ₇The sequence of IG, wherein Δ ₇Be the amino acid of non-tyrosine, and preferably Threonine, L-Ala or Histidine, more preferably be L-Ala or Histidine and most preferably be Histidine; And described nucleotide sequence also has the sequence of coding one of in the following subsequence group:

(g) described nucleotide sequence has coded amino acid subsequence T Δ ₁₆The sequence of G, wherein Δ ₁₆The leucic amino acid of right and wrong; And described nucleotide sequence also has coded amino acid subsequence YV Δ ₁₇The sequence of G, wherein Δ ₁₇Be the amino acid of non-L-Ala;

(h) KA Δ ₁₈F, wherein Δ ₁₈Be the amino acid of non-L-Ala;

(i) Q Δ ₁₉H, wherein Δ ₁₉The leucic amino acid of right and wrong.

17. the nucleic acid molecule according to claim 16, wherein said subsequence coded amino acid subsequence T Δ ₁₆G, wherein Δ ₁₆The leucic amino acid of right and wrong, Serine preferably; And described nucleotide sequence also has coded amino acid subsequence YV Δ ₁₇The sequence of G, wherein Δ ₁₇Be the amino acid of non-L-Ala, Threonine preferably.

18. nucleic acid molecule according to claim 1, its coding comprises the proporphyrinogen oxidase (protox) of the modification of plant protox, wherein the arginine that occurs on the position that is equivalent to SEQ ID NO:6 the 88th amino acids is by another kind of aminoacid replacement, preferably replaced by leucine, and the protox of wherein said modification tolerance can suppress the weedicide of the active consumption of naturally occurring protox.

19. the nucleic acid molecule according to claim 1, its coding comprises the proporphyrinogen oxidase (protox) of the modification of plant protox, and wherein the halfcystine that occurs on the position that is equivalent to SEQ ID NO:6 the 159th amino acids is replaced by leucine.

20. nucleic acid molecule according to claim 1, its coding comprises the proporphyrinogen oxidase (protox) of the modification of plant protox, wherein the L-Ala that occurs on the position that is equivalent to SEQ ID NO:6 the 175th amino acids is by another kind of aminoacid replacement, preferably by Xie Ansuan or Threonine replacement, and the protox tolerance of wherein said modification can suppress the weedicide of the active consumption of naturally occurring protox.

21. nucleic acid molecule according to claim 1, its coding comprises the proporphyrinogen oxidase (protox) of the modification of plant protox, wherein the leucine that occurs on the position that is equivalent to SEQ ID NO:6 the 337th amino acids is by another kind of aminoacid replacement, preferably replaced by Serine, and the protox of wherein said modification tolerance can suppress the weedicide of the active consumption of naturally occurring protox.

22. nucleic acid molecule according to claim 1, its coding comprises the proporphyrinogen oxidase (protox) of the modification of plant protox, and wherein the tyrosine that occurs on the position that is equivalent to SEQ ID NO:16 the 428th amino acids is replaced, preferably replaced by Histidine by Histidine or L-Ala.

23. nucleic acid molecule according to claim 1, its coding comprises the proporphyrinogen oxidase (protox) of the modification of plant protox, and wherein the L-Ala that occurs on the position that is equivalent to SEQ ID NO:2 the 220th amino acids is replaced by Isoleucine or tyrosine.

24. the nucleic acid molecule according to claim 1, its coding comprises the proporphyrinogen oxidase (protox) of the modification of plant protox, and wherein the tyrosine that occurs on the position that is equivalent to SEQ ID NO:2 the 426th amino acids is replaced by L-Ala.

25. one kind according to nucleic acid molecule any among claim 1 and the 8-9, its coding comprises the proporphyrinogen oxidase (protox) of the modification of plant protox, and especially a kind of coding comprises the dna molecular of the proporphyrinogen oxidase (protox) of the modification of plant protox; Described plant protox has the replacement of first seed amino acid and second seed amino acid replaces; First seed amino acid replaces the characteristic with generation inhibitor of anti-protox; And the replacement of second seed amino acid has raising is replaced the tolerance that produces by first seed amino acid characteristic; Wherein said plant is selected from corn, wheat, soybean, cotton, beet, rape, rice, Chinese sorghum, sugarcane and mouse ear mustard and belongs to the group of forming.

26. the nucleic acid molecule according to claim 25, wherein the replacement of first seed amino acid occurs on the position that is selected from following one group of position:

(a) be equivalent to the arginic position of SEQ ID NO:6 the 88th amino acids;

(c) be equivalent to the leucic position of SEQ ID NO:6 the 337th amino acids.

27. particularly preferably be a kind of dna molecular, wherein the replacement of first seed amino acid occurs on the position that is selected from following one group of position:

(a) be equivalent to the arginic position of SEQ ID NO:6 the 88th amino acids;

28. the nucleic acid molecule according to claim 27, wherein the replacement of first seed amino acid occurs on the position that is selected from following one group of position:

(a) be equivalent to the arginic position of SEQ ID NO:6 the 88th amino acids;

Wherein second seed amino acid replaces on the position that occurs in the group that is selected from following one group of position composition:

29. the nucleic acid molecule according to claim 1, it has dual aminoacid replacement, and one of them aminoacid replacement occurs in and is equivalent on the 347th leucic position of SEQ ID NO:6; And wherein second aminoacid replacement occurs on the position that is equivalent to the 453rd L-Ala of SEQ ID NO:6.

30. the nucleic acid molecule according to claim 29, it has dual aminoacid replacement and is wherein replaced by Serine at the leucine that is equivalent to occur on SEQ ID NO:6 the 347th amino acids position; And wherein replaced by Threonine at the L-Ala that is equivalent to occur on SEQ ID NO:6 the 453rd amino acids position.

31. according to any described nucleic acid molecule among the claim 1-30, wherein said plant is selected from the group that mouse ear mustard genus, corn, wheat, soybean, cotton, beet, rape, rice, Chinese sorghum and sugarcane are formed, preferably corn or cotton.

32. one kind has the active modifying enzyme of proporphyrinogen oxidase (protox), wherein said modifying enzyme by among the claim 1-30 any one described nucleotide sequence coded.

33. one kind is included in the mosaic gene that has the active promotor that is operably connected with any described nucleic acid molecule among the claim 1-30 in the plant.

34. recombinant vectors that comprises the described mosaic gene of claim 33.

35. vegetable cell that comprises any described nucleic acid molecule among the claim 1-30.

36. a kind of plant, plant tissue, vegetable cell or plant seed, comprise its filial generation, they comprise any described nucleic acid molecule among the claim 1-30, and wherein said nucleic acid molecule is expressed in described plant, plant tissue, vegetable cell or plant seed or its filial generation thus produces tolerance to the active inhibitor of naturally occurring protox.

37. the plant according to claim 36, plant tissue, vegetable cell or plant seed, comprise its filial generation, wherein said plant, plant tissue, vegetable cell or plant seed or its filial generation are selected from the group that mouse ear mustard genus, sugarcane, soybean, barley, cotton, tobacco, beet, rape, corn, wheat, Chinese sorghum, rye, oat, mud grass and forage grass, millet, forage grass and rice are formed.

38. the plant according to claim 36, plant tissue, vegetable cell or plant seed, comprise its filial generation, wherein said plant, plant tissue, vegetable cell or plant seed or its filial generation are selected from the group that mouse ear mustard genus, soybean, cotton, beet, rape, corn, wheat, Chinese sorghum, rice and sugarcane are formed.

39. a kind of plant, plant tissue, vegetable cell or plant seed, comprise its filial generation, they comprise any described nucleic acid molecule among the claim 1-30, and wherein said plant, plant tissue, vegetable cell or plant seed or its filial generation are selected from the group that mouse ear mustard genus, soybean, cotton, beet, rape, corn, wheat, Chinese sorghum, rice and sugarcane are formed.

40. the mosaic gene according to claim 33, it be included in work in the plant plastid operationally with claim 1-30 in any promotor that described nucleic acid molecule is connected.

41. the mosaic gene according to claim 40, the described promotor that wherein works in plant plastid is the clpP gene promoter.

42. plastid conversion carrier that comprises any described nucleic acid molecule among the claim 1-30.

43. plant plastid that comprises the described plastid conversion carrier of claim 42.

44. a kind of plant, plant tissue, vegetable cell or plant seed, comprise its filial generation, they comprise the plant plastid of claim 43, and the plant protox enzyme of wherein said modification is expressed in described plant, plant tissue, vegetable cell or plant seed and described plant, plant tissue, vegetable cell or plant seed produce tolerance to the active inhibitor of naturally occurring protox thus.

45. a method that is used to control unwanted vegetation growth, this method comprise the step of weedicide of using the inhibition protox of significant quantity to the plant population of containing vegetable cell any in claim 35-39 and 44.

46. a method that is used to control unwanted vegetation growth, this method comprise that the plant population to the plant plastid that contains claim 43 uses the step of weedicide of the inhibition protox of significant quantity.

47. one kind according to method any in claim 45 and 46, the weedicide of wherein said inhibition protox is selected from aryi-uracile (aryluracil), phenyl ether, oxidiazole, imide, phenylpyrazole, pyridine derivate, 3-and replaces-2-aryl-4,5,6, the O-Phenylpyrrolidine of 7-tetrahydrochysene indazole, phenopyrate and described phenopyrate also-and the similar thing of piperidino-(1-position only) carbamate.

48. the method according to claim 47, the weedicide of wherein said inhibition protox are the imide with general formula V, VI, VII, VIIa, VIII, IX, IXa or IXb.

49. the method according to claim 47, the weedicide of wherein said inhibition protox are the pyridyl pyrazoles with general formula X XIIIa or XXIIIb.

50. the method according to claim 47, the weedicide of wherein said inhibition protox are the phenylpyrazoles with general formula X XIV.

51. one kind is used for the method for weed growth that selectivity suppresses to contain the field of the farm crop of crop seeds of plantation or plant, this method comprises the following steps:

(a) farm crop or the crop seeds of plantation herbicide-resistant, they comprise the vegetable cell any in claim 35-39 and 44 or the plant plastid of claim 43; With

52. nucleic acid molecule, it comprises that coding has the nucleotide sequence of the active modifying enzyme of proporphyrinogen oxidase (protox), wherein said modifying enzyme tolerates the inhibitor of the natural existence form of described enzyme, and wherein said modifying enzyme comprises at least a amino acid subsequence that is selected from following one group of amino acid subsequence:

(a) KA Δ ₁₈F, wherein Δ ₁₈Be the amino acid of non-L-Ala;

(b) Q Δ ₁₉H, wherein Δ ₁₉The leucic amino acid of right and wrong;

(c) AP Δ ₁F, wherein Δ ₁It is leucine;

(d) Δ ₇IG, wherein Δ ₇It is Histidine;

(ii) wash among 2X SSC, the 1%SDS under 50 ℃.

53. according to the described nucleic acid molecule of claim 52, the nucleotide sequence of the natural existence form of the described enzyme of wherein encoding derives from a kind of plant.

54. one kind has the active modifying enzyme of proporphyrinogen oxidase (protox), wherein said modifying enzyme is described nucleotide sequence coded by claim 52.