CN1820073A - Plants with increased levels of one or more amino acids - Google Patents
Plants with increased levels of one or more amino acids Download PDFInfo
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- CN1820073A CN1820073A CN 200480019478 CN200480019478A CN1820073A CN 1820073 A CN1820073 A CN 1820073A CN 200480019478 CN200480019478 CN 200480019478 CN 200480019478 A CN200480019478 A CN 200480019478A CN 1820073 A CN1820073 A CN 1820073A
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Abstract
The present invention provides DNA constructs comprising exogenous polynucleotides encoding a threonine deaminase and/or AHAS. Transgenic plants transformed with the constructs, as well as seed and progeny dervied from these plants, are also provided. The transgenic plants have an increased level of one or more amino acids as compared to a non-transgenic plant of the same species.
Description
The present invention requires the U.S. Provisional Application No.60/468 with submission on May 7th, 2003, and 727 as right of priority, in this mode by reference this application is included in this paper.
The field of the invention is Agricultural biotechnologies.More specifically, the present invention relates to increase the biotechnological means of amino acid levels in the plant.
A variety of important crops comprise soybean and corn, all do not contain sufficient amount or reach the some seed amino acids that nutrition is perfect that make of correct balance.(Branched Chained amino acids, BCAA) leucine, Isoleucine and Xie Ansuan are especially in this way for branched amino acid.BCAA is necessary amino acid, because the mankind can not synthesize these molecules, therefore must obtain from meals.Isoleucine is synthetic band branched-chain amino acid from Threonine.Threonine self is synthetic next from aspartic acid again.Synthesis path between aspartic acid and the BCAA relates to some kinds of enzymes, and the allosteric that described enzyme is subjected to some seed amino acids suppresses.Comprise E.C. 2.7.2.4. (AK), difunctional E.C. 2.7.2.4.-homoserine dehydrogenase (AK-HSDH), isopropylmalate synthase, threonine deaminase (TD) and acetohydroxy acid synthase (AHAS) at enzyme to use in BCAA synthetic.Particularly, threonine deaminase (EC 4.2.1.16) (TD, threonine dehydra(ta)se; L-Threonine lytic enzyme (deaminizating)) and acetohydroxy acid synthase (AHAS; Acetolactate synthase (EC 4.1.3.18)) be the key enzyme in the BCAA biosynthetic process.
In E.coli, threonine deaminase exists with independent biosynthesizing form and biological degradation form.The biosynthesizing form of threonine deaminase is a gene ilvA coding, first committed step of band branched-chain amino acid biosynthetic process in its catalysis plant and the microorganism.This step makes L-Threonine dehydration deamination, utilize pyridoxal 5 '-phosphoric acid (PryP) produces the 2-oxy butyrate.The allosteric that the threonine deaminase of biosynthesizing form is subjected to being undertaken by the L-Isoleucine is regulated (Umbarger, Science, 123:848 (1956); Umbarger, Protein Science, 1:1392 (1992); Changeux, Cold Spring Harbor Symp.Quant.Biol., 26:313 (1961); Monod et al., J.Mol.Biol., 6:306 (1963)).The some kinds of threonine deaminases that gone to regulate all exist in plant and bacterium.See Feldberg et al., Eur.J.Biochem., 21:438-446 (1971); Mourad et al., Plant Phys., 107:43-52 (1995); Fisher et al., J.Bact., 175:6605-6613 (1993); Taillonet al., Gene, 63:245-252 (1988); M ckel et al., Mol.Microbiol., 13:833-842 (1994); Guillouet et al., Appl Environ Microbiol., 65:3100-3107 (1999); Slater et al., Nature Biotechnology, 7:1011-1016 (1999).
Opposite with the biosynthesizing form, the biological degradation form of threonine deaminase can be activated by AMP, and its feedback regulation to the L-Isoleucine is insensitive, and it produces in the anaerobic mode containing high-purity amino acid and do not contain in the substratum of glucose.In addition, in E.coli, the biological degradation form of threonine deaminase is encoded by independent gene (tdcB).
The AHAS enzyme is all guarded in large number of biological, for example, and bacterium, yeast and plant (Singhet al., Proc.Natl.Acad.Sci., 88:4572-4576 (1991)).In E.coli and other enterobacteria, AHAS is by two big subunits and two allos tetramer protein (Weinstock et al., J.Bacteriol., 174:5560-6 (1992)) that small subunit is formed of being called as ilvG and ilvM respectively.These tetrameric whole enzymic activitys are all provided by big subunit.Small subunit is that the stability and the regulation and control purpose of enzyme is needed.In plant, the gathering situation between different plant species is different.In some plants, Arabidopsis thaliana for example, the AHAS enzyme is by single structure genes encoding (Andersson et al., Plant Cell Reports, 22:261-267 (2003)), and in other plant species, for example in the tobacco, a more than functional gene may just be arranged.The same with bacterium, the AHAS enzyme of plant also is subjected to feedback inhibition.The AHAS enzyme of plant is the target (U.S. patent 6,727,414) of some commercial herbicides.
To carrying out aspect the equilibrated as on the one hand leucine and Xie Ansuan and as the level of on the other hand Isoleucine, AHAS plays an important role.AHAS is very important for driving pyruvic acid to acetolactic conversion, and acetylactis is the precursor of leucine and Xie Ansuan.AHAS also drives the conversion of 2-oxy butyrate to the acetyl hydroxybutyric acid, and the latter is the precursor of Isoleucine.Because AHAS is higher than pyruvic acid for the substrate priority of 2-oxy butyrate, so the production of Isoleucine is preferentially carried out in enzyme reaction.The level of Isoleucine can be subjected to the control of Isoleucine to the feedback inhibition of TD, and AHAS is subjected to Xie Ansuan and leucic feedback inhibition.Leucic production also is subjected to the feedback inhibition of isopropylmalate synthetase.
Commercially producing of BCAA can be undertaken by directly extract amino acid from protein hydrolyzate.For example, the present production level of Isoleucine is less than annual 400 tonnes, and for the demand of Isoleucine in sustainable growth.Therefore, for remedying the shortage through isolating BCAA, and provide its more economical source, people need carry out engineered to plant, so that the synthetic level raising of amino acid.
Summary of the invention
The present invention includes a kind of DNA construct, described construct comprises a plurality of expression of plants boxes, wherein first expression cassette is included in the promotor that function is arranged in the vegetable cell, it is operably connected to the exogenous polynucleotide of feeding back insensitive threonine deaminase with coding, second expression cassette is included in the promotor that function is arranged in the vegetable cell, and its exogenous polynucleotide with coding AHAS is operably connected.In one embodiment, DNA construct of the present invention comprises a plurality of expression of plants boxes, wherein first expression cassette is included in the promotor that function is arranged in the vegetable cell, it is operably connected to the exogenous polynucleotide of feeding back insensitive threonine deaminase with coding, second expression cassette comprises the big subunit of AHAS, the 3rd expression cassette is included in the promotor that function is arranged in the vegetable cell, and its exogenous polynucleotide with coding AHAS small subunit is operably connected.In one embodiment, each in the promotor all is seed enhancement type (seedenhanced) promotor.In another embodiment, each in the promotor all is selected from the following group that constitutes: napin, 7S alpha, 7S alpha ', 7S beta, USP 88, enhanced USP 88, Arcelin 5 and Oleosin.In one embodiment, have two kinds of different seed enhancement type promotors at least.
In one aspect of the invention, first box comprises coding to feeding back the polynucleotide of insensitive threonine deaminase, and it comprises SEQ ID NO:22.In one embodiment, these polynucleotide are SEQ ID NO:22.In another aspect of the present invention, first box comprises the exogenous polynucleotide of coding threonine deaminase variation allelotrope (variant allele) or its subunit, and described variation allelotrope or its subunit comprise the aminoacid replacement on L447F or L481F or L481Y or L481P or L481E or L481T or L481Q or L481I or L481V or L481M or the L481K position.Of the present invention aspect another, the coding threonine deaminase allelic polynucleotide that make a variation comprise SEQ ID NO:2.Aspect another, these polynucleotide are SEQ ID NO:2 of the present invention.
In one embodiment of the invention, first box also comprises the polynucleotide of the plastid transit peptides of encoding, and its polynucleotide with coding threonine deaminase, threonine deaminase variation allelotrope or its subunit are operably connected.
In another embodiment, second expression cassette comprises the polynucleotide of the big subunit of coding AHAS.In one embodiment, the polynucleotide of the big subunit of coding AHAS comprise SEQ IDNO:16.In one embodiment, these polynucleotide are SEQ ID NO:16.In another embodiment, the polynucleotide of coding plastid transit peptides are operably connected with the polynucleotide of the big subunit of coding AHAS.In one embodiment, the 3rd expression cassette comprises the polynucleotide of coding AHAS small subunit.In another embodiment, the polynucleotide of coding AHAS small subunit comprise SEQ ID NO:17.In one embodiment, these polynucleotide are SEQ ID NO:17.In another embodiment, the polynucleotide of coding plastid transit peptides are operably connected with the polynucleotide of coding AHAS small subunit.
In one aspect, DNA construct comprises a plurality of expression of plants boxes, wherein first expression cassette is included in the promotor that function is arranged in the vegetable cell, it is operably connected to the exogenous polynucleotide of feeding back insensitive threonine deaminase with coding, second expression cassette is included in the promotor that function is arranged in the vegetable cell, and its exogenous polynucleotide with the big subunit of coding AHAS is operably connected.In yet another aspect, each in the promotor all is seed enhancement type promotor.In yet another aspect, each in the promotor all is selected from the following group that constitutes: napin, 7Salpha, 7S alpha ', 7S beta, USP 88, enhanced USP 88, Arcelin 5 and Oleosin.In yet another aspect, have two kinds of different seed enhancement type promotors in the construct at least.
In one embodiment, first box comprises coding to feeding back the polynucleotide of insensitive threonine deaminase, and it comprises SEQ ID NO:22.In one embodiment, these polynucleotide are SEQ ID NO:22.In another embodiment, first box comprises threonine deaminase variation allelotrope, and it comprises the aminoacid replacement on L447F or L481F or L481Y or L481P or L481E or L481T or L481Q or L481I or L481V or L481M or the L481K position.In another embodiment, the coding threonine deaminase allelic polynucleotide that make a variation comprise SEQ ID NO:2, and it comprises the aminoacid replacement on L447F or L481F or L481Y or L481P or L481E or L481T or L481Q or L481I or L481V or L481M or the L481K position.In one embodiment, these polynucleotide are SEQ ID NO:22.In one aspect of the invention, first box comprises the polynucleotide of the plastid transit peptides of encoding, and its described polynucleotide with the coding threonine deaminase are operably connected.In yet another aspect, second expression cassette comprises the polynucleotide of the big subunit of coding AHAS.In yet another aspect, the polynucleotide of the big subunit of coding AHAS comprise SEQ ID NO:16.In one embodiment, these polynucleotide are SEQ ID NO:16.Aspect another, the polynucleotide of coding plastid transit peptides are operably connected with the described polynucleotide of the big subunit of the described AHAS of coding.
In one embodiment, DNA construct comprises a plurality of expression of plants boxes, and one of them expression cassette is included in the promotor that function is arranged in the vegetable cell, and its exogenous polynucleotide with coding monomer A HAS is operably connected.In another embodiment, DNA construct comprises a plurality of expression of plants boxes, wherein first expression cassette is included in the promotor that function is arranged in the vegetable cell, its exogenous polynucleotide with the big subunit of coding AHAS is operably connected, second expression cassette is included in the promotor that function is arranged in the vegetable cell, and its exogenous polynucleotide with coding AHAS small subunit is operably connected.In another embodiment, each in the promotor all is seed enhancement type promotor.In another embodiment, each in the promotor all is selected from the following group that constitutes: napin, 7S alpha, 7S alpha ', 7S beta, USP 88, enhanced USP 88, Arcelin 5 and Oleosin.In another embodiment, have two kinds of different seed enhancement type promotors at least.In one embodiment, first box comprises the big subunit of AHAS, and it comprises SEQ ID NO:16.In one embodiment, these polynucleotide are SEQ ID NO:16.In another embodiment, first box comprises the polynucleotide of the plastid transit peptides of encoding, and its described polynucleotide with the big subunit of the described AHAS of coding are operably connected.In another embodiment, second box comprises the polynucleotide of coding AHAS small subunit.In another embodiment, second box comprises the polynucleotide of coding AHAS small subunit, and it comprises SEQ ID NO:17.In one embodiment, these polynucleotide are SEQ ID NO:17.In another embodiment, second box comprises the polynucleotide of the plastid transit peptides of encoding, and its described polynucleotide with the described AHAS small subunit of coding are operably connected.
The present invention also provides a kind of method, be used for preparing the transgenosis dicotyledons that the seed amino acid levels increases, described level increase is relatively from the seed of the non-transgenic plant of identical plant species, described method comprises the steps: a) to introduce the transgenosis that comprises construct to dicotyledons renewable (regenerable) cell, and described construct comprises coding to feeding back the polynucleotide of insensitive threonine deaminase; B) make that described reproducible cell regeneration is dicotyledons; C) obtain seed from described plant; D) select one or many seeds of the amino acid levels with increase, described level is relatively from the seed of the non-transgenic plant of identical plant species; And e) plant described seed, wherein, if Isoleucine exists with the level that increases, so at least a extra amino acid levels also has been increased.In one embodiment, described dicotyledons is soybean plants (soybeanplant).In one embodiment, amino acid whose level increase comprises following amino acid whose concentration increases: a) one or more among Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe and Ile; Or b) one or more among Arg, Asn, Asp, His, Met, Leu, Val, Gln, Tyr, Thr, Lys, Ala, Ser and the Phe.The present invention includes the genetically engineered soybean plant of making by present method.
The present invention includes the method for the transgenosis dicotyledons of preparation aminoacids content increase, comprise the steps: a) to introduce the transgenosis that comprises construct to the regenerable cell of dicotyledons, described construct comprises the polynucleotide of coding monomer A HAS, and perhaps described construct comprises the polynucleotide of the big subunit of coding AHAS and the polynucleotide of coding AHAS small subunit; B) make that described reproducible cell regeneration is dicotyledons; C) obtain seed from described plant; D) select one or many seeds of the amino acid levels with increase, described level is relatively from the seed of the non-transgenic plant of identical plant species; And e) plants described seed.In one embodiment, described dicotyledons is soybean plants or rape (canola) plant.In one embodiment, amino acid whose level increase comprises the concentration increase of Ser or Val.In one embodiment, the present invention includes the genetically engineered soybean plant of making by present method.
The present invention also comprises the break chop of making from genetically engineered soybean (meal).
The invention still further relates to the container that contains seed of the present invention.The seed of plant of the present invention can be placed in the container, for example, and in the sack.Container used herein is any object that can accommodate above-mentioned seed.Preferably, contain in the container and surpass about 1,000, about 5,000 or about 25,000 seeds, wherein, about at least 10%, about 25%, about 50%, about 75% or about 100% in the described seed is seed of the present invention.Preferably, when seed of the present invention was soybean, described container is sack preferably, wherein contained about 60 pounds or about 130,000 bean or pea.
The invention still further relates to the foodstuff products of animal or human's class that the part (for example, seed) from transgenic plant of the present invention or plant produces.This type of foodstuff products can from, for example, grain, cereal meal (meal), flour, seed, cereal wait and prepare, and comprise from the intermediate product of this type of material preparation.
Description of drawings
Fig. 1 is the restriction map of plasmid pMON53905.
Fig. 2 is the restriction map of plasmid pMON25666.
Fig. 3 is the restriction map of plasmid pMON53910.
Fig. 4 is the restriction map of plasmid pMON53911.
Fig. 5 is the restriction map of plasmid pMON53912.
Fig. 6 has showed the threonine deaminase (diamond symbols) of Arabidopsis and the dynamics of E.coli threonine deaminase (circle symbol) by the original speed figure of wild-type enzyme to Threonine concentration is provided.
Fig. 7 provides the enzyme of the relative Isoleucine concentration of E.coli L481 allelotrope chart of percentage comparison alive.
Fig. 8 is the restriction map of plasmid pMON69657.
Fig. 9 is the restriction map of plasmid pMON69659.
Figure 10 is the restriction map of plasmid pMON69660.
Figure 11 is the restriction map of plasmid pMON69663.
Figure 12 is the restriction map of plasmid pMON69664.
Figure 13 is the restriction map of plasmid pMON58143.
Figure 14 is the restriction map of plasmid pMON58138.
Figure 15 is the restriction map of plasmid pMON58159.
Figure 16 is the restriction map of plasmid pMON58162.
Explanation to nucleic acid and peptide sequence
SEQ ID NO:1 has showed the polynucleotide sequence of wild-type E.coli threonine deaminase.
SEQ ID NO:2 has showed the aminoacid sequence of wild-type E.coli threonine deaminase.
SEQ ID NO:3 has showed the aminoacid sequence (Ilv219) that replaces the wild-type E.coli threonine deaminase of Leu at 447 Phe.
SEQ ID NO:4 has showed the aminoacid sequence (Ilv466) that replaces the wild-type E.coli threonine deaminase of Leu at 481 Phe.
SEQ ID NO:5 has showed the aminoacid sequence that replaces the wild-type E.coli threonine deaminase of Leu at 481 Tyr.
SEQ ID NO:6 has showed the aminoacid sequence that replaces the wild-type E.coli threonine deaminase of Leu at 481 Pro.
SEQ ID NO:7 has showed the aminoacid sequence that replaces the wild-type E.coli threonine deaminase of Leu at 481 Glu.
SEQ ID NO:8 has showed the aminoacid sequence that replaces the wild-type E.coli threonine deaminase of Leu at 481 Thr.
SEQ ID NO:9 has showed the aminoacid sequence that replaces the wild-type E.coli threonine deaminase of Leu at 481 Gln.
SEQ ID NO:10 has showed the aminoacid sequence that replaces the wild-type E.coli threonine deaminase of Leu at 481 Ile.
SEQ ID NO:11 has showed the aminoacid sequence that replaces the wild-type E.coli threonine deaminase of Leu at 481 Val.
SEQ ID NO:12 has showed the aminoacid sequence that replaces the wild-type E.coli threonine deaminase of Leu at 481 Met.
SEQ ID NO:13 has showed the aminoacid sequence that replaces the wild-type E.coli threonine deaminase of Leu at 481 Lys.
SEQ ID NO:14 has showed the aminoacid sequence that replaces the L447F E.coli threonine deaminase of Leu at 447 Phe.
SEQ ID NO:15 has showed the aminoacid sequence that replaces the L481F E.coli threonine deaminase of Leu at 481 Phe.
SEQ ID NO:16 has showed the polynucleotide sequence of the big subunit of ilvG AHAS.
SEQ ID NO:17 has showed the polynucleotide sequence of ilvM AHAS small subunit.
SEQ ID NO:18 has showed ilvG 5 '-segmental polynucleotide sequence.
SEQ ID NO:19 has showed the polynucleotide sequence of Arabidopsis SSUlA plastid transit peptides.
SEQ ID NO:20 has showed ilvG 3 '-segmental polynucleotide sequence.
SEQ ID NO:21 has showed the variant amino acid sequence body of wild-type E.coli threonine deaminase.
SEQ ID NO:22 has showed the polynucleotide sequence of Arabidopsis OMR1 threonine deaminase.
Detailed Description Of The Invention
The invention provides a kind of genetically modified plants, its genome has following nucleic acid through separating, and described nucleic acid coding threonine deaminase (TD) or its subunit comprise mutant and subunit with enzyme function. Preferably, this type of threonine deaminase or threonine deaminase subunit have resistance to the inhibition that the amino acid analogue of free ILE or isoleucine causes. Perhaps, in another preferred embodiment, a kind of nucleic acid of encode threonine deaminase or its subunit is provided, its expression way so that: the content of one or more in the plant among Ile content and Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe increases, no matter threonine deaminase or the dynamics of its subunit or the similar and different part of rejection characteristic of natural and external source. For example, use technology well known in the art, can make external source threonine deaminase predominant expression in the cellular compartment that is different from the native enzyme whereabouts. The expression of threonine deaminase or its subunit can be with the level of Ile in the plant and one or more the level rise among Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe to the level that surpasses when not existing this type of to express. Described nucleic acid other enzyme that the isoleucine biosynthesis relates to of can also encoding, for example, aspartokinase, difunctional aspartokinase-homoserine dehydrogenase or acetohydroxy acid synthase.
The invention still further relates to a kind of method that obtains plant, one or more among Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe that the free Ile that the described plant energy level of production is enhanced and level are enhanced. This type of excessive production is through the introducing of the nucleic acid of the coding threonine deaminase that separates and the result of expression. In addition, natural soybean threonine deaminase is responsive to the feedback inhibition of ILE, and has consisted of the regulatory site to biosynthesis pathway. Method provided by the invention also can be used to: this type of feedback inhibition is had the nucleic acid introducing of the threonine deaminase of resistance by encoding, one or more among Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe that the free Ile that the level of production is enhanced in plant and level are enhanced. The nucleic acid of this type of coding threonine deaminase can be introduced in a series of plants, comprises dicotyledon (for example beans) and monocotyledon (such as grain).
Definition
In this context, a large amount of terms will be used. Term " polynucleotides ", " polynucleotide sequence ", " nucleotide sequence ", " nucleic acid fragment " all are used interchangeably in this article with " through the nucleic acid fragment that separates ". Above-mentioned term has comprised nucleotide sequence etc. Polynucleotides can be RNA or the DNA condensates of two strands or strand, and alternatively, it contains synthetic, the non-natural or nucleotide base through changing. The polynucleotides that occur with DNA condensate form can comprise one or more segments in cDNA, genomic DNA, synthetic DNA or its mixture.
Used herein in through the plant, plant tissue, plant part or the plant cell that transform " through what change " level of one or more among Ile and Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe refer to than in the corresponding higher level of finding in through the plant, plant tissue, plant part or the plant cell that transform or lower. Usually, " through what change " level of one or more among Ile and Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe will be higher than the level of finding in corresponding not plant, plant tissue, plant part or the plant cell through conversion.
Term " with ... complementation " be used to indicate in this article nucleic acid chains sequence can with all or part of hybridization of the polynucleotide sequence of conduct reference. For the purpose of explaining is given an example, nucleotide sequence " TATAC " and canonical sequence 5 '-TATAC-3 ' have 100% homogeny (identity), but itself and canonical sequence 5 '-GTATA-3 ' 100% complementation.
Term " corresponding " is used for the expression polynucleotide sequence in this article, for example nucleic acid at least in part with all or part of identical (unnecessary strict evolution is relevant) of the polynucleotide sequence of conduct reference.
" gone regulate enzyme " used herein refers to adorned enzyme, such as what undertaken by mutagenesis, brachymemma etc., so that metabolin is lowered the feedback inhibition degree of this enzymatic activity, thereby this enzyme can show the activity higher than not modified enzyme in the situation that metabolin exists.
The used phrase of the part relevant with threonine deaminase " its domain " has comprised the structural or functional segment of total length threonine deaminase herein. Structural domain comprises (identifiable) structure that threonine deaminase inside can be identified. The example of structural domain comprises alpha spiral, beta laminated structure, avtive spot, substrate or inhibitor binding site etc. The functional structure territory comprises the threonine deaminase segment that shows the function that can be identified, for example isoleucine binding pocket, avtive spot or substrate or inhibitor binding site. The functional structure territory of threonine deaminase comprise threonine deaminase can catalysis the part of a step in the isoleucine biosynthesis pathway. Therefore, the functional structure territory comprises the fragment with enzymatic activity and the domain of threonine deaminase. The mutant structure territory of threonine deaminase also is included. Wild type threonine deaminase nucleic acid for the manufacture of the mutant structure territory comprises, for example, any coding is from the nucleic acid of the threonine deaminase domain of Escherichia coli (Escherichia coli), salmonella typhimurium (Salmonella typhimurium) or arabidopsis (Arabidopsis thaliana).
" external source " used herein threonine deaminase is the threonine deaminase by the nucleic acid coding that is introduced in the process separation in the host cell. This type of " external source " threonine deaminase usually with this cell natural, the unconverted state in any dna sequence dna of existing not identical. " endogenous " or " natural " threonine deaminase is the natural threonine deaminase that is present in host cell or the biology.
In this article, " increase " or " being enhanced " level of in plant cell, plant tissue, plant part or the plant among free Ile and Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe one or more be unconverted plant cell, plant tissue, plant part or plant (namely, its genome is not transformed by the existence of external source threonine deaminase nucleic acid or its domain) in about 2 to 100 times level of the level found, be preferably about 5 to 50 times, more preferably about 10-30 doubly. For example, with through among free Ile in the vegetable seeds that conversion and Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe one or more level and unconverted the mother plant seed or compare with their level in the seed of chimeric (chimeric) plant unconverted. Discovery also has in the present invention described several amino acid whose title, trigram and single-letter abbreviation and the DNA codon that it is encoded is all listed in the table 1 in plant.
The several amino acid whose title that table 1. is found in plant, trigram and single-letter abbreviation and to its DNA codon of encoding
| Amino acid |
| 3 letter abbreviations | The single-letter abbreviation | The DNA codon | |
| Alanine | Ala | A | GCT，GCC，GCA，GCG |
| Arginine | Arg | R | CGT，CGC，CGA，CGG，AGA，AGG |
| Asparagine | Asn | N | AAT，AAC |
| Aspartic acid | Asp | D | GAT，GAC |
| Cysteine | Cys | C | TGT，TGC |
| Glutamic acid | Glu | E | GAA，GAG |
| Glutamine | Gln | Q | CAA，CAG |
| Glycine | Gly | G | GGT，GGC，GGA，GGG |
| Histidine | His | H | CAT，CAC |
| Isoleucine | Iso | I | ATT，ATC，ATA |
| Leucine | Leu | L | CTT，CTC，CTA，CTG，TTA，TTG |
| Lysine | Lys | K | AAA，AAG |
| Methionine | Met | M | ATG |
| Phenylalanine | Phe | F | TTT，TTC |
| Proline | Pro | P | CCT，CCC，CCA，CCG |
| Serine | Ser | S | TCT，TCC，TCA，TCG，AGT，AGC |
| Threonine | Thr | T | ACT，ACC，ACA，ACG |
| Tryptophan | Trp | W | TGG |
| Tyrosine | Tyr | Y | TAT，TAC |
| Valine | Val | V | GTT，GTC，GTA，GTG |
The nucleic acid of coding threonine deaminase and coding transit peptides (transit peptide) or the nucleic acid of mark/reporter gene are " through isolating ", because they from its natural origin, no longer are in their common cells that exists.This type of can pass through at least partially in external preparation or operation through isolating nucleic acid, for example, from their common found cells it is separated, and it is carried out purifying and amplification.Under situation about combining with exogenous nucleic acid, this type of can also be " reorganization " through isolating nucleic acid.For example, recombinant DNA can be to be the process separated DNA that endogenous promotor is operably connected with exogenous promoter or for the host cell of selecting for use.
" natural (native) " gene used herein or nucleic acid represent that this gene or nucleic acid do not pass through external change or operation, that is, it is " wild-type " gene or the nucleic acid that did not pass through in-vitro separation, purifying, amplification or sudden change.
Term " plastid " refers to the organoid of a class vegetable cell, and it comprises amyloplast, chloroplast(id), chromoplastid, elaiosome, eoplast, leucoplastid (etioplast), leukoplast and proplastid.Above-mentioned organoid is a self-replicating, and contains the ring-shaped DNA molecule that is commonly called " plastom ", and its size specifically depends on the kind of plant in about scope of 120 to about 217kb, and it contains reverse repeat region usually.
" polypeptide " used herein refers to the amino acid whose successive chain that all connects together by peptide bond, and only except the N-that has amino and carboxyl respectively the terminal and C-end amino acid, they are continuous with peptide bond.Polypeptide can be any length, and can be by post transcriptional modificaiton, for example, and the modification of being undertaken by glycosylation or phosphorylation.
" inhibition of the amino acid analogue of Isoleucine is had resistance or tolerance " used herein vegetable cell, plant tissue or plant are following vegetable cell, plant tissue or plant: wherein, under the situation that the analogue of L-Isoleucine or L-Isoleucine exists, the threonine deaminase specific activity corresponding wild type threonine deaminase of reservation is wanted as many as about less 10%.Usually; the vegetable cell that " inhibition of Isoleucine had resistance or tolerance ", plant tissue or plant can grow under the environment that has a certain amount of Isoleucine amino acid analogue; the amount of the amino acid analogue of described Isoleucine can suppress the growth of vegetable cell, plant tissue or the plant of unconverted usually, and this measures by methods known in the art.For example, with coding the dna molecular that the amino acid analogue of Isoleucine has the threonine deaminase of sufficient resistance or tolerance is gone to transform the inbreeding plant of isozygotying and backcrossing and transforming, the plant that obtains can grow meeting causes the environment of Isoleucine amino acid analogue of amount of inhibition to (promptly highly isogenic) regenerated inbreeding plant-growth accordingly under.
" inhibition of the amino acid analogue of Isoleucine or Isoleucine is had resistance or tolerance " used herein threonine deaminase is following threonine deaminase: when described when having the amino acid analogue of the threonine deaminase of tolerance/resistance and Isoleucine that the wild-type threonine deaminase is exposed to isodose or Isoleucine, the activity that described threonine deaminase with tolerance/resistance keeps is much more about more than 10% than corresponding " wild-type " or natural responsive type threonine deaminase.Preferably, the specific activity that keeps of described threonine deaminase with resistance or tolerance is much more about more than 20% than corresponding " wild-type " or natural responsive type threonine deaminase.
General notion
The threonine deaminase nucleic acid of electing in advance must at first pass through and separate, and, if not plant origin, modified external, in vegetable cell, carry out the needed adjustment signal of genetic expression to comprise.This foreign gene can be modified, to add the sequence of coding plastid transit peptide sequence, so that gene product is directed in the above-mentioned organoid.
The biosynthesizing of one or more among Ile and Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe for a change, the nucleic acid (" gene ") that coding has the threonine deaminase of resistance must directly or indirectly be incorporated in the vegetable cell, and identifies through cell transformed above-mentioned.This gene can comprise in the genome of vegetable cell into with being stabilized.The gene transcription signal should be able to be discerned by vegetable cell, and can have function in vegetable cell.This means that this gene must be transcribed into messenger RNA(mRNA), mRNA must be stable in plant nucleolus, can intactly be translated in the transporte to cells matter.This gene can have suitable translation signals to carry out correct translation by the identification of vegetable cell ribose body and to it.The polypeptide gene product must be avoided significant proteolysis attack in the tenuigenin, and the three-dimensional conformation that can have the enzymic activity of giving.Threonine deaminase can also act in the biosynthetic process of Isoleucine and derivative thereof further; This means that it can be positioned near the natural plant enzyme of synthetic (supposing in plastid) flank step of catalysis biological,, and transmit suitable product with the substrate that need to obtain.
Even above-mentioned all conditions can both reach, be not predictable thing to one or more the successfully excessive productions among Ile and Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe yet.This other controlling mechanism that need not have to compensate the minimizing of threonine deaminase step regulation and control exists.This means not only not exist other mechanism that does not also exist the ruined speed of the amino acid that can make accumulation to increase to biosynthetic inhibition.Among Ile and Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe one or more must be with can not be to the toxigenous level of plant by excessive production.At last, the characteristic that is introduced into should be stable, heritable, to allow to carry out business development and application.
The separation and the evaluation of polynucleotide molecule to the coding threonine deaminase
Can identify and separate the nucleic acid of coding threonine deaminase by standard method, as Sambrook et al., Molecular Cloning:A Laboratory Manual, ColdSpring Harbor, NY (2001) is described.The nucleic acid of coding threonine deaminase can be from any protokaryon or eucaryon species.For example, can be by the genome dna library from any species be screened, or, identify the nucleic acid of coding threonine deaminase or its subunit by the cDNA library that is made by the nucleic acid from particular cell types, clone, primary cell (primarycell) or tissue is screened.Can be used for identifying that the example with the library of separating threonine deaminase includes but not limited to, from A.tumefaciens strains A 348, cDNA library (the Stratagene of corn inbred lines B73, La Jolla, California, Cat.#937005, Clontech, Palo Alto, California, Cat.#FL1032a, #FL1032b and FL1032n), from the genomic library of corn inbred lines Mo17 (Stratagene, Cat.#946102), from the genomic library (Clontech of corn inbred lines B73, Cat.#FL1032d), or from the genomic library of the convenient bacterial strain of Escherichia coli or Salmonella typhimurium.
The example that can be used for implementing threonine deaminase polynucleotide of the present invention or peptide molecule is described in the table 2.E.coli wild-type threonine deaminase gene (ilvA) (SEQ ID NO:1; Gi:146450, number K03503, version K03503.1) and the variation allelotrope of corresponding peptide sequence (SEQ ID NO:2) or coding SEQ ID NO:21 be the basic gene that obtains following table 2 described all other mutation alleles.
Nucleic acid with sequence relevant with above-mentioned threonine deaminase nucleic acid molecule can obtain by standard method, comprise clone or polymerase chain reaction (PCR), use and threonine deaminase sequence area complementary Oligonucleolide primers provided herein among the described PCR.Can verify by hybridization, partial sequence analysis or by in proper host cell, expressing through the sequence of isolating threonine deaminase nucleic acid.
The aminoacid replacement situation of E.coli ilv threonine deaminase in table 2. mutation allele
| The threonine deaminase sudden change | Description to mutation allele | SEQ ID NO： |
| E.coli(wt ilvA) | Wild-type E.coli | 1 |
| E.coli(wt ilvA) | Wild-type E.coli | 2 |
| L447F(ilvA219) | The 447th Leu replaced by | 3 |
| L481F(ilvA466) | The 481st Leu replaced by | 4 |
| L481Y | The 481st Leu replaced by | 5 |
| L481P | The 481st Leu replaced by | 6 |
| L481E | The 481st Leu replaced by Glu | 7 |
| L481T | The 481st Leu replaced by Thr | 8 |
| L481Q | The 481st Leu replaced by | 9 |
| L481I | The 481st Leu replaced by Ile | 10 |
| L481V | The 481st Leu replaced by | 11 |
| L481M | The 481st Leu replaced by | 12 |
Screening to all or part of dna fragmentation of coding threonine deaminase sequence can be undertaken by PCR, perhaps by the hybridization means plaque from genome or cDNA library is screened and carries out.Probe can come the threonine deaminase gene since nucleic acid acquisition provided herein, perhaps from other biology.Perhaps, can at its with can with the combining of threonine deaminase specificity bonded antibody, the plaque from the cDNA expression library is screened.Can with from the dna fragmentation of other biological threonine deaminase probe hybridization and/or carry the plaque that has immunoreactive dna fragmentation with threonine deaminase antibody, can be advanced carrier by subclone and checked order, and/or, all or part of other cDNA or the genome sequence of coding target threonine deaminase gene are identified as probe.
The cDNA library can prepare by following method: separating mRNA, produce cDNA, and cDNA is inserted in the suitable carriers.Available have specific antibody or probe screens containing the segmental library of cDNA to threonine deaminase.The dna fragmentation of the part of coding threonine deaminase gene can be by subclone, and is checked order, and is used as probe, so that genome threonine deaminase nucleic acid is identified.By measuring the homology with other known threonine deaminase gene, or by with the hybridization of threonine deaminase specificity messenger RNA(mRNA), can verify the dna fragmentation of the part of coding protokaryon or eucaryon threonine deaminase.In case obtained the cDNA fragment of coding threonine deaminase 5 ' terminal portions, middle portion and 3 ' terminal portions, just they can be used as probe, to identify and to clone the complete genome group copy of threonine deaminase gene from genomic library.
Can isolate the part of the single or multiple genome copies of threonine deaminase gene by the polymerase chain reaction or by genomic library is screened.Positive colony can be checked order; can identify 5 ' end of gene by standard method; described standard method comprises to the nucleic acid homology of other threonine deaminase gene or by RNAase protection analysis to be carried out; as Sambrook et al.; Molecular Cloning:A Laboratory Manual; Cold Spring Harbor, NY (1989 and 2001) is described.To 3 ' and 5 ' of the target gene terminal also available known threonine deaminase coding region in location,, the genomic sequence data storehouse undertaken by being carried out computer search.In case identified the part of gene, obtain the complete copy of threonine deaminase gene with regard to the available standards method, described standard method comprises that clone or polymerase chain reaction (PCR) synthesize, and wherein use 5 ' or 3 ' terminal nucleic acid complementary Oligonucleolide primers with this gene.Can express by hybridization, partial sequence analysis or by threonine deaminase, verify the existence of the isolating total length copy of process of threonine deaminase gene.
Following mutant is wanted, described mutant threonine deaminase is active to be increased, the susceptibility of the feedback inhibition that Isoleucine or its analogue are caused reduces, and/or can produce one or more and Ile among Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe that quantity increases in plant.The said mutation body can have its active type of showing or the functional variation on the level, some the time be called as " derivative " of wild-type threonine deaminase nucleic acid and polypeptide.
Yet, the invention still further relates to the threonine deaminase nucleic acid and the threonine deaminase varient that have " silence " sudden change.Silent mutation used herein is meant the nucleotide sequence that has changed threonine deaminase but the sudden change that can not cause the aminoacid sequence of coded threonine deaminase to change to some extent.The varient threonine deaminase is by the mutant nucleic acid coding, and described varient has the amino acid change that one or more can not cause remarkable change to the threonine deaminase activity than corresponding wild type threonine deaminase.The present invention relates to all these analog derivatives, varient and have the threonine deaminase nucleic acid of silent mutation.
Can obtain to encode by several different methods and pass through the DNA of the threonine deaminase that suddenlys change, described threonine deaminase has resistance and/or tolerance to the amino acid analogue of L-Isoleucine or Isoleucine.Described method includes but not limited to:
1. idiovariation and in culture, carry out direct screening mutant;
2. the method that the tissue culture of plant or tissue, seed or any cell type is carried out direct or indirect mutagenesis;
3. the threonine deaminase gene that the clone is obtained suddenlys change, use therein method for example, chemomorphosis; Locus specificity or site-directed mutagenesis (Sambrook et al., as previously mentioned); Transposon-mediated mutagenesis (Berg et al., Biotechnology, 1:417 (1983)) and deletion mutantion (Mitra et al., Molec.Gen.Genetic., 215:294 (1989)).
4. appropriate design is carried out in the sudden change of Key residues; And
5.DNA reorganization (DNA shuffling) is to comprise interested sudden change into some kinds of threonine deaminase nucleic acid.
For example, can be used to the threonine deaminase mutant is reasonably designed from proteinic heredity of obtainable threonine deaminase and/or protein structure information, described mutant have the active of increase or the possibility that reduces to the susceptibility of Isoleucine or Isoleucine analogue very high.This type of protein structure information is obtainable, for example, and to (Gallagher et al., Structure, the 6:465-475 (1998)) of E.coli threonine deaminase.To the appropriate design of sudden change can by the threonine deaminase aminoacid sequence that will select with from known structure (for example, E.coli) the threonine deaminase aminoacid sequence of threonine deaminase is compared and is carried out.In conjunction with understanding, can predict proteinic Isoleucine combination of threonine deaminase and catalysis region to structural information and sequence homology.For example, the residue in the Isoleucine binding pocket can be confirmed as the possible candidate of following sudden change, and described sudden change is used to change enzyme to Isoleucine feedback inhibition resistance.Use this type of structural information, can Isoleucine in conjunction with involved site or structural domain in, appropriate design goes out some kinds of E.coli threonine deaminase mutant.More specifically, the Isoleucine feedback inhibition is had the still less sudden change of the active threonine deaminase of susceptibility for being used to make, with the similar amino acid of L481 in the E.coli threonine deaminase may be useful residue.The present invention relates to any aminoacid replacement or insertion in any above-mentioned position.Perhaps, the amino acid of above-mentioned any position can be lacked and is substituted.
Site-directed mutagenesis can be used to make aminoacid replacement, disappearance and the insertion on a series of sites.The example of the specific sudden change of making in Escherichia coli threonine deaminase coding region comprises following sudden change:
Near 447, replace Leu (seeing, for example SEQ ID NO:3) with Phe;
Near 481, replace Leu (seeing, for example SEQ ID NO:4) with Phe;
Near 481, replace Leu (seeing, for example SEQ ID NO:5) with Tyr;
Near 481, replace Leu (seeing, for example SEQ ID NO:6) with Pro;
Near 481, replace Leu (seeing, for example SEQ ID NO:7) with Glu;
Near 481, replace Leu (seeing, for example SEQ ID NO:8) with Thr;
Near 481, replace Leu (seeing, for example SEQ ID NO:9) with Gln;
Near 481, replace Leu (seeing, for example SEQ ID NO:10) with Ile;
Near 481, replace Leu (seeing, for example SEQ ID NO:11) with Val;
Near 481, replace Leu (seeing, for example SEQ ID NO:12) with Met; Or
Near 481, replace Leu (seeing, for example SEQ ID NO:13) with Lys;
Compare by aminoacid sequence and E.coli threonine deaminase aminoacid sequence, can carry out similar sudden change in the similar position of any threonine deaminase to the threonine deaminase that will be suddenlyd change.The example of the E.coli threonine deaminase aminoacid sequence that can be used for comparing is SEQ ID NO:1.
Can also select to identify useful mutant by the mutagenesis and the heredity of classics.Can be by enzyme being exposed to the amino acid analogue of free L-Isoleucine or Isoleucine, activity to the enzyme of genes encoding is surveyed, perhaps, come detecting function to sexually revise by using Restriction Enzyme collection of illustrative plates or dna sequence analysis that the variation of dna molecular is surveyed.
For example, can from clone, isolate the gene of the threonine deaminase that coding fully tolerates Isoleucine to Isoleucine analogue tolerance.In brief, under the situation that is exposed to low-level Isoleucine analogue continuously, the plant cell cultures of part differentiation is grown and gone down to posterity cultivation.Cultivate the concentration that increases the Isoleucine analogue on the interval gradually some going down to posterity then.Exist under the situation of analogue, the cell or tissue of growing under the situation to the analogue that is generally toxic level in the existence cultivation that repeats to go down to posterity, and its feature analyzed.Analyze by the following method by the stability of the resistance characteristics of cultured cells: under the situation that does not have analogue, the clone of selecting is carried out the cultivation of different time sections, analyze then tissue is exposed to growing state behind the analogue.Identify by the clone that has enzymic activity under the situation to the Isoleucine analogue that is generally toxic (that is, growth inhibitor) level in existence, thereby select the clone that relies on this advantage of threonine deaminase to have tolerance with change.
The available standards method, for example (by reference the document is comprised into this paper) described in the U.S. patent 4581847, to the threonine deaminase gene that obtains of clone from the clone that the Isoleucine analogue is had resistance, carry out about to same or other the detection of tolerance of amino acid analogue.
Clone with threonine deaminase that Isoleucine analogue susceptibility is reduced can be used to separate the threonine deaminase with feedback resistance.Can create the DNA library that has the clone of tolerance to the Isoleucine analogue, by hybridizing, can identify part or all dna fragmentation of coding threonine deaminase gene with the cDNA probe of a coding threonine deaminase gene part.By cloning process, or, can obtain the complete copy of reformed gene by using the PCR that suitable primer carries out synthetic.Separation to the reformed gene of coding threonine deaminase can be verified through in the plant transformed cell, and this carries out by the following method: measure the threonine deaminase of expressing and whether remain with enzymic activity when being exposed to the Isoleucine analogue that is generally toxic level.For example, see Anderson et al., U.S. patent 6,118,047.
For at selected biology, for example to express in the host cell of Xuan Ding plant or other type, the coding region of any dna molecular provided herein can be optimised.
U.S patent 5,492,660 and 5 at Gruys et al., 958,745, Asrar et al., U.S. patent 6,091,002 and 6,228,623 and Slater et al., Nature Biotechnology, among the 17:1011 (1999), also have making the description that Isoleucine removes the threonine deaminase varient regulated.
Transgenosis (transgene) and carrier
In case obtain and amplify the nucleic acid of coding (for example) threonine deaminase or its structural domain, it can be operably connected on the promotor, and, randomly, link to each other, to form transgenosis with other element.
It is promotor and the zone that can regulate and control genetic expression that most of genes have known.Typically, in prokaryotic cell prokaryocyte and eukaryotic cell, promoter region is found in the upstream of encoding sequence.Promoter sequence provides the regulation and control that the downstream gene sequence is transcribed, and, typically, comprise from about 50 to about 2000 nucleotide bases right.Promoter sequence also contains regulating and controlling sequence, enhancer sequence for example, and it can influence the level of genetic expression.Some can provide the genetic expression of heterologous gene (that is, with genes natural or that homologous gene is different) through isolating promoter sequence.Also known, promoter sequence can be strong or weak or induction type.Strong promoter can provide high-caliber genetic expression, and weak promoter only provides very low-level genetic expression.Inducible promoter is the promotor of following type: it responds to the reagent of external source adding or responds to environment or grow stimulates, and allows to open or close genetic expression.Promotor also can provide tissue specificity or developmental regulation.The genetic expression of enough levels can be provided, and can make the process cell transformed be easy to be detected with selecteed strong promoter and may be good.In addition, if necessary, this type of strong promoter can provide high-caliber genetic expression.
Promotor in transgenosis of the present invention can provide the expression to target gene, for example, and by the expression of nucleic acid threonine deaminase of coding threonine deaminase.Preferably, encoding sequence is expressed, make vegetable cell increase, thereby make the total content of one or more and Ile among Arg, Asn in the cell, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe increase the tolerance of the feedback inhibition of free L-Isoleucine.Promotor can also be an induction type, thereby can open or close genetic expression by the reagent that external source adds.With coding region with can in plant, provide the promotor of tissue specific expression or developmental regulation genetic expression to combine, also might make us expecting.
Can be used for promotor of the present invention includes but not limited to: the promotor of virus, plastid, bacterium, bacteriophage or plant.Useful promotor comprises: CaMV 35S promoter (Odellet al., Nature, 313:810 (1985)), CaMV 19S (Lawton et al., Plant Mol.Biol., 9:31F (1987)), nos (Ebert et al., Proc.Nat.Acad.Sci. (U.S.A.), 84:5745 (1987)), Adh (Walker et al., Proc.Nat.Acad.Sci (U.S.A.), 84:6624 (1987)), sucrose synthase (Yang et al., Proc.Nat.Acad.Sci (U.S.A.), 87:4144 (1990)), alpha-tubulin, napin, Actin muscle (Wang et al., Mol.Cell.Biol., 12:3399 (1992)), cab (Sullivan et al., Mol.Gen.Genet., 215:431 (1989)) PEPCase promotor (Hudspeth et al., Plant Mol Biol., 12:579 (1989)), 7S α ' ball polyprotein (conglycinin) promotor (Beachy et al., EMBOJ., 4:3047 (1985)), perhaps relevant those (Chandler et al. with the R gene complex, The Plant Cell, 1:1175 (1989)).Preferred promotor comprises seed enhancement type promotor, for example the 7s α ' of soybean, 7s α, lea9, Arabidopsis perl and Brassica napusnapin.Consider that other useful promotor also is that those skilled in the art are obtainable in enforcement of the present invention.
Can also use the plastid promotor.Most of plastogenes contain the promotor of RNA polymerase of the nuclear coding of the RNA polymerase (PEP) of the plastid coding that is useful on many subunits and single subunit.The consensus sequence of polysaccharase (NEP) promotor of nuclear coding can be at Hajdukiewicz et al. with the tabulation that is used for the specific promoter sequence of some kinds of natural plastogenes, EMBO J., 16:4041-4048 finds in (1997), and the document is all comprised into this paper by reference.
The example of spendable plastid promotor comprises Zea mays plastid RRN (ZMRRN) promotor.When having Arabidopsis thaliana plastid RNA polymerase, the ZMRRN promotor can drive expression of gene.Similarly can be used for promotor of the present invention is Glycinemax plastid RRN (SOYRRN) and Nicotiana tabacum plastid RRN (NTRRN) promotor.Above-mentioned whole three kinds of promotors can be discerned by Arabidopsis plastid RNA polymerase.The general feature of RRN promotor is described in U.S. patent 6218145 to some extent.
In addition, the repetition of transcriptional enhancer or enhanser can be used to increase the expression from specific promotor.The example of this type of enhanser includes but not limited to, from the element (Last et al., U.S. patent 5290924) of CaMV 35S promoter and octopine synthase gene.For example, expect, can the carrier that use according to the present invention be made up, to comprise into ocs enhancer element.This element is (the Ellis et al. that conduct is identified out from the palindrome (palindromic) enhanser of the 16bp of octopine synthase (ocs) gene of Agrobacterium at first, EMBOJ., 6:3203 (1987)), it is present in (Bouchez etal. at least 10 kinds of other promotors, EMBO J., 8:4197 (1989)).Propose, when being used for the monocotyledons conversion, use enhancer element, for example a plurality of copies of ocs element, particularly this element can improve the transcriptional level from promotor-proximal.Include but not limited to (the Conkling et al. of tissue-specific promoter of root cells promotor, Plant Physiol., 93:1203 (1990)) and (the Fromm et al. of tissue-specific enhancer, The Plant Cell, 1:977 (1989)), also be considered to useful especially, inducible promoter, for example ABA-and expansion-inducible promoter etc. also are like this.
In view of the dna sequence dna between transcription initiation site and the encoding sequence starting point (homing sequence that promptly is not translated) can influence genetic expression, people also may wish to use specific homing sequence.Preferred homing sequence be considered to comprise following these: comprise that expectation can guide those of sequence of the optimum expression of appended gene, promptly comprise and to increase or to keep mRNA stability also can prevent to translate by incorrect initial preferred total (consensus) homing sequence (Joshi, Nucl.Acid Res., 15:6643 (1987)).Those skilled in the art can carry out the selection to this type of sequence at an easy rate.From dicotyledons particularly the sequence that obtains of the soybean camber gene of expressing be preferred.
The nucleic acid of coding target gene (being threonine deaminase) also can comprise plastid transit peptides, with assistance the threonine deaminase polypeptide is transported in the plastid, for example, in the chloroplast(id).The nucleic acid of the plastid transit peptides that coding is selected is connected in mode in the frame (in-frame) with the encoding sequence of threonine deaminase usually.But plastid transit peptides can place the N-end or the C-end of threonine deaminase.
Construct also will comprise target nucleic acid and be positioned at 3 ' terminal nucleic acid that the latter brings into play the signal effect, stops transcribing, and allows the mRNA that obtains that poly-adenosineization takes place.The example of 3 ' element comprises those (the Bevan etal. from Agrobacterium tumefaciens octopine synthase gene, Nucl.Acid Res., 11:369 (1983)), suppress sub-I or suppress 3 ' end of sub-II gene from the terminator of the T7 transcription of Agrobacterium tumefaciens octopine synthase gene and from the proteolytic enzyme of potato or tomato, though other 3 ' element well known by persons skilled in the art also is admissible.If necessary, can also comprise controlling element, Adh introne 1 (Callis et al. for example, Genes Develop., 1:1183 (1987)), sucrose synthase intron (Vasil et al., Plant Physiol, 91:5175 (1989)) or TMV omega element (Galli et al., The Plant Cell, 1:301 (1989)).Can be according to An, Methodsin Enzymology, 153:292 (1987) is described, obtain these 3 ' untranslated regulating and controlling sequences, perhaps, above-mentioned sequence has been present in can be from the plasmid that commercial source obtains, for example from Clontech, Palo Alto, California obtains.Can 3 ' untranslated regulating and controlling sequence be operably connected with 3 '-end of threonine deaminase gene by standard method.Can be used for other this type of controlling element of the invention process and be to those skilled in the art obtaining spendable.
Selective marker gene or reporter gene also can be used for the present invention.This genoid is given unique phenotype to the cell of expressing this marker gene, therefore makes this type of can be come with the cell differentiation that does not contain this sign by cell transformed.The selective marker gene has given such specific character: people can pass through chemical process, promptly by using selective reagent (for example, weedicide, microbiotic etc.) to carry out " selection ".The gene that reporter gene maybe can screen is then given a specific character, makes people or to observe by test, promptly identifies by " screening " (for example R-locus characteristic).Certainly, a lot of examples of suitable marker gene all are known in the art, and can be used to enforcement of the present invention.
May be used for selective key of the present invention and include but not limited to, neo gene (Potrykus etal., Mol.Gen.Genet., 199:183 (1985)), its neomycin resistance of encoding can use Xin Meisu, card to receive mycin, G418 and wait and select; The bar gene, two propylamine phosphine (bialaphos) resistances of its coding; The gene of the EPSP synthase protein (Hinchee et al., Biotech., 6:915 (1988)) that the coding process changes, thus glyphosate resistance can be brought; Nitrilase gene, for example from the bxn of Klebsiella ozaenae, it can bring the resistance (Stalker et al., Science, 242:419 (1988)) to bromoxynil; Mutant acetolactic acid sy nthase gene (ALS), it can bring the resistance (EP 0154204) to imidazolone, sulfonylurea or other ALS inhibition chemical substance; Rheumatrex resistance DHFR gene (Thillet et al., J.Biol.Chem., 263:12500 (1988)); Dalapon dehalogenation enzyme gene, it can bring the resistance to sterilant dalapon; Or the threonine deaminase gene through suddenling change, it can bring the resistance to 5-methyl Isoleucine.When using the mutant epsp synthase gene, suitable plastid transit peptides or chloroplast transit peptides (CTP) should merge with the EPSPS coding region.
In one embodiment, selective key has resistance to the N-phosphonomethyl-glycine that is commonly called glyphosate.Glyphosate can suppress to cause aromatics (comprising amino acid and VITAMIN) is carried out biosynthetic shikimic acid pathway.Particularly, glyphosate suppresses the conversion to 5-enol pyruvic acid-3-phosphoric acid shikimic acid of phosphoenolpyruvic acid and 3-phosphoric acid shikimic acid by suppressing 5-enol pyruvic acid-3-phosphoric acid shikimic acid synthase (EPSP synthase or EPSPS).Show, can be incorporated in the Plant Genome, produce the plant (Shah et al., Science, 233:478-481 (1986)) of glyphosate tolerant by the ability that will produce higher levels of EPSP synthase (this enzyme preferably glyphosate tolerant).Isolated the varient that glyphosate is had the wild-type EPSPS enzyme of tolerance, described tolerance is the reformed result of EPSPS amino acid coding.See Kishore et al., Ann.Rev.Biochem., 57:627-663 (1988); Schulz et al., Arch.Microbiol., 137:121-123 (1984); Sost et al., FEBS Lett., 173:238-241 (1984); Kishore et al., Fed.Proc., 45:1506 (1986).
The nucleic acid of coding glyphosate tolerant EPSP synthase or glyphosate degrading enzyme is incorporated into makes plant that glyphosate is had tolerance in the plant.The method of making the glyphosate tolerant plant is obtainable, for example, U.S. patent 5776760 and 5627061 and WO 92/00377 in, the content of these several pieces of documents is comprised into herein by reference.
The another kind of illustrative embodiment that can be used for the selective marker gene of system that transformant is selected is the gene of the careless ammonium phosphinothricin acetyl transferring enzyme of coding, for example from the bar gene of Streptomyces hygroscopicus or from the pat gene (U.S. patent 5550318) of Streptomycesviridochromogenes.Grass ammonium phosphinothricin acetyl transferring enzyme (PAT) can make the two propylamine phosphines of weedicide, careless ammonium phosphine (phosphinothricin, PPT) the activeconstituents inactivation in.PPT suppresses glutamine synthetase (Murakami et al., Mol.Gen.Genet., 205:42 (1986); Twell et al., Plant Physiol., 91:1270 (1989)), cause the run-up of ammonia, and necrocytosis.
The spendable sign that screens includes but not limited to, beta-glucuronic acid Glycosylase or uidA gene (GUS), and the enzyme of this genes encoding is known multiple different chromogenic substrate; R-seat gene, the product (Dellaporta et al., in Chromosome Structure and Function, pp.263-282 (1988)) that its coding is regulated the generation of anthocyania pigment in the plant tissue (redness); β-Nei Xiananmei gene (Sutcliffe, Proc.Nat.Acad.Sci. (U.S.A.), 75:3737 (1978)), the known enzyme (for example, PADAC, colour developing cephamycin) that multiple different chromogenic substrates are arranged of its coding; XylE gene (Zukowsky et al., Proc.Nat.Acad.Sci. (U.S.A.), 80:1101 (1983)), its coding catechol deoxygenase, this enzyme can transform the colour developing catechol; Alpha-amylase gene (Ikuta et al., Biotech., 8:241 (1990)); Tyrosinase cdna (Katz et al., J.Gen.Microbiol., 129:2703 (1983)), its coding can be oxidized to tyrosine the enzyme of DOPA and DOPA quinone, and its then condensation again forms the compound melanochrome that detects easily; Beta-galactosidase gene, there is the enzyme of chromogenic substrate in its coding; Luciferase (lux) gene (Ow et al., Science, 234:856 (1986)), it makes noclilucence detect to carry out; Perhaps or even aequorin gene (Prasher et al., Biochem.Biophys.Res.Comm., 126:1259 (1985)), it can be used to calcium sensitive bioluminescent detection, or green fluorescence protein gene (Niedz et al., Plant Cell Reports, 14:403 (1995)).Use, for example, x-ray film, scintillation counting, fluorescent spectroscopy, low-illuminance cameras, photon counting Kamera or porous luminous detection, the detectable existence of lux gene in cell transformed.It is also envisioned that, can develop, use it for noctilcent population screening (population screening), for example in tissue culturing plate, perhaps even be used for the screening of whole plants this system.
In addition, transgenosis also can be fabricated and be used to: gene product is targeted to intracellular compartment in the vegetable cell, and perhaps pilot protein matter is to extracellular environment.This can engage with the encoding sequence of specific gene by the nucleic acid of will encode transhipment or signal peptide sequence usually and obtain.Transhipment that obtains or signal peptide can be transported in the specific cell respectively protein or the point of destination, extracellular.Under many circumstances, after assisting protein to be transported cellular compartment, remove transhipment or signal peptide.Transhipment or signal peptide play a role by intracellular film by assisting protein, for example, and vacuole, vesicle, plastid and mitochondrial membrane, and signal peptide pilot protein matter is by extracellular film.By assisting protein transduction to transport in the cell or in the outer compartment, above-mentioned sequence can increase the accumulation of gene.
A specific example of this type of purposes relates to, and with interested gene, for example threonine deaminase is directed in the specific organoid, for example plastid but not tenuigenin.This can be by being used for carrying out illustration to making of Arabidopsis SSU1A transit peptides, and it makes albumen mass-energy be targeted to plastid specifically.Perhaps, transgenosis can comprise the nucleic acid of the plastid transit peptides of encoding, the nucleic acid of rbcS (RuBISCO) transit peptides of perhaps encoding, its be operably connected to promotor and the coding threonine deaminase nucleic acid between (referring to, Heijne et al., Eur.J.Biochem., 180:535 (1989); Keegstra et al., Ann.Rev.Plant Physiol.Plant Mol.Biol., 40:471 (1989)).If transgenosis will be introduced in the vegetable cell, this transgenosis can also contain Transcription Termination and the polyadenylation signal of plant, and translation signals, and 3 '-end of described signal and plant threonine deaminase gene links together.
The external source plastid transit peptides can use, and it is not that to be encoded in the natural phant threonine deaminase intragenic.Typically, plastid transit peptides is 40 to 70 amino acid longs, plays a role after translation, and pilot protein matter enters plastid.Can enter plastid with produce mature protein during or and then enter plastid after, transit peptides is scaled off.The complete copy of the gene of coded plant threonine deaminase can contain the plastid transit peptide sequence.In this case, just the plastid transit peptides combined sequence that external source obtains must not suffered to transgenosis.
The encoding sequence of external source plastid transit peptides can obtain from multiple different plant nucleus gene, as long as gene product is as comprising the preceding protein expression of N-terminal transit peptides, and is transported in the selected plastid and goes.The known example of the plant gene product of this type of transit peptide sequence that comprises includes but not limited to, small subunit, ferredoxin, the chlorophyll a/b of rrna bisphosphate carboxylase is conjugated protein, the chloroplast ribosome albumen of nuclear gene encoding, some heat shock protein(HSP), amino acid biosynthetic enzymes (for example, acetolactate synthase, 3-enol pyruvic acid phosphoric acid shikimic acid synthase, dihydrodi pyridine synthase (dihydrodipicolinate) etc.).Perhaps, can be from the known array of transit peptides, for example, and above listed those, whole or partly the dna fragmentation of coding transit peptides is carried out chemosynthesis.
Do not consider the to encode source of dna fragmentation of transit peptides, it should comprise translation initiation codon, and can be expressed as can be by identification of the plastid of host plant and the aminoacid sequence that can correctly play a role therein.Should also be noted that the aminoacid sequence of junction between transit peptides and the threonine deaminase, cut, to produce sophisticated enzyme at this.Some conservative aminoacid sequence is identified, be can be used as guidance.The accurate fusion of sequence and the threonine deaminase coding region of coding transit peptides may require wherein one or whole two nucleic acid are operated, with introducing restriction site easily for example.This can finish by following method, and described method comprises site-directed mutagenesis, inserts the oligonucleotide joint of chemosynthesis etc.
In case obtained the plastid transit peptide sequence, can be by standard method, it suitably is connected on the promotor and threonine deaminase coding region in the transgenosis.Can construct or obtain the plasmid contain the promotor that function is arranged and have the downstream multiple clone site vegetable cell from commercial source.Use Restriction Enzyme, the plastid transit peptide sequence can be inserted into the downstream of promotor.Then, the zone of coding threonine deaminase can be inserted into the downstream of plastid transit peptide sequence 3 '-end, and in its reading frame, the mode that makes plastid transit peptides to translate is fused to the N-terminal of threonine deaminase.In a single day transgenosis forms, just can be with its subclone in other plasmid or carrier.
Consider that the intracellular region chamber that gene product is targeted in the vegetable cell also can obtain by gene directly is transported to the intracellular region chamber.For example, P.Maliga (CurrentOpinion in Plant Biology, 5:164-172 (2002); Heifetz (Biochimie, 82:655-666 (2000)); Bock (J.Mol.Biol., 312:425-438 (2001)); The plastid that andDaniell et al., (Trends in Plant Science, 7:84-91 (2002)) have described plant transforms.
The transgenosis that contains threonine deaminase gene and/or other interested gene can be incorporated into this box in the vegetable cell after making up and finishing then.The DNA of coding threonine deaminase is incorporated in the vegetable cell, can give its tolerance to the amino acid analogue of Isoleucine or Isoleucine, and the isoleucine content in the change vegetable cell, this depends on the activity of the enzyme of the level of kind, genetic expression of vegetable cell and this genes encoding.
Some kinds of constructs that are included among the present invention have been described in the table 3.
Table 3. comprises construct in the present invention
| Species | Promotor | Encoding sequence | Terminator |
| Soybean | Lea9 | ilvA466 | NOS |
| Soybean | Per1 | ilvA466 | |
| Soybean | Lea | ||
| 9 | ilvA219 | NOS | |
| Soybean | Per1 | ilvA219 | NOS |
| A.Thaliana | 7s | ilvAL481Q | NOS |
| A.Thaliana | 7s | ilvAL481F | NOS |
| A.Thaliana | 7s | ilvAL481P | NOS |
| A.Thaliana | 7s | ilvAL481Y | NOS |
| Species | Promotor | Encoding sequence | Terminator |
| Species | pMON | Describe | Make up |
| Soybean | 53910 | 7Sα′-ilvAwt-NOS | |
| Soybean | 53911 | 7Sα′-ilvA219-NOS | |
| Soybean | 53912 | 7Sα′-ilvA466-NOS | |
| Soybean | 58028 | napin-ilvA219-NOS | |
| Soybean | 58029 | Napin-ilvA219-NOS, convergent | |
| A.thaliana | 58031 | napin-ilvA219-NOS | |
| Soybean/A. thaliana | 58117 | napin-OMR-1(TD-FBR)-NOS | |
| The TD-threonine deaminase | |||
| The AHAS-acetohydroxy acid synthase | |||
| The AK-E.C. 2.7.2.4. | |||
| The HSDH-homoserine dehydrogenase | |||
| FBR-feeds back resistance | |||
| Arc-Arcelin | |||
| Per1-peroxidation redox protein | |||
| The later stage enrichment takes place in the Lea-embryo | |||
Use the combination of nucleic acid
One embodiment of the present invention relate to the nucleic acid and the ilvG of the E.Coli of coding AHAS (acetohydroxy acid synthase) and/or the combination of ilvM gene of the threonine deaminase of encoding.This type of acetohydroxy acid synthase gene is not subjected to the amino acid feedback inhibition, and the 2-ketone butyric acid as substrate is had priority.In one embodiment, this activity is limited on the wall scroll fusion polypeptide.Another kind of embodiment relates to, to the insensitive E.C. 2.7.2.4.-homoserine dehydrogenase of amino acid (AK-HSDH) and threonine deaminase and possible and combination AHASII.In one embodiment, the mutant (Omori andKomatubara, J.Bact., 175:959 (1993)) from the thrA1 gene of S.marcescens is an AK-HSDH allelotrope.Above-mentioned nucleic acid can interpretative system be fused on the plastid transit peptides.
The AHAS enzyme is known to be present in all higher plants, and is found in the multiple different microorganism, among for example yeast Saccharomyces cerevisiae, and enterobacteria E.coli and the Salmonella typhimurium (U.S. patent 5731180).Be used for also having obtained good analysis in the hereditary basis that a large amount of above-mentioned species are produced common AHAS.For example, in E.coli and Salmonella typhimurium, have three kinds of isozymes of AHAS; Wherein two kinds to herbicide sensitive, and the third is insensitive.Above-mentioned isozyme all has big and a little protein subunit; Be positioned on the collection of illustrative plates on IlvIH, IlvGM and the IlvBN operon.In the yeast, single AHAS isozyme is navigated on the ILV2 locus.In each case, identified form responsive and that have resistance, some allelic sequences have also obtained mensuration (Friden et al., Nucl. AcidRes., 13:3979-3998 (1985); Lawther etal., PNAS USA, 78:922-928 (1982); Squires et al., Nucl.Acids Res., 811:5299-5313 (1983); Wek et al., Nucl.Acids Res., 13:4011-4027 (1985); Falco and Dumas, Genetics, 109:21-35 (1985); Falco et al., Nucl.Acids Res., 13:4011-4027 (1985)).
In tobacco, the function of AHAS is by two not chain genes, SuRA and SuRB coding.The height homogeny is arranged between these two genes, show on the amino acid levels of nucleotide level and mature protein, though the N-end, the variation of the transshipment area of inferring bigger (Leeet al., EMBO J., 7:1241-1248 (1988)).On the other hand, Arabidopis thaliana has wall scroll AHAS gene, and it is by order-checking (Mazur et al., Plant Physiol.85:1110-1117 (1987)) fully.Some zone of this sequence that relatively demonstrates to AHAS gene order in the higher plant has high-caliber conservative property; Particularly, have 10 zones at least with sequence conservation.Supposed in the past that these conservative regions were crucial to the function of enzyme, and sufficient sequence conservation is depended in the reservation of function.Therefore, the present invention relates to the overexpression of AHAS in plant, with increase wherein the Ile level and one or more the level among Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe.
E.C. 2.7.2.4. (AK) is the enzyme of catalysis Threonine, Isoleucine, Methionin and first step of methionine(Met) biosynthesizing.In the plant, the amino acid whose biosynthesizing of aspartate family betide in the plastid (see, Bryan (1980) In:The Biochemistry ofPlants, Vol.5, B.Miflin (Ed.) Acedamic Press, NY, p.403).Showed in the past, the Threonine overexpression that goes to regulate can make the interior level of cell of free L-Threonine increase by 55% (Shaul and Galili in blade, Plant Physiol., 100:1157 (1992)), in seed, increase by 15 times of (karchi et al., Plant J., 3:721 (1993)).
The overexpression of the E.C. 2.7.2.4. that wild-type or gone is regulated will increase the amount in available free threonine storehouse in the plastid.When combination has wild-type, mutant or during the threonine deaminase that gone to regulate, the amount that is converted to the Threonine of Isoleucine can be increased.Except that E.C. 2.7.2.4. (AK), homoserine dehydrogenase (HSD) and threonine synthase also can be used to further increase the level of free threonine.
Can be used for the E.C. 2.7.2.4. that is gone to regulate of the present invention and can have the Threonine insensitivity of certain level, make that the aspartokinase enzymic activity that shows is than high by 10% under the test conditions that does not have Threonine under the Km concentration of the aspartic acid when having the 0.1mM Threonine.Can be used for the homoserine dehydrogenase that is gone to regulate of the present invention and preferably have the Threonine insensitivity of certain level, make that the activity that this enzyme shows is than high by 10% under the test conditions that does not have Threonine under the Km concentration of 0.1mM Threonine and aspartic acid semialdehyde.The Vmax value of E.C. 2.7.2.4. and homoserine dehydrogenase can be between 0.1-100 times of its corresponding wild type enzyme.The Km value of E.C. 2.7.2.4. and homoserine dehydrogenase can be between 0.01-10 times of its corresponding wild type enzyme.
Threonine synthase is to participate in the enzyme that phosphohomoserine transforms to Threonine, it demonstrates: in Methylobacillus glycogenes during overexpression, the relative endogenous levels of the level of Threonine can be increased about 10 times (Motoyama et al., Appl.Microbiol.Biotech., 42:67 (1994)).In addition, the E.coli threonine synthase of overexpression makes in the tobacco cell culture: from the looked long (Muhitch of 10 multiplications of Threonine level of 6 multiplications of threonine synthase gross activity, Plant Physiol., 108 (2Suppl.): 71 (1995)).Therefore, the present invention relates to the overexpression of threonine synthase in plant, to increase Threonine level wherein.This can be used for the present invention, increases with the supply of guaranteeing Threonine, one or more the production to Ile and Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and Phe that is used for that threonine deaminase carries out.
The conversion of host cell
Comprise target gene, for example the transgenosis of threonine deaminase gene can be by subclone in known expression vector, can survey and/or quantitatively the expression of threonine deaminase.This screening method can be used for identifying the threonine deaminase expression of gene, and the expression of threonine deaminase in process plant transformed cytosome.
Plasmid vector comprises the extra nucleic acid that following function can be provided, described function comprises: be easy to screening, increase and transgenosis is transformed in protokaryon and the eukaryotic cell, described carrier is the pUC derivative vector for example, for example pUC8, pUC9, pUC18, pUC19, pUC23, pUC119 and pUC120, pSK derivative vector, pGEM derivative vector, pSP derivative vector or pBS derivative vector.Extra nucleic acid comprises: be used for carrier host bacterium carry out replication orgin, optimized encoding microbiotic or the Herbicid resistant of self-replicating the selective marker gene, be provided for inserting the nucleic acid of encoding in the transgenosis or gene a plurality of sites uniqueness multiple clone site and strengthen protokaryon and sequence that eukaryotic cell transforms.
Being used in the another kind of carrier of expressing in plant and the prokaryotic cell prokaryocyte is binary Ti-plasmids (binary Ti plasmid), and as Schilperoort et al., U.S. patent 4940838 is disclosed, and carrier pGA582 is its example.This binary Ti-plasmids carrier draws exhausted An by preamble in the past and carried out sign.This binary Ti carrier can for example duplicate among E.coli or the Agrobacterium on the protokaryon bacterium.The Agrobacterium plasmid vector also can be used to the transgenosis body is shifted into vegetable cell.Binary Ti carrier preferably includes nopaline T DNA right margin and the left margin replicon with multiple clone site, colE1 replication orgin and the broad host range of uniqueness that effective vegetable cell conversion, selective marker gene, T borderline region are provided.Carry genetically modified binary Ti carrier of the present invention and can be used to transform protokaryon and eukaryotic cell, but be preferred for transformed plant cells, for example, see Glassman et al., U.S. patent 5258300.
Can expression vector be incorporated in protokaryon or the eukaryotic cell by obtainable method then.The special effective conversion method of dicotyledons is included but not limited to: to the microparticle bombardment (U.S. patent 5990390) or the II type embryo generation callus cell of immature embryo, as W.J.Gordon-Kamm et al., Plant Cell, 2:603 (1990); M.E.Fromm et al., Bio/Technology, 8:833 (1990); And D.A.Walters et al., Plant MolecularBiology, 18:189 (1992) is described; Perhaps pass through electroporation to I type embryo generation callus, as D ' Halluin et al., The Plant Cell, 4:1495 (1992) is described, or as Krzyzek, U.S. patent 5384253 is described.By mixing vegetable cell is transformed (Coffee et al., U.S. patent 5302523) and by also being operable with cellular exposure to the conversion that the liposome that contains DNA carries out with the tungsten whisker concussion that is enclosed with DNA.
Screen the strategy of the clone of excessive production Isoleucine
Use tissue culture technique, the effective screening of wanting of varient that the Isoleucine analogue is had an excessive production Isoleucine of resistance is needed the careful screening conditions of determining.Above-mentioned condition is optimised, so that: the cell that Isoleucine or Isoleucine analogue is had the excessive production Isoleucine of resistance can grow in substratum and accumulate, and the growth of cell colony major part (bulk ofthe cell population) can be suppressed.The viability height of individual cells depends on the viability of adjacent cells in the colony, and this fact becomes complicated with this state.
The condition that cell culture is exposed to Isoleucine or Isoleucine analogue is to be decided by the interactional characteristics between compound and the tissue.This type of factor, for example toxic degree and inhibiting rate should be considered.Cell also needs to be considered to the accumulation and the persistence and the stability of compound in substratum and cell of compound in the culture.
Isoleucine or Isoleucine analogue are carried out careful assessment to the influence of culture viability and form.The particularly important is, select the vegetable regeneration capacity of culture is not had the analogue exposure condition of influence.To the selection of analogue exposure condition also can be subjected to analogue can cell killing the still only influence that suppressed of pair cell division.
Above-mentioned consideration is depended in the selection of screening scheme.The hereinafter simple scheme of describing can be used to screening process.For example, for selecting the cell that growth-inhibiting that Isoleucine or its analogue are caused has resistance, will in the fluid suspension culture thing, be exposed to high-caliber Isoleucine or its analogue at short notice by the meticulous cell that separates.Reclaim or collect the cell of survival then, again it is exposed the longer time subsequently.Perhaps, be exposed to continuously under the situation that is initially low-level free L-Isoleucine or its analogue, the cell culture of organized part differentiation is grown and gone down to posterity cultivation.In some cultivations at interval of going down to posterity, increase concentration gradually then.Though such scheme can be used to screening process, the present invention is not limited to these processes.
Screening and analysis to clone with resistance
Screen, up to being recovered to following cell or tissue, described cell or tissue can be observed under the situation that has the Isoleucine analogue level that can cause inhibition usually, can well-grown.Under the situation that has one or more Isoleucine analogues, " be " to carry out the cultivation of going down to posterity of extra several times to above-mentioned cell, removing the cell that does not have resistance, and then characterize.By under the situation that has multiple analogue concentration, the growing state of more above-mentioned clone and unscreened cell or tissue can determine the amount of acquired resistance.Stability by the resistance feature of cultured cells can be assessed in the following way: under the situation that does not have analogue, the clone of selecting is carried out the cultivation of different time simply, and then tissue is exposed to analogue analyzes growing state.Can also use external chemical research to assess clone with resistance,, in threonine deaminase, and/or whether there is sudden change to form and formed which type of sudden change and make that the inhibition susceptibility that the Isoleucine analogue is caused is lower with checking analogue reaction site.
Can survey with quantitative through moment (transient) expression to the threonine deaminase gene in the cell transformed.Can analyze by ThermoScript II polymerase chain reaction (RT-PCR), quantitatively the Western marking analyze (using threonine deaminase to have specific antibody) to being cloned or by under the situation of the amino acid analogue that has Isoleucine or Isoleucine to enzyme work survey, come genetic expression is carried out quantitatively.Can separate by immunochemistry dyeing process (wherein using threonine deaminase to have specific antibody) or subcellular fractionation being cloned, and biochemistry subsequently and/or immunoassay, the tissue and the Subcellular Localization of coming definite threonine deaminase of being cloned.The threonine deaminase of being cloned also can be determined to the susceptibility of reagent.Then, can use the transgenosis of the expression of the threonine deaminase that threonine deaminase is provided or the inhibition that the amino acid analogue of free L-Isoleucine or Isoleucine causes is had tolerance to transform unifacial leaf and/or dicotyledons histocyte and make plant transformed and seed regeneration.Can come at the existence of selective marker gene or reporter gene for example to be undertaken by Herbicid resistant to screening through cell transformed.Can use the threonine deaminase to being cloned to have specific antibody, perhaps analyze by RT-PCR, the moment expression to the threonine deaminase gene in transgenic embryos generation callus is surveyed.
Be used for the gene that plant is modified
As mentioned before, the gene that plays a role as selective marker gene and reporter gene can operationally make up with the nucleic acid of coding threonine deaminase or its structural domain in transgenosis of the present invention, carrier and plant.In addition, also can add other agronomy characteristic to transgenosis of the present invention, carrier and plant.This class feature includes but not limited to, insect-resistant or tolerance; Disease resistance or tolerance (virus, bacterium, fungi, nematode); Stress resistance or tolerance, its example comprise arid, hot, cold, freezing, too humidity, salt stress, oxidative stress resistance or tolerance; The output that increases; Food content and composition; Physical appearance; Male sterile; Mummification (drydown); Standing property (standability); Yielding ability; Starch property; The quality and quantity of oil etc.The gene that one or more can be had above-mentioned characteristic is inserted in the plant of the present invention.
To environment or resistance of coercing or tolerance
Can realize the raising of plant by genetic expression to multiple environmental stress-tolerance ability.For example, can be by introducing " freeze proof " albumen, Winter Flounder described (Cutler et al., J Plant Physiol., 135:351 (1989)) for example, perhaps its synthetic gene derivative increases the resistance to freezing temperature.Enhanced can obtain (Wolter et al., EMBO J., 11:4685 (1992)) by the expression that increases glycerol-3-phosphate acyltransferase in plastid to the tolerance of cold.Resistance to oxidative stress can obtain (Gupta et al. by the expression of superoxide-dismutase, Proc.Natl.Acad.Sci. (U.S.A.), 90:1629 (1993)), and can be improved (Bowler et al. by glutathione reductase, Ann Rev.Plant Physiol., 43:83 (1992)).
Consider, can strengthen the tolerance of plant, so will be useful arid to the water-content of plant, the expression of gene of the always flow of water, osmotic potential and the favourable influence of expansion.For example, propose, coding has the biosynthetic expression of gene of the solute of osmotically active, may give the protection at arid.This genoid comprises the gene of coding mannitol dehydrogenase (Leeand Saier, J.Bacteriol., 258:10761 (1982)) and trehalose-6-phosphate synthase (Kaasen et al., J.Bacteriol., 174:889 (1992)).
Similarly; the function of other metabolite possibility protective enzyme or integrity (the Loomis etal. of film; J.Expt.Zoology; 252:9 (1989)), therefore the encode biosynthetic expression of gene of these compounds may provide resistance to arid with or complementary mode similar with N.F,USP MANNITOL.Have osmotically active and/or can between arid and/or dry epoch, provide other examples of the naturally occurring metabolite of some direct protective action to comprise: fructose, tetrahydroxybutane, Sorbitol Powder, melampyrum, glycerol-glucose, sucrose, stachyose, raffinose, proline(Pro), glycine, trimethyl-glycine, awns handle alcohol (ononitol) and pine camphor.For example, see U.S. patent 6281411.
Based on structural similarity, found three class embryo generation late proteinses (referring to, Dureet al., Plant Molecular Biology, 12:475 (1989)).Can bring tolerance from the expression of the structure gene of whole three groups of LEA to arid.The protein of derivative other type during water is coerced that comes in handy comprises thiol proteinase, zymohexase, transmembrane transporter, and they can bring the multiple different protectiveness and/or the function of reparation property during drought stress.For example, see PCT/CA99/00219 (Na+/H+ exchanges polypeptide gene).Realize the biosynthetic gene of lipid bring arid resistance aspect also may be useful.
It also may be useful relating to the expression of gene that can increase the specific modality characteristic of absorption water from arid soil.The gene that strengthens breeding fitness (reproductivefitness) during coercing also may be useful.Also propose, during coercing, make amount that the minimized expression of gene of seeds abortion can increase the cereal that will gather in the crops also so valuable.
By the introducing and the expression of gene, make plant can more effectively utilize water, be not in the limiting factor in the obtaining degree of soil moisture, can improve the general performance of plant.By introducing the gene can improve the following ability of plant, can realize the consistence of yielding stability or output performance, described ability is for utilizing the maximized ability of degree to water in all relevant with the water availability coercing.
Plant component or quality
Ingredients of vegetable can be changed, for example, to improve amino acid whose balance, this can be undertaken by several different methods, forms bad protein expression, changes the component of natural protein or introduces the brand-new proteinic gene that coding has better component comprising the expression that increases natural protein, reduction.See, for example, U.S. patent 6160208 (to the change of seed storage protein matter expression).The gene that introducing can change the content of oil in the plant also is valuable.For example, see U.S. patent 6069289 and 6268550 (ACCase gene).Can introduce the gene that increases plant amylum composition nutritive value, for example, be undertaken, thereby make the usability of starch in ox improve by delaying metabolism by increasing degree of branching.
Plant agronomy feature
Determine that it is average day normal temperature during the season of growth and the time span between the frost that plant can grow in the factor where two.The expression of gene that relates in the adjusting that development of plants is carried out may be useful, for example the no tip of a leaf (liguleless) that has been identified in corn and the gene of coarse sheath.
The gene that can improve standing property (standability) and other plant growth characteristics can be introduced in the corn.Can make that stem is strongr, to be enhanced the expression of gene that maybe can prevent or reduce the fringe loss will be valuable for the peasant to the root system system.
Nutritional utilization
Ability concerning obtainable nutrient utilization may be a restrictive factor for plant-growth.Change the nutrition absorption by introducing gene, to the tolerance of pH ultimate, flowing in the plant, the availability of storage (storage pools) and metabolic activity is possible.Above-mentioned modification can make plant can more effectively utilize obtainable nutritive substance.For example, the activity of enzyme that is present in the plant usually and relates to the utilization of nutritive substance increases the availability of the material that can have additional nutrients.Example of this zymoid is a phytase.
Male sterile
Male sterile is useful for producing cenospecies, can obtain male sterile by genetic expression.Introducing TUFR-13 by transforming, is possible so that male sterile is separated with disease susceptibility.See Levings, (Science, 250:942-947, (1990)).Because may must make male recovery fertility, therefore can also introduce coding and make male fertility restorative gene in order to breed purpose and cereal production.
Plant regeneration and to the production of seed
Embryo's generation callus that process transforms, meristematic tissue (meristemate tissue), embryo, leaf dish etc. can be used to produce transgenic plant, and described plant can show the stable heredity of the threonine deaminase gene that process is transformed.Pass through methods known in the art, the plant cell that the amino acid analogue or the free L-Isoleucine that can show Isoleucine has satisfied tolerance level is used the plant regeneration scheme, the maturation plant and the seed that give expression to the tolerance feature with acquisition (for example, are seen U.S. patent 5990390 and 5489520; And Laursen etal., Plant Mol.Biol., 24:51 (1994)).The plant regeneration scheme allows body embryo (somaticembryo) to grow, and sends out roots subsequently and bud.
For confirming that the tolerance feature representation is in the plant organ through differentiation, but not only limit to without in the cell culture that breaks up, the plant that is reproduced is detected, detection is carried out at Ile level that exists in a plurality of parts of plant and one or more the level among Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe, and described level is for the plant of regenerated unconverted.Use standard method, can be from following through obtaining transgenic plant and seed cell transformed and the tissue, described cell and tissue show one or more the variation of content among Ile content and Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe, perhaps to the resistance of Isoleucine analogue.In blade or the seed, the increase of one or more content among Ile content and Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe is particularly preferred.Exist under the situation of the Isoleucine of amount of suppression or its analogue, the change of the specific activity of enzyme can be by measuring the enzymic activity measurement in the process cell transformed, as Widholm, Biochimica et Biophysica Acta, 279:48 (1972) is described.The change of the total content of one or more among Ile and Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe can also detect by standard method, for example, as Jones et al., Analyst, 106:968 (1981) is described.
Can obtain sophisticated plant from the known clone that can express above-mentioned characteristic then.If possible, the plant that is reproduced is from pollination.In addition, the pollen that obtains from the plant that is reproduced can be by the plant that by seed grown of hybridization to inbred lines important on the agronomy.In some cases, the pollen from the plant of above-mentioned inbred lines can be used for to the plant pollination that is reproduced.By the separation case of this characteristic in the first-generation and the progeny plants is assessed, can come this characteristic is characterized from the genetics angle.If described characteristic is to can be used for commercial use, so, the heredity of the characteristic of selecting from tissue culture in plant and expression are particularly importants.
If can obtain various cross combination is used for selling, in soybean, other beans, cereal and other plant, can reach maximum to one or more commercial values of carrying out excessive production among Ile and Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe.Typically, the more than a kind of hybrid plant of peasant planting is based on ripening stage, standing property or other agronomy characteristic.In addition, be applicable to that the hybrid plant in a part of rural area is not suitable for another part because above-mentioned characteristic, for example the ripening stage, have different to the resistance of disease and insect.Therefore, must will introduce a large amount of parent's inbred lines, thereby obtain multiple cross combination one or more the excessive production among Ile and Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe.
By with initial excessive production system and common original seed strain (elite line) hybridization, and then offspring and common parent backcrossed, carry out a kind of transition process (backcrossing).Offspring from this hybridization will separate, thereby some vegetation zones are useful on the gene of excessive production, and some are not with.The plant that has said gene will hybridize with common parent once more, obtain again carrying out isolating offspring according to excessive production and common production.This is carried out repetition, change the strain of the excessive production of energy into, and have other all important quality of in common parent, finding at first up to initial common parent.To being converted into one or more all the original seed strains of carrying out the strain of excessive production among Ile and Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe are carried out the independent program of backcrossing.
After backcrossing, new excessive production system and the appropriate combination that can produce the strain of the commercial hybrid plant of getting well are carried out assessment about excessive production and one group of important agronomy characteristic.The type of producing initial common system and hybrid plant is correct excessive production system and hybrid plant.This need get off to assess a series of envrionment conditionss (wherein, described system and hybrid plant are grown by commercialization usually).For production has one or more soybean among high-load Ile and Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe, following condition may be essential: isozygoty aspect one or more the characteristic of two parents of hybrid plant seed in high-content Ile and Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and Phe.The cenospecies production method of use standard, the parent who increases the gratifying hybrid plant of performance is to use it for the production of hybrid plant.
The transgenic plant that our expectation produces herein can be used for multiple commerce and research purposes.Can be in traditional agriculture application aims and producing transgenic plant, described plant has following characteristic, described characteristic is for be good the human consumer of the cereal of gathering in the crops from plant (for example, the nutraceutical content that increases in human foods or the animal-feed).In this type of purposes, normally be that the purposes of its cereal in the mankind or animal-derived food product comes plant is grown.But the other parts of described plant comprise that stem, shell, root, stem tuber, flower, Food ﹠ Nutrition Department grade and may purposes be arranged also, comprise a part that is used as animal silage, fermentation feed, biocatalysis, perhaps are used for ornamental use.
Transgenic plant also can be used for protein or other molecule are carried out commercial production, wherein molecule (s) of interest extract or purifying from the part of plant, seed etc.Also can cultivate cell or tissue from plant, extracorporeal culture or the fermentation to produce this quasi-molecule.
Transgenic plant also can be used for commercial breeding plan, or can hybridize with the plant of relevant crop species or breed.Improvement by recombinant DNA coding can be transferred, and the cell for example from soya cells to other thing for example merges by protoplastis.
Following embodiment is used to further set forth some aspect of the present invention.
Present embodiment relates to the structure to following plant expression vector, and described carrier contains the polynucleotide allelic variant of the threonine deaminase of encoding.
Particularly, select for use amino acid L481 that the threonine deaminase that is gone to regulate is reasonably designed.Produce some mutation alleles, wherein each all has than the higher or lower IC of ilvA L481F mutation allele
50 IleValue.Above-mentioned allelotrope is used to measure the feedback insensitivity scope of threonine deaminase, is used for transgenic plant.Table 2 (above) has been listed the aminoacid replacement that carries out in the 481st amino acids of ilvA.
In embodiment as herein described, the dna modification enzyme comprises Restriction Enzyme, all available from NewEngland Biolabs (Beverly, MA).Oligonucleolide primers is by Invitrogen LifeTechnologies (Carlsbad, California) synthetic.All other chemical substance all available from Sigma-Aldrich (St Louis, MO).Protein determination is to carry out according to (Bradford, Anal.Biochem., 72:248-254 (1976)) is described.
The ilvA allelotrope that uses derives from the E.coli ilv threonine deaminase gene (SEQ ID NO:1) of wild-type, and its coding can (be numbered K03503 from the SEQ ID NO:2 that the GenBank database obtains; Lawther et al., Nucleic Acids Res., 15:2137 (1987)).Produce Isoleucine and remove the threonine deaminase varient regulated by E.coli being carried out mutagenesis and separating, according to (Gruys et al., U.S. patent 5942660; Asrar et al., U.S. patent 6091002 and 6228623; With Slater et al., Nature Biotechnology, 7:1011-1016 (1999)) described.The nucleotide sequence through the E.coli of mutagenesis threonine deaminase gene that contains ilvA219 (L447F) sudden change is SEQ ID NO:14, and the peptide sequence through translation of its correspondence is SEQ ID NO:3.The nucleotide sequence through the E.coli of mutagenesis threonine deaminase gene that contains ilvA466 (L481F) sudden change is SEQ ID NO:15.All sudden changes are all verified by dna sequence analysis.
With restriction enzyme BamHI plasmid pMON53905 (Fig. 1) is digested, produce the 5.9Kbp backbone segments.The general backbone segments of this fragment conduct construct hereinafter described.
With BamHI plasmid pMON25666 (Fig. 2) is digested, be respectively 3.8 and two bar segment of 2.8Kbp with generation.The fragment of 2.8Kbp is connected on the backbone segments from the 5.9Kbp of pMON53905, produces the plasmid (Fig. 3) of pMON53910 by name.This plasmid contains wild-type ilvA gene (SEQ ID NO:1), is used as contrast.
With BamHI plasmid pMON25694 is digested, be respectively 3.8 and two bar segment of 2.8Kbp with generation.The fragment of 2.8Kbp is connected on the backbone segments of (from pMON53905's) 5.9Kbp, produces the plasmid (Fig. 4) of pMON53911 by name.This plasmid contains the E.coli threonine deaminase gene through mutagenesis, ilvA219 (L447F) (SEQ IDNO:14).
With BamHI plasmid pMON25695 is digested, be respectively 3.8 and two bar segment of 2.8Kbp with generation.The fragment of 2.8Kbp is connected on the backbone segments of 5.9Kbp, produces the plasmid (Fig. 5) of pMON53912 by name.This plasmid contains the E.coli biosynthesizing threonine deaminase gene through mutagenesis, ilvA466 (L481F) (SEQ ID NO:15).
In with transgenic plant, carry out before the further transformation experiment through isolating ilvA allelotrope, with each allelotrope overexpression in E.coli all, to measure its kinetic parameter.Table 4 has been showed the dynamics data of the threonine deaminase that contains various mutations, and with can be from the comparison of the data that obtain from the threonine deaminase of Arabidopsis.E.coliilvA481 varient subclone is advanced pSE380, and (invitrogen, Carlsbad California), came abduction delivering with 0.2mM IPTG in 3 hours 37 ℃ of processing.Shown in SDS-PAGE, the allelic expression of E.coli is very high, and quite consistent.Every kind of varient threonine deaminase has accounted for more than 50% of soluble protein total amount among the E.coli.Unique exception is a L481K varient threonine deaminase, and it is expressed seldom, and enzyme is lived also very low.
By under the situation that has and do not exist the L-Isoleucine, carrying out the stable state dynamical analysis, the influence of the aminoacid replacement on the Leu481 among the ilvA is estimated.The threonine deaminase polypeptide that is used for using at external dynamics research extracts from the E.coli cell, be extracted in the test damping fluid and carry out, described damping fluid contains the ethylenediamine tetraacetic acid (EDTA) of 50mM potassiumphosphate (pH7.5), 1mM dithiothreitol (DTT) (DTT) and 0.5mM.The method of using long run test at the 230nm place to α-ketone butyro-formation directly monitor (ε
230(pH7.5)=540M
-1Cm
-1, and Threonine absorbs can ignore (～1%)).By in the test chamber that contains L-Threonine (2.5mM to 50mM), adding the crude extract of 20 μ l through the 1:20v/v dilution, come starting tests, final volume is 1mL.For causing the L-Isoleucine to suppress, add the L-Isoleucine between the 0mM to 20mM.(Erithacus Software, Surrey UK), with data point substitution formula, measure kinetic parameter to use GraFit 4.0 softwares.For comparing, with the allelic k of L481
CatValue is normalized to the k of wild-type IlvA enzyme
CatValue.The results are shown in Fig. 6 and 7 of above-mentioned analysis.The enzyme of listing among Fig. 7 is: wild-type E.coli threonine deaminase (circle), L481Y TD enzyme (rhombus), L481F TD enzyme (trilateral) and L481T TD enzyme (square).Table 4 is also summarized the kinetic parameter of the varient threonine deaminase of multiple E.coli ilvA allelotrope production.
Some is expressed in the dynamics data of the threonine deaminase among the E.coli table 4.
| pMON | The TD polypeptide | K m Thr (mM) | IC 50 Ile(μM) |
| NA | Wild-type Arabidopsis | 2.8 | 10 |
| NA | Mutant Arabidopsis (OMR1) | 3.6 | 500 |
| 25858 | E.coli(wt)(SEQ ID NO：2) | 8.3 | 56 |
| 25859 | L447F(ilvA219)(SEQ ID NO：3) | 1.7 | ＞20,000 |
| 25857 | L481F(ilvA466)(SEQ ID NO：4) | 4 | 800 |
| 25868 | L481Y(SEQ ID NO：5) | 2 | 1,600 |
| 25864 | L481P(SEQ ID NO：6) | 8.8 | 650 |
| 25860 | L481E(SEQ ID NO：7) | 3.9 | 445 |
| 25866 | L481T(SEQ ID NO：8) | 3.4 | 449 |
| 25865 | L481Q(SEQ ID NO：9) | 8.8 | 188 |
| 25861 | L481I(SEQ ID NO：10) | 7.6 | 134 |
| 25867 | L481V(SEQ ID NO：11) | 7.1 | 97 |
| 25863 | L481M(SEQ ID NO：12) | 6.4 | 100 |
All L481 allelotrope all show with substrate and are combined into positive cooperativity (S type curve), and the threonine deaminase of Arabidopsis demonstrates and do not have dependent activity (typical hyperbolic line) (Fig. 6).(pMON25868 is L481Y) to 1.6 (pMON25865, L481Q 1.1 for the synergitic degree of mutant (Hill coefficient); PMON25861, L481I) between (table 4).What is interesting is that ((SAS Institute, Cary NC), can be classified as hyperbolic line with the dynamics data curve (n=1.1) about L481Y, and degree of confidence is 99% in the JMP statistical software by the F-detection.When having Isoleucine, the activity of L481 mutant enzyme can be suppressed, IC
50(pMON25867 is L481V) to 1600 μ M (pMON25868, L481Y) (Fig. 7 and table 4) from 97 μ M for value.There is not a kind of meeting in the L481 mutant at higher IC
50Value down, to substrate in conjunction with affinity (K
m) generation detrimentally affect (table 4).Therefore, by comparison, substrate is in conjunction with affinity (K
m) be not subjected to the influences that 481 of residues are gone up the sudden change of Isoleucine binding pockets.Different with the L481 mutant, L447F ilvA219 mutant has demonstrated negative cooperativity (n=0.5), though this mutant only is subjected to the slight inhibition (IC of Isoleucine
50＞20,000 μ M).
Based on above-mentioned dynamics data, IC
50 IleBeing selected to Arabidopsis at 100 μ M (L481M) to four kinds of L481 allelotrope in 1600 μ M (L481Y) scopes transforms.
Then every kind of L481 allelotrope is cloned into the plant expression plasmid of seed-specific from the E.coli expression plasmid Central Asia described in the last table 4, is used for transforming to the Arabidopsis plant.E.coli ilvA481 allelotrope is downcut from the listed E.coli expression plasmid of table 4, be cloned in the intermediate carrier as box, described intermediate carrier contains: seed enhancement type promotor (7S α '; Doyle et al., J.Biol.Chem., 261:9228-9238 (1986)); The open reading frame of coding ArabidopsisSSUIA transit peptides (Stark et al., Science, 258:287 (1992)), described transit peptides merges with the open reading frame that contains one of five kinds of ilvA481 allelotrope; And 3 ' untranslated zone (NOS; Depicker et al., J.Mol.Appl.Genet., 1 (4): 361-370 (1982)).Intrusion reaction (infiltration) by the Agrobacterium mediation, with the binary Plant Transformation plasmid pMON69657 (L481P) that obtains (Fig. 8), pMON69659 (L481Y) (Fig. 9), pMON69660 (L481F) (Figure 10), pMON69663 (L481I) (Figure 11) and pMON69664 (L481M) (Figure 12) be transformed into (Beachtold et al. among the Arabidopsis, C.R.Acad.Sci.Ser.111,316:1194-1199 (1993)).Under the situation that 50 μ M glyphosates exist, transformant is screened.
With colorimetric terminal point test (Szamosi et al., Plant Phys., 101:999-1004 (1993)), the extract through plant transformed is carried out about the active screening of threonine deaminase.The terminal point test is carried out in the reaction buffer that contains 100mM Tris-HCl (pH9.0), 100mM KCl, 12.5mM L-Threonine.By adding 50 μ l zyme extract to final volume is 333 μ l, comes initial action.Be reflected at 37 ℃ of following incubations 30 minutes, the 0.05%DNPH (dinitrophenylhydrazine) that is among the 1N HCl with 333 μ l stops.At room temperature incubation is 10 minutes, and the NaOH with 333 μ l 4N neutralizes afterwards.Reaction product is transferred in the disposable tubule (Sarstedt), read at the 540nm place with the HP8453 diode array spectrophotometer.Some independent eventss (event) produce, and wherein contain various L481 allelotrope.The conversion of (Fig. 8) carrying out with pMON69657 (L481P) has unusual low transformation frequency.Lower transformation efficiency quilt is owing to the transformation and selection condition, but not used specific threonine deaminase allelotrope (data not shown).The various L481 allelotrope of the process of all survivals transformed plants can't be distinguished with contrast on phenotype, they all have the rate of setting seeds normally (seed set), this shows that the allelic expression of threonine deaminase can not produce harmful effect to the health of plant.
For measuring the concentration of Isoleucine in plant transformed, collect sophisticated Arabidopsis seed and other nutritive issue that becomes dry, it is carried out the standard amino acid analysis.In brief, by mixing 15 minutes, in 100 μ l5% trichoroacetic acid(TCA)s, the unseeded plant tissue of 5mg is extracted in the room temperature vibration.16,000g carries out 15 minutes centrifugal to extracting solution, and supernatant liquor is forwarded in the HPLC pipe, is used for the analysis of carrying out according to Agilent (TechnicalPublication, in April, 2000).By fluorescence spectrum, be 340nm and be emitted as the 450nm place in excitation wavelength, amino acid concentration is measured.
For measuring the amino acid concentration in the seed, with zirconium/silicon-dioxide bead of the sophisticated Arabidopsis seed of 20mg, 500 μ l 0.5mm (Boise Products, Inc.) and 400 μ L extract damping fluids (100mM potassiumphosphate (pH7.4), 5mM magnesium chloride, 1mM EGTA, 2mMDTT, 2mM 4-2-amino-ethyl benzene sulfonyl fluorine (AEBSF), 100 μ M leupeptins, 10% glycerine) and divide in the pipe that installs to 2mL band screw-cap.At 4 ℃, pearl mill formula tissue grinder (Biospec Products, Inc.) in, the highest setting down carried out twice grindings of 45 seconds to seed.At 4 ℃, under the 16000g, 10 minutes centrifugal carried out in pair cell homogenate, by fluorescence spectrum, is 340nm and is emitted as the 450nm place in excitation wavelength, and supernatant liquor is analyzed.
Table 5A-5B shown pMON69659 (L481Y) (Fig. 9), pMON69660 (L481F) (Figure 10), pMON69663 (L481I) (Figure 11) and pMON69664 (L481M) (Figure 12) R2 of incident for the Isoleucine accumulation (ppm) in the seed.The same with expection presents distribution widely from the accumulation of Isoleucine in the transgenic plant of different event.Ile average out to 85.9 ± 37.4ppm that the incident that transforms with pMON69659 (L481Y) produces, scope is 38.1 to 153.9ppm.Ile average out to 319.6 ± 397.4ppm that the incident that transforms with pMON69660 (L481F) produces, scope is 41.4 to 2592ppm.Ile average out to 204.3 ± 159.1ppm that the incident that transforms with pMON69663 (L481I) produces, scope is 55.4 to 728.2ppm.Ile average out to 168.1 ± 232.0ppm that the incident that transforms with pMON69664 (L481M) produces, scope is 42.3 to 1308.6ppm.The contrast incident of the gene transformation of process coding threonine deaminase does not produce the Ile of average out to 73.75 ± 2.5ppm.A kind of incident, it is based on L481F (ilvA466) allelotrope, and can produce increases by 23 times Ile, and this is observed maximum increasing.
Most of transformant all can't accumulate Isoleucine, with respect to contrast increase level.In addition, IC
50 ILeAnd do not demonstrate any association between the amount of the Isoleucine that accumulates in the transgenic plant.For example, the strain that transformed with pMON69659 (L481Y) has the highest IC
50 ILe, but the incident that any Isoleucine level significantly increases does not appear.
The concentration (ppm) of Ile in the Arabidopsis plant that table 5A. transformed with four kinds of different threonine deaminase constructs
| pMON | Incident | Ile(ppm) |
| NA | Contrast | 70.0 |
| 69659 | 8263 | 38.1 |
| 69659 | 8284 | 38.3 |
| 69659 | 8275 | 39.0 |
| 69659 | 8261 | 43.5 |
| 69659 | 8277 | 50.9 |
| 69659 | 8271 | 52.0 |
| 69659 | 8262 | 55.6 |
| 69659 | 8265 | 62.3 |
| 69659 | 8266 | 68.2 |
| 69659 | 8279 | 74.7 |
| 69659 | 8276 | 76.2 |
| 69659 | 8286 | 78.2 |
| 69659 | 8269 | 81.3 |
| 69659 | 8268 | 847 |
| 69659 | 8270 | 87.5 |
| 69659 | 8278 | 94.6 |
| 69659 | 8258 | 97.6 |
| 69659 | 8287 | 100.4 |
| 69659 | 8264 | 116.7 |
| 69659 | 8260 | 125.0 |
| 69659 | 8259 | 143.4 |
| 69659 | 8272 | 150.1 |
| 89659 | 8273 | 150.8 |
| 69659 | 8274 | 153.9 |
| pMON | Incident | Ile(ppm) |
| NA | Contrast | 75.0 |
| 69660 | 7946 | 41.4 |
| 69660 | 8301 | 92.5 |
| 69660 | 8309 | 102.9 |
| 69660 | 8300 | 116.1 |
| 69660 | 7943 | 118.2 |
| 69660 | 8298 | 119.3 |
| 69660 | 8292 | 128.9 |
| 69660 | 8314 | 136.9 |
| 69660 | 8307 | 139.6 |
| 69660 | 8312 | 151.6 |
| 69660 | 8296 | 164.2 |
| 69660 | 8308 | 167.8 |
| 69660 | 8295 | 174.1 |
| 69660 | 8297 | 189.2 |
| 69660 | 8294 | 198.1 |
| 69660 | 8306 | 198.2 |
| 69660 | 8290 | 205.5 |
| 69660 | 8317 | 218.1 |
| 69660 | 8310 | 224.0 |
| 69660 | 8311 | 236.1 |
| 69660 | 8316 | 258.5 |
| 69660 | 8313 | 265.8 |
| 69660 | 8289 | 324.9 |
| 69660 | 8288 | 336.4 |
| 69660 | 8299 | 346.8 |
| 69660 | 8291 | 403.3 |
| 69660 | 8305 | 451.4 |
| 69660 | 8303 | 485.1 |
| 69660 | 8302 | 540.0 |
| 69660 | 8304 | 590.7 |
| 69660 | 8293 | 809.7 |
| 69660 | 8315 | 2292.0 |
The concentration (ppm) of Ile in the Arabidopsis plant that table 5B. transformed with four kinds of different threonine deaminase constructs
| pMON | Incident | Ile(ppm) |
| NA | Contrast | 75.0 |
| 69663 | 8452 | 55.4 |
| 69663 | 8459 | 80.0 |
| 69663 | 8453 | 81.7 |
| 69663 | 8445 | 82.2 |
| 69663 | 8443 | 92.1 |
| 69663 | 8447 | 92.1 |
| 69663 | 8465 | 93.9 |
| 69663 | 8444 | 95.5 |
| 69663 | 8467 | 98.4 |
| 69663 | 8450 | 104.2 |
| 69663 | 8460 | 111.4 |
| 69663 | 8442 | 112.9 |
| 69663 | 8439 | 131.7 |
| 69663 | 8463 | 133.4 |
| 69663 | 8451 | 156.6 |
| 69663 | 8457 | 174.4 |
| 69663 | 8441 | 177.4 |
| 69663 | 8438 | 190.9 |
| 69663 | 8461 | 196.1 |
| 69663 | 8455 | 197.8 |
| 69663 | 8446 | 212.9 |
| 69663 | 8458 | 223.8 |
| 69663 | 8456 | 247.4 |
| 69663 | 8449 | 287.6 |
| 69663 | 8448 | 307.5 |
| 69663 | 8440 | 309.1 |
| 69663 | 8466 | 410.2 |
| 69663 | 8464 | 496.5 |
| 69663 | 8454 | 578.1 |
| 69663 | 8462 | 728.2 |
| pMON | Incident | Ile(ppm) |
| NA | Contrast | 75.0 |
| 69664 | 8492 | 42.3 |
| 69664 | 8468 | 44.8 |
| 69664 | 8469 | 47.0 |
| 69664 | 8493 | 53.4 |
| 69664 | 8475 | 62.0 |
| 69664 | 8481 | 64.2 |
| 69664 | 8490 | 78.8 |
| 69664 | 8478 | 85.0 |
| 69664 | 8477 | 86.7 |
| 69664 | 8494 | 90.3 |
| 69664 | 8470 | 94.0 |
| 69664 | 8484 | 98.4 |
| 69664 | 8473 | 100.0 |
| 69664 | 7982 | 114.3 |
| 69664 | 8480 | 119.7 |
| 69664 | 8471 | 125.8 |
| 69664 | 8488 | 126.5 |
| 69664 | 8496 | 135.6 |
| 69664 | 8487 | 140.9 |
| 69664 | 8479 | 141.4 |
| 69664 | 8495 | 150.2 |
| 69664 | 8483 | 183.2 |
| 69664 | 8489 | 183.4 |
| 69664 | 8486 | 184.0 |
| 69664 | 8485 | 187.5 |
| 69664 | 8472 | 197.8 |
| 69664 | 8491 | 220.2 |
| 69664 | 8482 | 502.3 |
| 69664 | 8476 | 1308.6 |
Between relative expression's level for the level of the Isoleucine that determine to produce and threonine deaminase whether any association is arranged, the some kinds of strains that have the homoisoleucine accumulation and hang down the Isoleucine accumulation are carried out the Western marking and enzyme activity assay.In brief, with sample on the soluble crude extractive of about 10 μ g to the SDS-PAGE gel (Zaxis) of 4%-20% gradient.Protein transduction is moved on on the pvdf membrane (Biorad).The marking is carried out 1 hour sealing with 5% milk among the TBST (the Tris buffer saline has 0.05%Tween 20).With (with the TBST that the contains 0.5%BSA) rabbit anteserum (MR324) that diluted at 1: 3000 that contains at the polyclonal antibody of the enzyme of purifying, the marking carried out 1 hour detection (probe).Sew after antibody surveys with anti-rabbit alkaline phosphatase, (Sigma, St.Louis MO) develop the color to film with Sigma Fast BCIP/NBT tablet.
The result shows, not significantly association (data not shown) between expression, activity and the Isoleucine accumulation.Only in the strain that contains the accumulation of highest level threonine deaminase, activity can be detected, the Western positive signal can be accumulated though all L481 allelotrope all shows.For the activity in the strain of surveying lower expression, can use more responsive test method (Gruyset al., 1999).
Present embodiment has been showed a kind of method that increases Isoleucine and Xie Ansuan concentration in the Arabidopsis plant, described method is threonine deaminase (the TD) (ilvA466 by Isoleucine being gone regulate, SEQ ID NO:15) with Xie Ansuan and Isoleucine biosynthetic pathway in the other enzyme that relates to combine and realize, described other enzyme promptly, coding E.coli ilvG acetolactate synthase big subunit (EC:2.2.1.6; SEQ ID NO:16) and ilvM acetolactate synthase II small subunit (EC:2.2.1.6; SEQ ID NO:17) polynucleotide molecule.
With SmaI and PvuII Restriction Enzyme, from pMON53912, downcut threonine deaminase E.coli IlvA466 allelotrope (SEQ ID NO:15), be connected on the SamI and PmeI restriction site on the carrier is carrier pMON38207, produce pMON58143.Carrier pMON58143 (Figure 13) is used for conversion by Agrobacterium mediation, and described conversion is carried out under card is received the mycin screening.
By polymerase chain reaction (PCR), isolate the gene of coding ilvG and ilvM, wherein use based on it and divide the primer of other primary sequence right.PMON58131 contains ilvG gene (SEQ ID NO:16).With SEQ ID NO:16 be connected to the pGEM-Teasy carrier (Promega Corporation, USA) on, obtain carrier TTFAGA018992.With BspHI and KpnI Restriction Enzyme, the 5 ' polynucleotide passage (SEQ ID NO:18) from TTFAGA018992 downcuts the ilvG gene is connected to and contains Arabidops SSU1A transit peptides (SEQID NO:9; Stark et al., Science, 258:287 (1992)) intermediate carrier on, produce pMON58145.Use KpnI and NcoI Restriction Enzyme then, downcut the SSU1A transit peptides (SEQ ID NO:19) and the ilvG gene fragment (SEQ ID NO:18) that are operably connected, be connected among the pMON58132.Use BglII and KpnI Restriction Enzyme then, from pMON58132, downcut the SEQ ID NO:18 and 19 that is operably connected, be connected among the shuttle vectors pMON36220, cut, be connected among the pMON58146 with SamI and KpnI Restriction Enzyme.With KpnI and EcoRI Restriction Enzyme, downcut remaining 3 ' ilvG polynucleotide passage (SEQ ID NO:20) from TTFAGA018992, be connected among the pMON58146 that is operably connected with SEQ ID NO:18 and 19, produce pMON58147.Use NotI and EcoRI Restriction Enzyme then, downcut SSU1A transit peptides (SEQ ID NO:19) and complete ilvG coding region (SEQ ID NO:16), be connected among the pMON64205 from pMON58147.Then with PmeI and BglII, downcut SSU1A transit peptides/ilvG box from pMON64205, then it is operably connected on 7s-alpha promotor (U.S.Publication No.2003/0093828) and arcelin53 ' the untranslated zone (WO02/50295-A2), produces pMON58136.Downcut complete box with NotI and BspHI from pMON58136, be connected on the conversion carrier pMON38207, produce pMON58138.
PMON58133 contains ilvM polynucleotide sequence (SEQ ID NO:17).SEQID NO:17 is connected among the pGEM-Teasy (Promega sees above), produces pMON58137.Use BspHI and NotI Restriction Enzyme then, downcut SEQ ID NO:17, be connected to pMON58129 (digesting with PmeI and NcoI earlier) from pMON58137.This can make SEQ ID NO:17 be operably connected on Napin promotor (U.S. patent 5420034), Arabidopsis SSU1A transit peptides and ADR123 '-untranslated zone (the U.S. patent 5981841).This plasmid is called as pMON58140.With BspHI and NotI Restriction Enzyme, downcut expression cassette, be connected among the plant conversion carrier pMON38207 (digesting with Restriction Enzyme NotI earlier), produce pMON58151.
Downcut the ilvM box with NotI and BspHI Restriction Enzyme from middle carrier pMON58140, be connected among the pMON58138 that contains ilvG box and Plant Transformation main chain, produce pMON58159.In addition, downcut ilvA466 from pMON53912, it is operably connected on the ilvG and ilvM box from pMON58159 generation pMON58162 (Figure 16) with PvuII and SmaI Restriction Enzyme.
Intrusion reaction by the Agrobacterium mediation, with the binary Plant Transformation plasmid pMON58143 (ilvA466) that obtains (Figure 13), pMON58159 (ilvG+ilvM) (Figure 14) and pMON58162 (ilvA466+ilvG+ilvM) (Figure 15) be transformed into (Beachtold et al. among the Arabidopsis, C.R.Acad.Sci.Ser.111,316:1194-1199 (1993)).Transformant is screened under the situation of mycin existing card to receive.
For measuring amino acid whose concentration in the seed, the sophisticated seed tissue of 5mg is ground to form fine powder, mix by vibration, in room temperature, in the trichoroacetic acid(TCA) of 100 μ l 5%, described powder carried out 15 minutes extraction.16, under the 000g extracting solution is carried out 15 minutes centrifugal, supernatant liquor is transferred in the HPLC pipe, analyze according to manufacturer is described (AgilentTechnologies, USA).By fluorescence spectrum, be 340nm and be emitted as the 450nm place in excitation wavelength, amino acid concentration is measured.
For every kind of construct, some kinds of independently incidents have been produced.For every kind of incident, the sophisticated scattered Arabidopsis seed of collecting exsiccation is used it for amino acid analysis as the storehouse.Seed with ilvA466 (pMON58143) transformed plants has the Isoleucine level of increase, and the average Isoleucine level that it is found in the seed with the ilvA466 plant transformed not has about 69 times growth (table 6A).Also observed, positive association with other total free aminoacids (comprising arginine, glutamine, leucine, Methionin, Threonine, tyrosine, phenylalanine and Xie Ansuan) concentration, it is defined as Pearson ' s incidence coefficient (r) is 0.60 or higher (Snedecor and Cochran, In:Statistical Methods, 1980).
The seed of ilvG, ilvM (pMON58159) transformed plants of using by oneself contains the Xie Ansuan level of rising, it is about than high about 15 times of the contrast seed that does not contain ilvG and ilvM, and has positive association (r＞0.60) (table 6B) with tryptophane, L-Ala, arginine, glutamine, glycine, Serine, phenylalanine, leucine, Methionin, Threonine and tyrosine.
The seed of ilvG, ilvM and ilvA466 (pMON58162) transformed plants of using by oneself contains the Isoleucine (having increased by 15 times) and Xie Ansuan (having increased by the 19 times) level of rising, and, Isoleucine and Methionin, phenylalanine, Threonine, tyrosine and Xie Ansuan have positive association (r＞0.60) relatively, Xie Ansuan has positive association (r＞0.60) (table 6C) with L-Ala, glutamine, Serine, Threonine, Isoleucine and tyrosine relatively.
Table 6A express in the allelic Arabidopsis plant of E.coli ilvA466 amino acid concentration and with the cognation of Ile concentration
| Amino acid | Construct | Mean value | Standard variance | r(Ile) |
| Trp | pMON58143 | 54.1 | 47.4 | 0.377 |
| Ile | pMON58143 | 2624.9 | 625.7 | NA |
| Ala | pMON58143 | 198.4 | 53.3 | 0.538 |
| Arg | pMON58143 | 2364.3 | 727.0 | 0.676 |
| Asn | pMON58143 | 1125.1 | 414.2 | 0.518 |
| Asp | pMON58143 | 234.2 | 55.6 | 0.589 |
| Gln | pMON58143 | 1179.8 | 290.5 | 0.665 |
| Glu | pMON58143 | 841.1 | 158.6 | 0.163 |
| Gly | pMON58143 | 30.8 | 13.5 | 0.406 |
| His | pMON58143 | 335.8 | 207.6 | 0.026 |
| Leu | pMON58143 | 192.0 | 71.5 | 0.925 |
| Lys | pMON58143 | 292.3 | 77.1 | 0.806 |
| Met | pMON58143 | 29.4 | 9.4 | 0.505 |
| Phe | pMON58143 | 100.3 | 20.9 | 0.665 |
| Ser | pMON58143 | 116.8 | 33.3 | 0.217 |
| Thr | pMON58143 | 184.8 | 54.3 | 0.677 |
| Tyr | pMON58143 | 108.2 | 28.6 | 0.627 |
| Val | pMON58143 | 356.3 | 131.1 | 0.829 |
Table 6B express in the Arabidopsis plant of ilvG and ilvM amino acid concentration and with the cognation of Ile and Val concentration
| Amino acid | Construct | Mean value | Standard variance | r(Val) |
| Trp | pMON58159 | 58.7 | 35.4 | 0.867 |
| Ile | pMON58159 | 118.3 | 140.8 | 0.030 |
| Ala | pMON58159 | 196.7 | 87.8 | 0.863 |
| Arg | pMON58159 | 753.7 | 405.8 | 0.771 |
| Asn | pMON58159 | 479.4 | 203.4 | 0.352 |
| Asp | pMON58159 | 178.4 | 54.8 | 0.515 |
| Gln | pMON58159 | 854.7 | 519.6 | 0.979 |
| Glu | pMON58159 | 501.5 | 196.6 | -0.217 |
| Gly | pMON58159 | 42.8 | 16.2 | 0.807 |
| His | pMON58159 | 99.6 | 56.7 | 0.530 |
| Leu | pMON58159 | 239.1 | 160.8 | 0.782 |
| Lys | pMON58159 | 195.9 | 89.4 | 0.920 |
| Met | pMON58159 | 10.2 | 4.5 | -0.448 |
| Phe | pMON58159 | 79.7 | 22.6 | 0.725 |
| Ser | pMON58159 | 968.0 | 608.8 | 0.976 |
| Thr | pMON58159 | 211.9 | 98.9 | 0.932 |
| Tyr | pMON58159 | 94.1 | 48.3 | 0.966 |
| Val | pMON58159 | 2525.3 | 1572.1 | NA |
Table 6C express in the Arabidopsis plant of ilvA466, ilvG and ilvM amino acid concentration and with the cognation of Ile and Val concentration
| Amino acid | Construct | Mean value | Standard variance | r(Ile) | r(Val) |
| Trp | pMON58162 | 284.3 | 852.2 | -0.324 | -0.512 |
| Ile | pMON58162 | 566.0 | 299.6 | NA | 0.604 |
| Ala | pMON58162 | 268.9 | 92.2 | 0.468 | 0.697 |
| Arg | pMON58162 | 1723.4 | 859.7 | 0.464 | 0.367 |
| Asn | pMON58162 | 1034.5 | 516.1 | 0.065 | 0.099 |
| Asp | pMON58162 | 261.9 | 127.9 | -0.148 | -0.270 |
| Gln | pMON58162 | 869.4 | 452.5 | 0.578 | 0.764 |
| Glu | pMON58162 | 743.0 | 215.8 | -0.148 | -0.414 |
| Gly | pMON58162 | 34.1 | 13.5 | -0.180 | -0.059 |
| His | pMON58162 | 255.2 | 135.0 | 0.467 | 0.315 |
| Leu | pMON58162 | 451.2 | 377.7 | 0.581 | 0.493 |
| Lys | pMON58162 | 280.2 | 87.5 | 0.662 | 0.585 |
| Met | pMON58162 | 20.3 | 16.2 | 0.204 | -0.157 |
| Phe | pMON58162 | 120.5 | 36.9 | 0.742 | 0.441 |
| Ser | pMON58162 | 486.9 | 319.5 | 0.298 | 0.632 |
| Thr | pMON58162 | 238.1 | 81.8 | 0.708 | 0.825 |
| Tyr | pMON58162 | 127.7 | 43.2 | 0.608 | 0.690 |
| Val | pMON58162 | 3196.3 | 1183.1 | 0.604 | NA |
Present embodiment has been showed the conversion of soybean plants being carried out with the expression vector that contains the threonine deaminase mutation allele, wherein uses the method for partickle bombardment and Agrobacterium (Agrobacterium) mediation.
To the obtainable soybean seeds of commerce (Asgrow A3244, the A4922) germination treatment of spending the night (approximately 16-24 hour), downcut the meristematic tissue explant.Primary leaf is moved down, and to expose meristematic tissue, explant is placed in the target substratum, and its mesomeristem is placed on the vertical direction of the direction of transporting with particle.Conversion carrier pMON53190, the pMON53900 and the pMON53912 that contain the different allelic coding regions of ilvA are used CaCl
2Be deposited on the trickle gold grain with spermidine, be suspended in the ethanol again subsequently.Suspension is applied on the Mylar lamella, then it is placed on the electric discharge device.By under about 60% capacitive conditions, discharging, particle is accelerated in the plant tissue.
After the bombardment, explant is placed Woody PlantMedium (WPM) (the McCown ﹠amp that is added with the 75mM glyphosate; Lloyd, Proc.International PlantPropagation Soc., 30:421 (1981)) in, place 5-7 week, make that genetically modified bud can screened and cultivation.About 5-7 week after the bombardment, results glyphosate male bud, be placed on the selectivity beans root media that is added with the 25mM glyphosate (Bean Rooting Media, BRM) in, place 2-3 week.The component of BRM is shown in the table 7.The bud that can send out roots is transferred in the greenhouse, plants in soil.The bud that to keep fit in screening process but can not produce root is transferred in the nonselective root media (the beans root media (" BRM ") that does not have glyphosate), places for 2 weeks again.The root that comes (off the selection) in the comfortable screening can produce any bud of root is carried out the test expressed about the glyphosate selective key, afterwards it is transferred to the greenhouse, plant in soil.Plant is held under the standard greenhouse experiment, and up to the results seed, this seed is defined as the R1 seed.
The component and the preparation of table 7. beans root media (BRM)
| Stock compound | The amount that is used for 4L |
| MS salt *** | 8.6g |
| Inositol (cell cultures level other) | 0.40g |
| SBRM VITAMIN storage liquid ** | 8.0ml |
| L-halfcystine (10mg/ml) | 40.0ml |
| Sucrose (ultrapure) | 120g |
| Regulate pH to 5.8 | |
| The agar of washing | 32g |
| Add behind the autoclaving: | |
| SBRM/TSG hormone storage liquid * | 20.0ml |
| *SBRM/TSG hormone storage liquid (be added among the 1L BRM, add following substances) 3.0ml IAA (0.033mg/ml) 2.0ml sterile purified water | |
| **SBRM VITAMIN storage liquid (every 1L storage liquid) glycine 1.0g nicotinic acid 0.25g pyridoxol HCl 0.25g thiamines HCl 0.25g | |
| ***MS salt (Murashige and Skoog, Physiol.Plant., 15:473-497 (1962)) | |
This substratum is used under the condition that adds or do not add glyphosate (typically, 0.025mM or 0.040mM).All the components once dissolves a kind of.With sterile purified water mixture is carried out constant volume, it is preserved in the bottle that tinfoil encases,, be no more than one month in 4 ℃.
Method for transformation (as Martinell et al., U.S. patent 6384301 is described) with the Agrobacterium mediation also transforms soybean plants with pMON58028, pMON58029 and pMON58031.For this method, use standard method well known by persons skilled in the art, the overnight culture that will contain the Agrobacterium tumefaciens of the plasmid that comprises target gene is cultured to the logarithm stage, and being diluted to final optical density then is 0.3 to 0.6.Above-mentioned culture is used to inoculate to the soybean embryo explants of preparation, and is as mentioned below.
In brief, this method is directly to carry out the conversion of kind of system in the individual soya cells in the soybean embryo meristematic tissue that downcuts.After surface sterilization and seed sprout, the soybean embryo is removed.Explant is placed on the OR substratum then, this is according to Barwal et al., Plants, the standard MS substratum of the described improvement of 167:473-481 (1986), wherein be added with the Pyocianil of 3mg/LBAP, 200mg/L, the cefotaxime of 62.5mg/L and the F-1991 (Benomyl) of 60mg/L, preserve down in 15 ℃ in the dark and spend the night.Second day, with scalpel blade explant is scratched, use according to the Agrobacterium culture of preparation mentioned above it is inoculated.To at room temperature cultivate 3 days through the explant of inoculation then.
Transform after the cultivation of back, then the meristematic tissue zone is placed on the standard plant tissue culture media and cultivate, wherein have herbicide glyphosate (Monsato Company, St.Louis, MO), glyphosate plays a dual role as the hormone of selective reagent and induced bud.At Martinell et al., the detailed description of pair nutrient media components and cultivation length is arranged in the U.S. patent 6384301.After 5 to 6 weeks, the explant that will have the survival of positive phenotype is transferred in the soil, under greenhouse experiment it is cultivated, up to maturation.
Isoleucine concentration (shown in embodiment 2) to 5 kinds of isolating R1 seeds of discrete is measured, and those incidents that will have high density grow into the R1 plant.For every kind of incident, plant 24 seeds.The R2 seed that obtains of results is measured Isoleucine concentration, has a situation analysis to genetically modified.R2 seed, R2 plant and R3 seed are carried out same analysis.
Present embodiment has been showed the evaluation to the soybean plants that transformed with the threonine deaminase gene construct.Be to measure the threonine deaminase activity, in 100 μ L 1X mill damping fluid (table 8) to simple grain (～100mg) seed is milled.Then mixture is carried out 2-3 minute centrifugal under top speed.By being applied to of the supernatant liquor desalination of Bio-Rad Bio-Gel P-30 desalting column to obtaining.
Be added in the 5X test mixture (table 8) through the protein extract (25-50 μ L) of desalination, to final volume be 100 μ L.Under 37 ℃, mixture carried out 30 minutes cultivation.Come termination reaction by 0.05% dinitrophenylhydrazine that adds among the 100 μ L 1N HCl, then cultivated again 10 minutes in room temperature.The aliquots containig that adds the 4N NaOH of 100 μ L is then measured absorption value by spectrophotometer at the 540nm place.
Table 8. is used for the damping fluid of threonine deaminase test
| 5X test mixture (for 1mL) | ||
| Composition | Aliquots containig | Concentration |
| 2M Tris-HCl，pH9.0 | 250μL | (100mM) |
| 1M KCl | 500μL | (100mM) |
| 0.5ML-Threonine | 25μL | (12.5mM) |
| 0.5mM DTT | ||
| H 2O | 225μL | |
| The 1X damping fluid (for 100mL) of milling | |
| Aliquots containig | Concentration |
| 5mL | (100mM) |
| 10mL | (100mM) |
| 0 | 0 |
| 4μL | (2mM) |
| 85mL | |
The seed middle reaches leucic concentration of divorcing is measured by the following method: pulverize the seed of about 50mg, the material that will pulverize places centrifuge tube, weighs then.The trichoroacetic acid(TCA) that in every sample hose, adds 1mL 5%.With the vibration mixed instrument, in room temperature sample is carried out 15 minutes mixing.Under 14000rpm, in micro-centrifuge tube, sample carried out 15 minutes rotating centrifugal then.Take out some in the supernatant liquor, place the HPLC pipe, and in the sealing.Before the analysis, sample is placed on 4 ℃.
All R1 soybean seeds are carried out sample analysis one time, 5 seeds of every kind of incident, every seed shot.For the offspring who represents R2 and R3 seed, use fairly large test, 10 seeds of every kind of incident, every kind of incident shot.
HPLC system with Agilent Technologies 1100 series analyzes sample.OPA reagent (Phthalyldicarboxaldehyde in the borate buffer and 3-thiohydracrylic acid with 2.5 μ L, Hewlett-Packard PN 5061-3335), in the 0.4N borate buffer (Hewlett-Packard PN 5061-3339) of the pH10.2 of 10 μ L, the aliquots containig of 0.5 μ L of sample is carried out derivatize.Derivative is expelled to Agilent Technologies Eclipse XDB-C183.5 μ m, and 4.6 * 75mm, flow velocity are 2mL/min.
Table 9.HPLC experiment condition
| Time (minute): | 0 | 9.8 | 12 | 12.5 | 14 |
| | 0 | 70 | 0 | 0 | 0 |
| | 0 | 30 | 100 | 0 | 0 |
HPLC buffer A: 95%40mM Na
2HPO
4, pH=7.8+5% buffer B+0.01%NaN
3
HPLC buffer B: 45%: 45%: 10%:: methyl alcohol: acetonitrile: water
Measure Isoleucine concentration (the 340nm place excites, the emission of 450nm place) with fluorescence detection, value is calculated by the typical curve in 10 to 800 μ g/mL scopes.
Assessment result to the leucine concentration of divorcing through the soybean plants middle reaches that transform shows, empty map middle reaches divorce leucine concentration is: about 100 μ g/g in the seed, and have the about 600 and 1300 μ g/g of surpassing respectively with ilvA219 and ilvA466 allelotrope transformed plants.Above-mentioned data presentation, than not passing through plant transformed, the plant middle reaches divorces leucine level of threonine deaminase gene transformation that spends adjusting is significantly higher.
For whether having threonine deaminase protein in the soybean plants of determining to transform through the threonine deaminase construct, the strain that the threonine deaminase mutant allele that goes to regulate from wild-type and Isoleucine produces obtains sophisticated soybean seeds, and it is carried out the analysis of the Western marking.With the soybean seeds drying, pulverize into powder.The 1XSDS-PAGE sample buffer that adds 200 μ l in the powder of 20mg is under rotation, in 4 ℃ of cultivations of mixture being carried out 4 hours.By boiling 5-10 minute, come termination reaction.Under 14000rpm, mixture is carried out 10 minutes centrifugal then.The supernatant liquor that obtains places on one side, repeats centrifugal.Supernatant liquor is partly merged, it is carried out about proteinic test with Bio-Rad protein test test kit (Bio-Rad).
Use 10% Tris-HCl damping fluid then, come the separation of supernatant part by SDS-PAGE.After adding sample staining agent (10%v/v), the sample of sample 1mL preparation on each sample well.In Tris-glycine-SDS damping fluid, 140 volts down, ran glue 1 hour.Then the protein transduction in the gel is moved on in advance on the pvdf membrane of crossing with methyl alcohol and transferring buffered liquid wetting.After film (cartridge) was gone up sample, in cold Tris-glycine-methyl alcohol damping fluid, 100 volts were carried out 1 hour transfer down.Seal step with 10% milk soln (the 5g skim-milk is in the TBS damping fluid that contains 0.1%Tween 20 (20mM Tris, pH7.5 and 150mM NaCl) of 50mL at cumulative volume).
One-level antibody is the anti-threonine deaminase antibody of multi-clone rabbit, with the TBS damping fluid that contains 1 %Tween 20 and 1% milk soln it is carried out 1: 1000 dilution.At room temperature cultivated 1 hour or 4 ℃ of following overnight incubation.Secondary antibodies is the anti-rabbit antibody of polyclone that obtains from Sigma Chemical Co..Carry out development step then: wash 3 times with the TBS that contains 1%Tween 20, each 10 minutes, then washed 10 minutes with TBS, dye then.
The R3 seed extract of the soybean plants that transforms hanging oneself carries out the analysis of the Western marking, wherein get the kernel maturing of three kinds of different stepss, heterozygosis strain and empty strain (nullline) are carried out, and the result shows that the concentration of mutein increases with seed maturity.In the gel that obtains, be visible corresponding to the proteinic band of mutant threonine deaminase position, this band comes across through the duct of plant transformed correspondence, does not then exist in the duct of empty strain correspondence.In addition, the intensity of band along with maturation by obviously increasing to late period in early days.
Present embodiment has been showed the result of the amino acid analysis of the R3 soybean seeds that the polynucleotide sequence through the coding threonine deaminase is transformed.The table 10A-10R provide use JMP statistical software (SAS Institute, Cary, NC, the result who USA) obtains, described result is: the statistical average value and the error of the amino acid concentration that records in the R3 soybean event that transformed with threonine deaminase.
Ile level in the soybean plants of table 10A. expression threonine deaminase
| Connectivity | Incident | pMON | Gene | N | Mean value | Standard variance | |
| Heterozygote | 13894 | 53911 | 7S alpha’- | 12 | 614.6 | 591.0 | |
| Heterozygote | 14202 | 53911 | 7S alpha’- | 11 | 380.5 | 331.1 | |
| Heterozygote | 14269 | 53912 | 7S alpha’-ilvA466 | 8 | 109.7 | 57.1 | |
| Homozygote | 13747 | 53910 | 7S alpha’- | 3 | 199.6 | 80.4 | |
| Homozygote | 14269 | 53912 | 7S alpha’- | 3 | 346.8 | 43.9 | |
| Empty | 13894 | 53911 | 7S alpha’- | 11 | 37.1 | 20.2 | |
| Empty | 14202 | 53911 | 7S alpha’-ilvA219 | 10 | 43.4 | 49.3 | |
| Empty | 14269 | 53912 | 7S alpha’- | 5 | 24.9 | 3.8 | |
| Empty | A4922 | | Basic germplasm | 6 | 30.7 | 11.4 |
Asp level in the soybean plants of table 10B. expression threonine deaminase
| Connectivity | Incident | pMON | Gene | N | Mean value | Standard variance | |
| Heterozygote | 13894 | 53911 | 7S alpha’- | 12 | 148.9 | 28.8 | |
| Heterozygote | 14202 | 53911 | 7S alpha’- | 11 | 171.7 | 35.6 | |
| Heterozygote | 14269 | 53912 | 7S alpha’-ilvA466 | 8 | 132.6 | 26.1 | |
| Homozygote | 13747 | 53910 | 7S alpha’- | 3 | 98.4 | 12.2 | |
| Homozygote | 14269 | 53912 | 7S alpha’- | 3 | 54.2 | 12.7 | |
| Empty | 13894 | 53911 | 7S alpha’- | 11 | 170.6 | 33 | |
| Empty | 14202 | 53911 | 7S alpha’-ilvA219 | 10 | 176.3 | 29 | |
| Empty | 14269 | 53912 | 7S alpha’- | 5 | 144.6 | 36.1 | |
| Empty | A4922 | | Basic germplasm | 6 | 178.8 | 19.7 |
Glu level in the soybean plants of table 10C. expression threonine deaminase
| Connectivity | Incident | pMON | Gene | N | Mean value | Standard variance | |
| Heterozygote | 13894 | 53911 | 7S alpha’- | 12 | 213.0 | 40.4 | |
| Heterozygote | 14202 | 53911 | 7S alpha’- | 11 | 225.0 | 40.3 | |
| Heterozygote | 14269 | 53912 | 7S alpha’-ilvA466 | 8 | 224.9 | 36.0 | |
| Homozygote | 13747 | 53910 | 7S alpha’- | 3 | 338.9 | 20.2 | |
| Homozygote | 14269 | 53912 | 7S alpha’- | 3 | 194.5 | 31.8 | |
| Empty | 13894 | 53911 | 7S alpha’- | 11 | 210.1 | 38.3 | |
| Empty | 14202 | 53911 | 7S alpha’-ilvA219 | 10 | 225.9 | 42.6 | |
| Empty | 14269 | 53912 | 7S alpha’- | 5 | 224.2 | 22.8 | |
| Empty | A4922 | | Basic germplasm | 6 | 236.2 | 21.5 |
Asn level in the soybean plants of table 10D. expression threonine deaminase
| Connectivity | Incident | pMON | Gene | N | Mean value | Standard variance | |
| Heterozygote | 13894 | 53911 | 7S alpha’- | 12 | 29.6 | 9.9 | |
| Heterozygote | 14202 | 53911 | 7S alpha’- | 11 | 28.3 | 9.4 | |
| Heterozygote | 14269 | 53912 | 7S alpha’-ilvA466 | 8 | 24.5 | 8.7 | |
| Homozygote | 13747 | 53910 | 7S alpha’- | 3 | 230.3 | 151.8 | |
| Homozygote | 14269 | 53912 | 7S alpha’- | 3 | 25.3 | 9.9 | |
| Empty | 13894 | 53911 | 7S alpha’- | 11 | 23.8 | 7.0 | |
| Empty | 14202 | 53911 | 7S alpha’-ilvA219 | 10 | 22.9 | 4.2 | |
| Empty | 14269 | 53912 | 7S alpha’- | 5 | 25.0 | 10.3 | |
| Empty | A4922 | | Basic germplasm | 6 | 24.3 | 3.3 |
Ser level in the soybean plants of table 10E. expression threonine deaminase
| Connectivity | Incident | pMON | Gene | N | Mean value | Standard variance | |
| Heterozygote | 13894 | 53911 | 7S alpha’- | 12 | 17.8 | 5.7 | |
| Heterozygote | 14202 | 53911 | 7S alpha’- | 11 | 16.2 | 3.6 | |
| Heterozygote | 14269 | 53912 | 7S alpha’-ilvA466 | 8 | 14.4 | 2.0 | |
| Homozygote | 13747 | 53910 | 7S alpha’- | 3 | 13.8 | 3.6 | |
| Homozygote | 14269 | 53912 | 7S alpha’- | 3 | 13.4 | 3.3 | |
| Empty | 13894 | 53911 | 7S alpha’- | 11 | 14.7 | 3.4 | |
| Empty | 14202 | 53911 | 7S alpha’-ilvA219 | 10 | 14.3 | 1.7 | |
| Empty | 14269 | 53912 | 7S alpha’- | 5 | 14.0 | 1.4 | |
| Empty | A4922 | | Basic germplasm | 6 | 15.0 | 1.5 |
Gln level in the soybean plants of table 10F. expression threonine deaminase
| Connectivity | Incident | pMON | Gene | N | Mean value | Standard variance | |
| Heterozygote | 13894 | 53911 | 7S alpha’- | 12 | 4.8 | 1.5 | |
| Heterozygote | 14202 | 53911 | 7S alpha’- | 11 | 4.5 | 0.8 | |
| Heterozygote | 14269 | 53912 | 7S alpha’-ilvA466 | 8 | 4.9 | 1.1 | |
| Homozygote | 13747 | 53910 | 7S alpha’- | 3 | 34.7 | 41.9 | |
| Homozygote | 14269 | 53912 | 7S alpha’- | 3 | 4.7 | 0.3 | |
| Empty | 13894 | 53911 | 7S alpha’- | 11 | 4.1 | 1.3 | |
| Empty | 14202 | 53911 | 7S alpha’-ilvA219 | 10 | 4.0 | 0.6 | |
| Empty | 14269 | 53912 | 7S alpha’- | 5 | 4.6 | 0.6 | |
| Empty | A4922 | | Basic germplasm | 6 | 4.3 | 0.4 |
His level in the soybean plants of table 10G. expression threonine deaminase
| Connectivity | Incident | pMON | Gene | N | Mean value | Standard variance | |
| Heterozygote | 13894 | 53911 | 7S alpha’- | 12 | 40.3 | 33.2 | |
| Heterozygote | 14202 | 53911 | 7S alpha’- | 11 | 28.0 | 19.5 | |
| Heterozygote | 14269 | 53912 | 7S alpha’-ilvA466 | 8 | 18.0 | 7.9 | |
| Homozygote | 13747 | 53910 | 7S alpha’- | 3 | 48.1 | 22.5 | |
| Homozygote | 14269 | 53912 | 7S alpha’- | 3 | 20.2 | 9.1 | |
| Empty | 13894 | 53911 | 7S alpha’- | 11 | 12.8 | 3.3 | |
| Empty | 14202 | 53911 | 7S alpha’-ilvA219 | 10 | 12.6 | 3.8 | |
| Empty | 14269 | 53912 | 7S alpha’- | 5 | 14.3 | 5.0 | |
| Empty | A4922 | | Basic germplasm | 6 | 13.5 | 1.9 |
Gly level in the soybean plants of table 10H. expression threonine deaminase
| Connectivity | Incident | pMON | Gene | N | Mean value | Standard variance | |
| Heterozygote | 13894 | 53911 | 7S alpha’- | 12 | 17.6 | 5.3 | |
| Heterozygote | 14202 | 53911 | 7S alpha’- | 11 | 17.8 | 4.7 | |
| Heterozygote | 14269 | 53912 | 7S alpha’-ilvA466 | 8 | 15.8 | 2.7 | |
| Homozygote | 13747 | 53910 | 7S alpha’- | 3 | 154.8 | 87.6 | |
| Homozygote | 14269 | 53912 | 7S alpha’- | 3 | 18.8 | 1.7 | |
| Empty | 13894 | 53911 | 7S alpha’- | 11 | 14.1 | 3.6 | |
| Empty | 14202 | 53911 | 7S alpha’-ilvA219 | 10 | 16.8 | 8.7 | |
| Empty | 14269 | 53912 | 7S alpha’- | 5 | 14.6 | 1.5 | |
| Empty | A4922 | | Basic germplasm | 6 | 14.7 | 1.2 |
Thr level in the soybean plants of table 10I. expression threonine deaminase
| Connectivity | Incident | pMON | Gene | N | Mean value | Standard variance | |
| Heterozygote | 13894 | 53911 | 7S alpha’- | 12 | 10.9 | 2.6 | |
| Heterozygote | 14202 | 53911 | 7S alpha’- | 11 | 10.8 | 1.9 | |
| Heterozygote | 14269 | 53912 | 7S alpha’-ilvA466 | 8 | 9.4 | 1.2 | |
| Homozygote | 13747 | 53910 | 7S alpha’- | 3 | 6.3 | 2.0 | |
| Homozygote | 14269 | 53912 | 7S alpha’- | 3 | 5.4 | 0.7 | |
| Empty | 13894 | 53911 | 7S alpha’- | 11 | 10.6 | 1.6 | |
| Empty | 14202 | 53911 | 7S alpha’-ilvA219 | 10 | 10.3 | 1.2 | |
| Empty | 14269 | 53912 | 7S alpha’- | 5 | 10.0 | 1.0 | |
| Empty | A4922 | | Basic germplasm | 6 | 10.8 | 0.5 |
Arg level in the soybean plants of table 10J. expression threonine deaminase
| Connectivity | Incident | pMON | Gene | N | Mean value | Standard variance | |
| Heterozygote | 13894 | 53911 | 7S alpha’- | 12 | 308.3 | 121.9 | |
| Heterozygote | 14202 | 53911 | 7S alpha’- | 11 | 299.3 | 126.2 | |
| Heterozygote | 14269 | 53912 | 7S alpha’-ilvA466 | 8 | 302.1 | 134.5 | |
| Homozygote | 13747 | 53910 | 7S alpha’- | 3 | 475.6 | 188.1 | |
| Homozygote | 14269 | 53912 | 7S alpha’- | 3 | 352.9 | 119.9 | |
| Empty | 13894 | 53911 | 7S alpha’- | 11 | 216.5 | 49.8 | |
| Empty | 14202 | 53911 | 7S alpha’-ilvA219 | 10 | 215.1 | 54.9 | |
| Empty | 14269 | 53912 | 7S alpha’- | 5 | 228.0 | 61.6 | |
| Empty | A4922 | | Basic germplasm | 6 | 190.9 | 29.3 |
Ala level in the soybean plants of table 10K. expression threonine deaminase
| Connectivity | Incident | pMON | Gene | N | Mean value | Standard variance | |
| Heterozygote | 13894 | 53911 | 7S alpha’- | 12 | 56.1 | 13.5 | |
| Heterozygote | 14202 | 53911 | 7S alpha’- | 11 | 64.9 | 8.8 | |
| Heterozygote | 14269 | 53912 | 7S alpha’-ilvA466 | 8 | 69.5 | 7.9 | |
| Homozygote | 13747 | 53910 | 7S alpha’- | 3 | 82.0 | 44.5 | |
| Homozygote | 14269 | 53912 | 7S alpha’- | 3 | 60.2 | 7.8 | |
| Empty | 13894 | 53911 | 7S alpha’- | 11 | 65.7 | 11.6 | |
| Empty | 14202 | 53911 | 7S alpha’-ilvA219 | 10 | 70.9 | 8.4 | |
| Empty | 14269 | 53912 | 7S alpha’- | 5 | 76.2 | 8.3 | |
| Empty | A4922 | | Basic germplasm | 6 | 72.8 | 8.7 |
Tyr level in the soybean plants of table 10L. expression threonine deaminase
| Connectivity | Incident | pMON | Gene | N | Mean value | Standard variance | |
| Heterozygote | 13894 | 53911 | 7S alpha’- | 12 | 15.5 | 53.6 | |
| Heterozygote | 14202 | 53911 | 7S alpha’- | 11 | 25.3 | 83.8 | |
| Heterozygote | 14269 | 53912 | 7S alpha’-ilvA466 | 8 | 0.0 | 0.0 | |
| Homozygote | 13747 | 53910 | 7S alpha’- | 3 | 0.0 | 0.0 | |
| Homozygote | 14269 | 53912 | 7S alpha’- | 3 | 71.0 | 123.0 | |
| Empty | 13894 | 53911 | 7S alpha’- | 11 | 22.9 | 75.9 | |
| Empty | 14202 | 53911 | 7S alpha’-ilvA219 | 10 | 18.3 | 57.8 | |
| Empty | 14269 | 53912 | 7S alpha’- | 5 | 41.1 | 91.9 | |
| Empty | A4922 | | Basic germplasm | 6 | 75.4 | 116.8 |
Val level in the soybean plants of table 10M. expression threonine deaminase
| Connectivity | Incident | pMON | Gene | N | Mean value | Standard variance | |
| Heterozygote | 13894 | 53911 | 7S alpha’- | 12 | 51.2 | 30.9 | |
| Heterozygote | 14202 | 53911 | 7S alpha’- | 11 | 39.3 | 12.2 | |
| Heterozygote | 14269 | 53912 | 7S alpha’-ilvA466 | 8 | 27.0 | 5.6 | |
| Homozygote | 13747 | 53910 | 7S alpha’- | 3 | 38.3 | 14.6 | |
| Homozygote | 14269 | 53912 | 7S alpha’- | 3 | 27.6 | 5.8 | |
| Empty | 13894 | 53911 | 7S alpha’- | 11 | 31.5 | 4.7 | |
| Empty | 14202 | 53911 | 7S alpha’-ilvA219 | 10 | 31.8 | 5.0 | |
| Empty | 14269 | 53912 | 7S alpha’- | 5 | 26.1 | 6.3 | |
| Empty | A4922 | | Basic germplasm | 6 | 31.3 | 6.3 |
Met level in the soybean plants of table 10N. expression threonine deaminase
| Connectivity | Incident | pMON | Gene | N | Mean value | Standard variance | |
| Heterozygote | 13894 | 53911 | 7S alpha’- | 12 | 13.7 | 3.8 | |
| Heterozygote | 14202 | 53911 | 7S alpha’- | 11 | 13.3 | 4.6 | |
| Heterozygote | 14269 | 53912 | 7S alpha’-ilvA466 | 8 | 12.6 | 3.9 | |
| Homozygote | 13747 | 53910 | 7S alpha’- | 3 | 35.0 | 10.9 | |
| Homozygote | 14269 | 53912 | 7S alpha’- | 3 | 21.3 | 4.4 | |
| Empty | 13894 | 53911 | 7S alpha’- | 11 | 9.4 | 1.7 | |
| Empty | 14202 | 53911 | 7S alpha’-ilvA219 | 10 | 9.7 | 1.7 | |
| Empty | 14269 | 53912 | 7S alpha’- | 5 | 9.1 | 1.6 | |
| Empty | A4922 | | Basic germplasm | 6 | 10.0 | 0.9 |
Trp level in the soybean plants of table 10O. expression threonine deaminase
| Connectivity | Incident | pMON | Gene | N | Mean value | Standard variance | |
| Heterozygote | 13894 | 53911 | 7S alpha’- | 12 | 172.7 | 38.1 | |
| Heterozygote | 14202 | 53911 | 7S alpha’- | 11 | 165.6 | 42.5 | |
| Heterozygote | 14269 | 53912 | 7S alpha’-ilvA466 | 8 | 166.6 | 38.2 | |
| Homozygote | 13747 | 53910 | 7S alpha’- | 3 | 152.5 | 75.4 | |
| Homozygote | 14269 | 53912 | 7S alpha’- | 3 | 163.9 | 4.3 | |
| Empty | 13894 | 53911 | 7S alpha’- | 11 | 127.6 | 20.7 | |
| Empty | 14202 | 53911 | 7S alpha’-ilvA219 | 10 | 121.0 | 18.0 | |
| Empty | 14269 | 53912 | 7S alpha’- | 5 | 131.7 | 12.6 | |
| Empty | A4922 | | Basic germplasm | 6 | 130.2 | 10.0 |
Phe level in the soybean plants of table 10P. expression threonine deaminase
| Connectivity | Incident | pMON | Gene | N | Mean value | Standard variance | |
| Heterozygote | 13894 | 53911 | 7S alpha’- | 12 | 26.2 | 8.5 | |
| Heterozygote | 14202 | 53911 | 7S alpha’- | 11 | 25.1 | 9.0 | |
| Heterozygote | 14269 | 53912 | 7S alpha’-ilvA466 | 8 | 20.2 | 4.7 | |
| Homozygote | 13747 | 53910 | 7S alpha’- | 3 | 16.7 | 2.5 | |
| Homozygote | 14269 | 53912 | 7S alpha’- | 3 | 29.1 | 0.4 | |
| Empty | 13894 | 53911 | 7S alpha’- | 11 | 17.1 | 2.6 | |
| Empty | 14202 | 53911 | 7S alpha’-ilvA219 | 10 | 17.8 | 2.6 | |
| Empty | 14269 | 53912 | 7S alpha’- | 5 | 16.7 | 2.9 | |
| Empty | A4922 | | Basic germplasm | 6 | 17.9 | 1.4 |
Leu level in the soybean plants of table 10Q. expression threonine deaminase
| Connectivity | Incident | pMON | Gene | N | Mean value | Standard variance | |
| Heterozygote | 13894 | 53911 | 7S alpha’- | 12 | 30.2 | 15.7 | |
| Heterozygote | 14202 | 53911 | 7S alpha’- | 11 | 24.7 | 11.2 | |
| Heterozygote | 14269 | 53912 | 7S alpha’-ilvA466 | 8 | 18.1 | 4.9 | |
| Homozygote | 13747 | 53910 | 7S alpha’- | 3 | 39.0 | 11.4 | |
| Homozygote | 14269 | 53912 | 7S alpha’- | 3 | 32.5 | 2.6 | |
| Empty | 13894 | 53911 | 7S alpha’- | 11 | 12.8 | 1.9 | |
| Empty | 14202 | 53911 | 7S alpha’-ilvA219 | 10 | 13.1 | 2.5 | |
| Empty | 14269 | 53912 | 7S alpha’- | 5 | 12.5 | 1.6 | |
| Empty | A4922 | | Basic germplasm | 6 | 12.6 | 1.2 |
Lys level in the soybean plants of table 10R. expression threonine deaminase
| Connectivity | Incident | pMON | Gene | N | Mean value | Standard variance | |
| Heterozygote | 13894 | 53911 | 7S alpha’- | 12 | 62.7 | 47.5 | |
| Heterozygote | 14202 | 53911 | 7S alpha’- | 11 | 36.5 | 22.0 | |
| Heterozygote | 14269 | 53912 | 7S alpha’-ilvA466 | 8 | 24.6 | 11.6 | |
| Homozygote | 13747 | 53910 | 7S alpha’- | 3 | 14.9 | 2.8 | |
| Homozygote | 14269 | 53912 | 7S alpha’- | 3 | 47.8 | 27.6 | |
| Empty | 13894 | 53911 | 7S alpha’- | 11 | 17.7 | 3.8 | |
| Empty | 14202 | 53911 | 7S alpha’-ilvA219 | 10 | 20.9 | 7.6 | |
| Empty | 14269 | 53912 | 7S alpha’- | 5 | 18.8 | 2.7 | |
| Empty | A4922 | | Basic germplasm | 6 | 19.7 | 3.1 |
That lists among the table 10A to 10R shows amino acid whose analytical results, and in the soybean plants that the polynucleotide with the coding threonine deaminase transformed, a large amount of amino acid whose concentration all increase to some extent.Connectivity makes data separating.Also provide a kind of process comprehensive assessment, wherein removed the influence of connectivity.Method (Snedecor and Cochran, In:Statistical Methods, 1982 with Pearson; The JMP statistical software (SAS Institute, Cary, NC, USA)), data are carried out correlation analysis.The value of numeral is being represented Pearson ' s incidence coefficient (r).0.60 it is or higher on the occasion of show amino acid concentration and Ile concentration positive association.Under the heterozygosis condition, amino acid Asn, Ser, His, Gly, Thr, Arg, Val, Met, Phe, Leu and Lys all with the horizontal positive association of Ile.Under the condition of isozygotying, Phe and Lys and Ile concentration positive association.
Table 11.Ile concentration and other amino acid whose related situation
| Amino acid | Heterozygosis | Isozygoty | Empty | Comprehensively (Pooled) |
| Asp | 0.0980 | -0.8622 | 0.4567 | -0.0809 |
| Glu | -0.0054 | -0.7400 | 0.4711 | -0.0134 |
| Asn | 0.6754 | -0.4476 | 0.3528 | 0.0925 |
| Ser | 0.7795 | 0.3465 | 0.2511 | 0.6755 |
| Gln | 0.3713 | -0.4478 | 0.3311 | 0.0297 |
| His | 0.9667 | -0.1998 | 0.5769 | 0.9074 |
| Gly | 0.7912 | -0.4385 | 0.1686 | 0.0812 |
| Thr | 0.6827 | 0.1135 | 0.2806 | 0.3667 |
| Arg | 0.6686 | 0.1362 | 0.4715 | 0.6214 |
| Ala | -0.0320 | -0.1327 | 0.3596 | -0.1400 |
| Tyr | 0.1155 | 0.1180 | 0.0082 | -0.0047 |
| Val | 0.9561 | -0.1752 | 0.4279 | 0.8940 |
| Met | 0.8940 | -0.2349 | 0.6515 | 0.4118 |
| Trp | 0.5249 | 0.4908 | 0.2077 | 0.6049 |
| Phe | 0.8488 | 0.8805 | 0.5792 | 0.8489 |
| Leu | 0.9703 | 0.0609 | 0.7700 | 0.8825 |
| Lys | 0.8287 | 0.6265 | 0.8070 | 0.8439 |
All publications and patent all by reference mode are comprised into this paper, this with comprise separately that by reference this paper's into is the same.Shown in the invention is not restricted to and described detail, and should understand like this, within the spirit and scope of the present invention that limited by statement, can carry out a variety of variations and improvement.
Sequence table
<110>Weaver，Lisa M
Mitsky，Timothy A
Rapp，William D
Gruys，Kenneth J
Liang，Jihong
＜120〉plant of one or more amino acid whose levels increases
<130>REN-00-095
<140>US 60/468,727
<141>2003-05-07
<160>22
<170>PatentIn version 3.2
<210>1
<211>1714
<212>DNA
＜213〉intestinal bacteria
<400>1
ctcgaggtga caaagcctgg acgccgaaaa atcgtgaacg tcaggtctcc tttgccctgc 60
gtgcttatgc cagcctggca accagcgccg acaaaggcgc ggtgcgcgat aaatcgaaac 120
tggggggtta ataatggctg actcgcaacc cctgtccggt gctccggaag gtgccgaata 180
tttaagagca gtgctgcgcg cgccggttta cgaggcggcg caggttacgc cgctacaaaa 240
aatggaaaaa ctgtcgtcgc gtcttgataa cgtcattctg gtgaagcgcg aagatcgcca 300
gccagtgcac agctttaagc tgcgcggcgc atacgccatg atggcgggcc tgacggaaga 360
acagaaagcg cacggcgtga tcactgcttc tgcgggtaac cacgcgcagg gcgtcgcgtt 420
ttcttctgcg cggttaggcg tgaaggccct gatcgttatg ccaaccgcca ccgccgacat 480
caaagtcgac cggctgcgcg gcttcggcgg cgaagtgctg ctccacggcg cgaactttga 540
tgaagcgaaa cgcaaagcga tcgaactgtc acagcagcag gggttcacct gggtgccgcc 600
gttcgaccat ccgatggtga ttgccgggca aggcacgctg gcgctggaac tgctccagca 660
ggacgcccat ctcgaccgcg tatttgtgcc agtcggcggc ggcggtctgg ctgcttgcgt 720
ggcggtgctg atcaaacaac tgatgccgca aatcaaagtg atcgccgtag aagcggaaga 780
ctccgcctgc ctgaaagcag cgctggatgc gggtcatccg gttgatctgc cgcgcgtagg 840
gctatttgct gaaggcgtag cggtaaaacg catcggtgac gaaaccttcc gtttatgcca 900
ggagtatctc gacgacatca tcaccgtcga tagcgatgcg atctgtgcgg cgatgaagga 960
tttattcgaa gatgtgcgcg cggtggcgga accctctggc gcgctggcgc tggcgggaat 1020
gaaaaaatat atcgccctgc acaacattcg cggcgaacgg ctggcgcata ttctttccgg 1080
tgccaacgtg aacttccacg gcctgcgcta cgtctcagaa cgctgcgaac tggtcgaaca 1140
gcgtgaagcg ttgttggcgg tgaccattcc ggaagaaaaa ggcagcttcc tcaaattctg 1200
ccaactgctt ggcgggcgtt cggtcaccga gttcaactac cgttttgccg atgccaaaaa 1260
cgcctgcatc tttgtcggtg tgcgcctgag ccgcggcctc gaagagcgca aagaaatttt 1320
gcagatgctc aacgacggcg gctacagcgt ggttgatctc tccgacgacg aaatggcgaa 1380
gctacacgtg cgctatatgg tcggcggacg tccatcgcat ccgttgcagg aacgcctcta 1440
cagcttcgaa ttcccggaat caccgggcgc gctgctgcgc ttcctcaaca cgctgggtac 1500
gtactggaac atttctttgt tccactatcg cagccatggc accgactacg ggcgcgtact 1560
ggcggcgttc gaacttggcg accatgaacc ggatttcgaa acccggctga atgagctggg 1620
ctacgattgc cacgacgaaa ccaataaccc ggcgttcagg ttctttttgg cgggttaggg 1680
aaaaatgcct gatagcgctt ccgttatcag gcct 1714
<210>2
<211>514
<212>PRT
＜213〉intestinal bacteria
<400>2
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr
1 5 10 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Arg Leu Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Arg Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Cys Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Gly Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Val Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Leu Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu
500 505 510
Ala Gly
<210>3
<211>514
<212>PRI
＜213〉intestinal bacteria
<400>3
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr
1 5 10 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Arg Leu Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Arg Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Cys Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Gly Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Val Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Phe Leu
435 440 445
Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Leu Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu
500 505 510
Ala Gly
<210>4
<211>514
<212>PRT
＜213〉intestinal bacteria
<400>4
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr
1 5 10 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Arg Leu Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Arg Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Cys Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Gly Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Val Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Phe Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu
500 505 510
Ala Gly
<210>5
<211>514
<212>PRT
＜213〉intestinal bacteria
<400>5
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr
1 5 l0 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Arg Leu Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Arg Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
l65 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Cys Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Gly Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Val Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Tyr Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu
500 505 510
Ala Gly
<210>6
<211>514
<212>PRT
＜213〉intestinal bacteria
<400>6
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr
1 5 10 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Arg Leu Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Arg Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Cys Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys TyrIle Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Gly Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Val Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Pro Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu
500 505 510
Ala Gly
<210>7
<211>514
<212>PRT
＜213〉intestinal bacteria
<400>7
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr
1 5 10 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Arg Leu Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Arg Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Cys Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Gly Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Val Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Glu Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu
500 505 510
Ala Gly
<210>8
<211>514
<212>PRT
＜213〉intestinal bacteria
<400>8
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr
1 5 10 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Arg Leu Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Arg Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Cys Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Gly Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Val Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Thr Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu
500 505 510
Ala Gly
<210>9
<211>514
<212>PRT
＜213〉intestinal bacteria
<400>9
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr
1 5 10 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Arg Leu Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Arg Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Cys Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Gly Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Val Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Gln Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu
500 505 510
Ala Gly
<210>10
<211>514
<212>PRT
＜213〉intestinal bacteria
<400>10
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr
1 5 10 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Arg Leu Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Arg Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Cys Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Gly Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Val Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Ile Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu
500 505 510
Ala Gly
<210>11
<211>514
<212>PRT
＜213〉intestinal bacteria
<400>11
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr
1 5 10 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Arg Leu Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Arg Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Cys Val Ala Val Lau Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Gly Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Val Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Val Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu
500 505 510
Ala Gly
<210>12
<211>514
<212>PRT
＜213〉intestinal bacteria
<400>12
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr
1 5 10 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Arg Leu Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Arg Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Cys Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Gly Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Val Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Met Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu
500 505 510
Ala Gly
<210>13
<211>514
<212>PRT
＜213〉intestinal bacteria
<400>13
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr
1 5 10 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Arg Leu Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Arg Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Cys Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Gly Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Val Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Lys Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu
500 505 510
Ala Gly
<210>14
<211>1544
<212>DNA
＜213〉artificial
<220>
＜223〉variation allelotrope
<400>14
atggctgact cgcaacccct gtccggtgct ccggaaggtg ccgaatattt aagagcagtg 60
ctgcgcgcgc cggtttacga ggcggcgcag gttacgccgc tacaaaaaat ggaaaaactg 120
tcgtcgcgtc ttgataacgt cattctggtg aagcgcgaag atcgccagcc agtgcacagc 180
tttaagctgc gcggcgcata cgccatgatg gcgggcctga cggaagaaca gaaagcgcac 240
ggcgtgatca ctgcttctgc gggtaaccac gcgcagggcg tcgcgttttc ttctgcgcgg 300
ttaggcgtga aggccctgat cgttatgcca accgccaccg ccgacatcaa agtcgaccgg 360
ctgcgcggct tcggcggcga agtgctgctc cacggcgcga actttgatga agcgaaacgc 420
aaagcgatcg aactgtcaca gcagcagggg ttcacctggg tgccgccgtt cgaccatccg 480
atggtgattg ccgggcaagg cacgctggcg ctggaactgc tccagcagga cgcccatctc 540
gaccgcgtat ttgtgccagt cggcggcggc ggtctggctg cttgcgtggc ggtgctgatc 600
aaacaactga tgccgcaaat caaagtgatc gccgtagaag cggaagactc cgcctgcctg 660
aaagcagcgc tggatgcggg tcatccggtt gatctgccgc gcgtagggct atttgctgaa 720
ggcgtagcgg taaaacgcat cggtgacgaa accttccgtt tatgccagga gtatctcgac 780
gacatcatca ccgtcgatag cgatgcgatc tgtgcggcga tgaaggattt attcgaagat 840
gtgcgcgcgg tggcggaacc ctctggcgcg ctggcgctgg cgggaatgaa aaaatatatc 900
gccctgcaca acattcgcgg cgaacggctg gcgcatattc tttccggtgc caacgtgaac 960
ttccacggcc tgcgctacgt ctcagaacgc tgcgaactgg tcgaacagcg tgaagcgttg 1020
ttggcggtga ccattccgga agaaaaaggc agcttcctca aattctgcca actgcttggc 1080
gggcgttcgg tcaccgagtt caactaccgt tttgccgatg ccaaaaacgc ctgcatcttt 1140
gtcggtgtgc gcctgagccg cggcctcgaa gagcgcaaag aaattttgca gatgctcaac 1200
gacggcggct acagcgtggt tgatctctcc gacgacgaaa tggcgaagct acacgtgcgc 1260
tatatggtcg gcggacgtcc atcgcatccg ttgcaggaac gcctctacag cttcgagttc 1320
ccggaatcac cgggcgcgtt cctgcgcttc ctcaacacgc tgggtacgta ctggaacatt 1380
tctttgttcc actatcgcag ccatggcacc gactacgggc gcgtactggc ggcgttcgaa 1440
cttggcgacc atgaaccgga tttcgaaacc cggctgaatg agctgggcta cgattgccac 1500
gacgaaacca ataacccggc gttcaggttc tttttggcgg gtta 1544
<210>15
<211>1544
<212>DNA
＜213〉artificial
<220>
＜223〉variation allelotrope
<400>15
atggctgact cgcaacccct gtccggtgct ccggaaggtg ccgaatattt aagagcagtg 60
ctgcgcgcgc cggtttacga ggcggcgcag gttacgccgc tacaaaaaat ggaaaaactg 120
tcgtcgcgtc ttgataacgt cattctggtg aagcgcgaag atcgccagcc agtgcacagc 180
tttaagctgc gcggcgcata cgccatgatg gcgggcctga cggaagaaca gaaagcgcac 240
ggcgtgatca ctgcttctgc gggtaaccac gcgcagggcg tcgcgttttc ttctgcgcgg 300
ttaggcgtga aggccctgat cgttatgcca accgccaccg ccgacatcaa agtcgaccgg 360
ctgcgcggct tcggcggcga agtgctgctc cacggcgcga actttgatga agcgaaacgc 420
aaagcgatcg aactgtcaca gcagcagggg ttcacctggg tgccgccgtt cgaccatccg 480
atggtgattg ccgggcaagg cacgctggcg ctggaactgc tccagcagga cgcccatctc 540
gaccgcgtat ttgtgccagt cggcggcggc ggtctggctg cttgcgtggc ggtgctgatc 600
aaacaactga tgccgcaaat caaagtgatc gccgtagaag cggaagactc cgcctgcctg 660
aaagcagcgc tggatgcggg tcatccggtt gatctgccgc gcgtagggct atttgctgaa 720
ggcgtagcgg taaaacgcat cggtgacgaa accttccgtt tatgccagga gtatctcgac 780
gacatcatca ccgtcgatag cgatgcgatc tgtgcggcga tgaaggattt attcgaagat 840
gtgcgcgcgg tggcggaacc ctctggcgcg ctggcgctgg cgggaatgaa aaaatatatc 900
gccctgcaca acattcgcgg cgaacggctg gcgcatattc tttccggtgc caacgtgaac 960
ttccacggcc tgcgctacgt ctcagaacgc tgcgaactgg tcgaacagcg tgaagcgttg 1020
ttggcggtga ccattccgga agaaaaaggc agcttcctca aattctgcca actgcttggc 1080
gggcgttcgg tcaccgagtt caactaccgt tttgccgatg ccaaaaacgc ctgcatcttt 1140
gtcggtgtgc gcctgagccg cggcctcgaa gagcgcaaag aaattttgca gatgctcaac 1200
gacggcggct acagcgtggt tgatctctcc gacgacgaaa tggcgaagct acacgtgcgc 1260
tatatggtcg gcggacgtcc atcgcatccg ttgcaggaac gcctctacag cttcgaattc 1320
ccggaatcac cgggcgcgct gctgcgcttc ctcaacacgc tgggtacgta ctggaacatt 1380
tctttgttcc actatcgcag ccacggcacc gactacgggc gcgtactggc ggcgttcgaa 1440
tttggcgacc atgaaccgga tttcgaaacc cggctgaatg agctgggcta cgattgccac 1500
gacgaaacca ataacccggc gttcaggttc tttttggcgg gtta 1544
<210>16
<211>1644
<212>DNA
＜213〉intestinal bacteria
<400>16
atggcgcaca gtgggtggta catgcgttgc gggcacaggg tgtgaacacc gttttcggtt 60
atccgggtgg cgcaattatg ccggtttacg atgcattgta tgacggcggc gtggagcact 120
tgctatgccg acatgagcag ggtgcggcaa tggcggctat cggttatgct cgtgctaccg 180
gcaaaactgg cgtatgtatc gccacgtctg gtccgggcgc aaccaacctg ataaccgggc 240
ttgcggacgc actgttagat tccatccctg ttgttgccat caccggtcaa gtgtccgcac 300
cgtttatcgg cactgacgca tttcaggaag tggatgtcct gggattgtcg ttagcctgta 360
ccaagcatag ctttctggtg cagtcgctgg aagagttgcc gcgcatcatg gctgaagcat 420
tcgacgttgc ctgctcaggt cgtcctggtc cggttctggt cgatatccca aaagatatcc 480
agttagccag cggtgacctg gaaccgtggt tcaccaccgt tgaaaacgaa gtgactttcc 540
cacatgccga agttgagcaa gcgcgccaga tgctggcaaa agcgcaaaaa ccgatgctgt 600
acgttggcgg tggcgtgggt atggcgcagg cagttccggc tttgcgtgaa tttctcgctg 660
ccacaaaaat gcctgccacc tgtacgctga aagggctggg cgcagtagaa gcagattatc 720
cgtactatct gggcatgctg gggatgcacg gcaccaaagc ggcaaacttc gcggtgcagg 780
agtgtgacct gctgatcgcc gtgggcgcac gttttgatga ccgggtgacc ggcaaactga 840
acaccttcgc gccacacgcc agtgttatcc atatggatat cgacccggca gaaatgaaca 900
agctgcgtca ggcacatgtg gcattacaag gtgatttaaa tgctctgtta ccagcattac 960
agcagccgtt aaatcaatat gactggcagc aacactgcgc gcagctgcgt gatgaacatt 1020
cctggcgtta cgaccatccc ggtgacgcta tctacgcgcc gttgttgtta aaacaactgt 1080
cggatcgtaa acctgcggat tgcgtcgtga ccacagatgt ggggcagcac cagatgtggg 1140
ctgcgcagca catcgcccac actcgcccgg aaaatttcat cacctccagc ggtttaggta 1200
ccatgggttt tggtttaccg gcggcggttg gcgcacaagt cgcgcgaccg aacgataccg 1260
ttgtctgtat ctccggtgac ggctctttca tgatgaatgt gcaagagctg ggcaccgtaa 1320
aacgcaagca gttaccgttg aaaatcgtct tactcgataa ccaacggtta gggatggttc 1380
gacaatggca gcaactgttt tttcaggaac gatacagcga aaccaccctt actgataacc 1440
ccgatttcct catgttagcc agcgccttcg gcatccatgg ccaacacatc acccggaaag 1500
accaggttga agcggcactc gacaccatgc tgaacagtga tgggccatac ctgcttcatg 1560
tctcaatcga cgaacttgag aacgtctggc cgctggtgcc gcctggcgcc agtaattcag 1620
aaatgttgga gaaattatca tgag 1644
<210>17
<211>285
<212>DNA
＜213〉intestinal bacteria
<400>17
tggggaattc tcatgatgca acatcaggtc aatgtatcgg ctcgcttcaa tccagaaacc 60
ttagaacgtg ttttacgcgt ggtgcgtcat cgtggtttcc acgtctgctc aatgaatatg 120
gccgccgcca gcgatgcaca aaatataaat atcgaattga ccgttgccag cccacggtcg 180
gtcgacttac tgtttagtca gttaaataaa ctggtggacg tcgcacacgt tgccatctgc 240
cagagcacaa ccacatcaca acaaatccgc gcctgataag aattc 285
<210>18
<211>1201
<212>DNA
＜213〉intestinal bacteria
<400>18
atggcgcaca gtgggtggta catgcgttgc gggcacaggg tgtgaacacc gttttcggtt 60
atccgggtgg cgcaattatg ccggtttacg atgcattgta tgacggcggc gtggagcact 120
tgctatgccg acatgagcag ggtgcggcaa tggcggctat cggttatgct cgtgctaccg 180
gcaaaactgg cgtatgtatc gccacgtctg gtccgggcgc aaccaacctg ataaccgggc 240
ttgcggacgc actgttagat tccatccctg ttgttgccat caccggtcaa gtgtccgcac 300
cgtttatcgg cactgacgca tttcaggaag tggatgtcct gggattgtcg ttagcctgta 360
ccaagcatag ctttctggtg cagtcgctgg aagagttgcc gcgcatcatg gctgaagcat 420
tcgacgttgc ctgctcaggt cgtcctggtc cggttctggt cgatatccca aaagatatcc 480
agttagccag cggtgacctg gaaccgtggt tcaccaccgt tgaaaacgaa gtgactttcc 540
cacatgccga agttgagcaa gcgcgccaga tgctggcaaa agcgcaaaaa ccgatgctgt 600
acgttggcgg tggcgtgggt atggcgcagg cagttccggc tttgcgtgaa tttctcgctg 660
ccacaaaaat gcctgccacc tgtacgctga aagggctggg cgcagtagaa gcagattatc 720
cgtactatct gggcatgctg gggatgcacg gcaccaaagc ggcaaacttc gcggtgcagg 780
agtgtgacct gctgatcgcc gtgggcgcac gttttgatga ccgggtgacc ggcaaactga 840
acaccttcgc gccacacgcc agtgttatcc atatggatat cgacccggca gaaatgaaca 900
agctgcgtca ggcacatgtg gcattacaag gtgatttaaa tgctctgtta ccagcattac 960
agcagccgtt aaatcaatat gactggcagc aacactgcgc gcagctgcgt gatgaacatt 1020
cctggcgtta cgaccatccc ggtgacgcta tctacgcgcc gttgttgtta aaacaactgt 1080
cggatcgtaa acctgcggat tgcgtcgtga ccacagatgt ggggcagcac cagatgtggg 1140
ctgcgcagca catcgcccac actcgcccgg aaaatttcat cacctccagc ggtttaggta 1200
c 1201
<210>19
<211>264
<212>DNA
＜213〉Arabidopis thaliana
<400>19
atggcttcct ctatgctctc ttccgctact atggttgcct ctccggctca ggccactatg 60
gtcgctcctt tcaacggact taagtcctcc gctgccttcc cagccacccg caaggctaac 120
aacgacatta cttccatcac aagcaacggc ggaagagtta actgcatgca ggtgtggcct 180
ccgattggaa agaagaagtt tgagactctc tcttaccttc ctgaccttac cgattccggt 240
ggtcgcgtca actgcatgca ggcc 264
<210>20
<211>443
<212>DNA
＜213〉intestinal bacteria
<400>20
catgggtttt ggtttaccgg cggcggttgg cgcacaagtc gcgcgaccga acgataccgt 60
tgtctgtatc tccggtgacg gctctttcat gatgaatgtg caagagctgg gcaccgtaaa 120
acgcaagcag ttaccgttga aaatcgtctt actcgataac caacggttag ggatggttcg 180
acaatggcag caactgtttt ttcaggaacg atacagcgaa accaccctta ctgataaccc 240
cgatttcctc atgttagcca gcgccttcgg catccatggc caacacatca cccggaaaga 300
ccaggttgaa gcggcactcg acaccatgct gaacagtgat gggccatacc tgcttcatgt 360
ctcaatcgac gaacttgaga acgtctggcc gctggtgccg cctggcgcca gtaattcaga 420
aatgttggag aaattatcat gag 443
<210>21
<211>514
<212>PRT
＜213〉artificial
<220>
＜223〉varient polynucleotide
<400>21
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr
1 5 10 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Ala Val Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Ala Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Gly Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Gly Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Gly Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Leu Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu
500 505 510
Ala Gly
<210>22
<211>592
<212>PRT
＜213〉Arabidopis thaliana
<400>22
Met Asn Ser Val Gln Leu Pro Thr Ala Gln Ser Ser Leu Arg Ser His
1 5 10 15
Ile His Arg Pro Ser Lys Pro Val Val Gly Phe Thr His Phe Ser Ser
20 25 30
Arg Ser Arg Ile Ala Val Ala Val Leu Ser Arg Asp Glu Thr Ser Met
35 40 45
Thr Pro Pro Pro Pro Lys Leu Pro Leu Pro Arg Leu Lys Val Ser Pro
50 55 60
Asn Ser Leu Gln Tyr Pro Ala Gly Tyr Leu Gly Ala Val Pro Glu Arg
65 70 75 80
Thr Asn Glu Ala Glu Asn Gly Ser Ile Ala Glu Ala Met Glu Tyr Leu
85 90 95
Thr Asn Ile Leu Ser Thr Lys Val Tyr Asp Ile Ala Ile Glu Ser Pro
100 105 110
Leu Gln Leu Ala Lys Lys Leu Ser Lys Arg Leu Gly Val Arg Met Tyr
115 120 125
Leu Lys Arg Glu Asp Leu Gln Pro Val Phe Ser Phe Lys Leu Arg Gly
130 135 140
Ala Tyr Asn Met Met Val Lys Leu Pro Ala Asp Gln Leu Ala Lys Gly
145 150 155 160
Val Ile Cys Ser Ser Ala Gly Asn His Ala Gln Gly Val Ala Leu Ser
165 170 175
Ala Ser Lys Leu Gly Cys Thr Ala Val Ile Val Met Pro Val Thr Thr
180 185 190
Pro Glu Ile Lys Trp Gln Ala Val Glu Asn Leu Gly Ala Thr Val Val
195 200 205
Leu Phe Gly Asp Ser Tyr Asp Gln Ala Gln Ala His Ala Lys Ile Arg
210 215 220
Ala Glu Glu Glu Gly Leu Thr Phe Ile Pro Pro Phe Asp His Pro Asp
225 230 235 240
Val Ile Ala Gly Gln Gly Thr Val Gly Met Glu Ile Thr Arg Gln Ala
245 250 255
Lys Gly Pro Leu His Ala Ile Phe Val Pro Val Gly Gly Gly Gly Leu
260 265 270
Ile Ala Gly Ile Ala Ala Tyr Val Lys Arg Val Ser Pro Glu Val Lys
275 280 285
Ile Ile Gly Val Glu Pro Ala Asp Ala Asn Ala Met Ala Leu Ser Leu
290 295 300
His His Gly Glu Arg Val Ile Leu Asp Gln Val Gly Gly Phe Ala Asp
305 310 315 320
Gly Val Ala Val Lys Glu Val Gly Glu Glu Thr Phe Arg Ile Ser Arg
325 330 335
Asn Leu Met Asp Gly Val Val Leu Val Thr Arg Asp Ala Ile Cys Ala
340 345 350
Ser Ile Lys Asp Met Phe Glu Glu Lys Arg Asn Ile Leu Glu Pro Ala
355 360 365
Gly Ala Leu Ala Leu Ala Gly Ala Glu Ala Tyr Cys Lys Tyr Tyr Gly
370 375 380
Leu Lys Asp Val Asn Val Val Ala Ile Thr Ser Gly Ala Asn Met Asn
385 390 395 400
Phe Asp Lys Leu Arg Ile Val Thr Glu Leu Ala Asn Val Gly Arg Gln
405 410 415
Gln Glu Ala Val Leu Ala Thr Leu Met Pro Glu Lys Pro Gly Ser Phe
420 425 430
Lys Gln Phe Cys Glu Leu Val Gly Pro Met Asn Ile Ser Glu Phe Lys
435 440 445
Tyr Arg Cys Ser Ser Glu Lys Glu Ala Val Val Leu Tyr Ser Val Gly
450 455 460
Val His Thr Ala Gly Glu Leu Lys Ala Leu Gln Lys Arg Met Glu Ser
465 470 475 480
Ser Gln Leu Lys Thr Val Asn Leu Thr Thr Ser Asp Leu Val Lys Asp
485 490 495
His Leu Arg Tyr Leu Met Gly Gly Arg Ser Thr Val Gly Asp Glu Val
500 505 510
Leu Cys Arg Phe Thr Phe Pro Glu Arg Pro Gly Ala Leu Met Asn Phe
515 520 525
Leu Asp Ser Phe Ser Pro Arg Trp Asn Ile Thr Leu Phe His Tyr Arg
530 535 540
Gly Gln Gly Glu Thr Gly Ala Asn Val Leu Val Gly Ile Gln Val Pro
545 550 555 560
Glu Gln Glu Met Glu Glu Phe Lys Asn Arg Ala Lys Ala Leu Gly Tyr
565 570 575
Asp Tyr Phe Leu Val Ser Asp Asp Asp Tyr Phe Lys Leu Leu Met His
580 585 590
Claims (49)
1. DNA construct, described construct comprises a plurality of expression of plants boxes, wherein first expression cassette is included in the promotor that function is arranged in the vegetable cell, this promotor is operably connected to the exogenous polynucleotide of feeding back insensitive threonine deaminase with coding, second expression cassette is included in the promotor that function is arranged in the vegetable cell, and this promotor is operably connected with the exogenous polynucleotide of coding AHAS.
2. DNA construct, described construct comprises a plurality of expression of plants boxes, wherein first expression cassette is included in the promotor that function is arranged in the vegetable cell, this promotor is operably connected to the exogenous polynucleotide of feeding back insensitive threonine deaminase with coding, second expression cassette comprises the big subunit of AHAS, the 3rd expression cassette is included in the promotor that function is arranged in the vegetable cell, and this promotor is operably connected with the exogenous polynucleotide of coding AHAS small subunit.
3. DNA construct as claimed in claim 1 or 2, each in the wherein said promotor all are seed enhancement type promotors.
4. DNA construct as claimed in claim 1 or 2, each in the wherein said promotor all are selected from the group that is made of napin, 7S alpha, 7S alpha ', 7S beta, USP 88, enhanced USP 88, Arcelin 5 and Oleosin.
5. wherein there are at least two kinds of different seed enhancement type promotors in DNA construct as claimed in claim 3.
6. DNA construct as claimed in claim 1 or 2, wherein, described first box comprises the polynucleotide of coding to the threonine deaminase that feeds back the insensitive SEQ of comprising ID NO:22.
7. DNA construct as claimed in claim 1 or 2, wherein, described first box comprises the exogenous polynucleotide of coding threonine deaminase variation allelotrope or its subunit, and described variation allelotrope or its subunit comprise the aminoacid replacement on L447F or L481F or L481Y or L481P or L481E or L481T or L481Q or L481I or L481V or L481M or the L481K position.
8. DNA construct as claimed in claim 1 or 2, wherein, the described coding threonine deaminase allelic polynucleotide that make a variation are SEQ ID NO:2, wherein comprise the aminoacid replacement on L447F or L481F or L481Y or L481P or L481E or L481T or L481Q or L481I or L481V or L481M or the L481K position.
9. DNA construct as claimed in claim 1 or 2, wherein, described first box also comprises the polynucleotide of the plastid transit peptides of encoding, and described polynucleotide are operably connected with the polynucleotide of the described threonine deaminase of coding.
10. DNA construct as claimed in claim 2, wherein, described second expression cassette comprises the polynucleotide of the big subunit of coding AHAS.
11. DNA construct as claimed in claim 10, wherein, the polynucleotide of the big subunit of described coding AHAS comprise SEQ ID NO:16.
12. DNA construct as claimed in claim 10, wherein, the polynucleotide of coding plastid transit peptides are operably connected with the described polynucleotide of the big subunit of the described AHAS of coding.
13. DNA construct as claimed in claim 2, wherein, described the 3rd expression cassette comprises the polynucleotide of coding AHAS small subunit.
14. DNA construct as claimed in claim 13, wherein, the polynucleotide of described coding AHAS small subunit comprise SEQ ID NO:17.
15. DNA construct as claimed in claim 13, wherein, the polynucleotide of coding plastid transit peptides are operably connected with the described polynucleotide of the described AHAS small subunit of coding.
16. DNA construct, described construct comprises a plurality of expression of plants boxes, wherein first expression cassette is included in the promotor that function is arranged in the vegetable cell, it is operably connected to the exogenous polynucleotide of feeding back insensitive threonine deaminase with coding, second expression cassette is included in the promotor that function is arranged in the vegetable cell, and its exogenous polynucleotide with the big subunit of coding AHAS is operably connected.
17. DNA as claimed in claim 16 builds body, each in the wherein said promotor all is a seed enhancement type promotor.
18. DNA construct as claimed in claim 17, each in the wherein said seed enhancement type promotor all is selected from the group that is made of napin, 7S alpha, 7S alpha ', 7S beta, USP88, enhanced USP 88, Arcelin 5 and Oleosin.
19., wherein have at least two kinds of different seed enhancement type promotors as claim 16 or 17 described DNA construct.
20. DNA construct as claimed in claim 16, wherein, described first box comprises the polynucleotide of coding to the threonine deaminase that feeds back the insensitive SEQ of comprising ID NO:22.
21. DNA construct as claimed in claim 16, wherein, described first box comprises threonine deaminase variation allelotrope, and described variation allelotrope comprises the aminoacid replacement on L447F or L481F or L481Y or L481P or L481E or L481T or L481Q or L481I or L481V or L481M or the L481K position.
22. DNA construct as claimed in claim 16, wherein, the described coding threonine deaminase allelic polynucleotide that make a variation are SEQ ID NO:2, wherein comprise the aminoacid replacement on L447F or L481F or L481Y or L481P or L481E or L481T or L481Q or L481I or L481V or L481M or the L481K position.
23. DNA construct as claimed in claim 16, wherein, described first box comprises the polynucleotide of the plastid transit peptides of encoding, and described polynucleotide are operably connected with the polynucleotide of described coding threonine deaminase.
24. DNA construct as claimed in claim 16, wherein, described second expression cassette comprises the polynucleotide of the big subunit of coding AHAS.
25. DNA construct as claimed in claim 24, wherein, the polynucleotide of the big subunit of described coding AHAS comprise SEQ ID NO:16.
26. DNA construct as claimed in claim 25, wherein, the polynucleotide of coding plastid transit peptides are operably connected with the described polynucleotide of the big subunit of the described AHAS of coding.
27. a DNA construct, described DNA construct comprise a plurality of expression of plants boxes, one of them expression cassette is included in the promotor that function is arranged in the vegetable cell, and its exogenous polynucleotide with coding monomer A HAS is operably connected.
28. DNA construct, described DNA construct comprises a plurality of expression of plants boxes, wherein first expression cassette is included in the promotor that function is arranged in the vegetable cell, its exogenous polynucleotide with the big subunit of coding AHAS is operably connected, second expression cassette is included in the promotor that function is arranged in the vegetable cell, and its exogenous polynucleotide with coding AHAS small subunit is operably connected.
29. DNA as claimed in claim 28 builds body, each in the wherein said promotor all is a seed enhancement type promotor.
30. DNA construct as claimed in claim 28, each in the wherein said seed enhancement type promotor all is selected from the group that is made of napin, 7S alpha, 7S alpha ', 7S beta, USP88, enhanced USP 88, Arcelin 5 and Oleosin.
31. wherein there are at least two kinds of different seed enhancement type promotors in DNA construct as claimed in claim 28.
32. DNA construct as claimed in claim 28, wherein, described first box comprises the big subunit of AHAS, and it is made of SEQ ID NO:16.
33. DNA construct as claimed in claim 29, wherein, described first box comprises the polynucleotide of the plastid transit peptides of encoding, and its described polynucleotide with the described big subunit of coding AHAS are operably connected.
34. DNA construct as claimed in claim 28, wherein, described second box comprises the polynucleotide of coding AHAS small subunit.
35. DNA construct as claimed in claim 28, wherein, described second box comprises the polynucleotide of coding AHAS small subunit, and it is made of SEQ ID NO:17.
36. DNA construct as claimed in claim 35, wherein, described second box comprises the polynucleotide of the plastid transit peptides of encoding, and its described polynucleotide with the described AHAS small subunit of coding are operably connected.
37. method, be used for preparing the transgenosis dicotyledons that the seed amino acid levels increases, described level increase is relatively from the seed of the non-transgenic plant of identical plant species, and described method comprises the steps: a) to introduce the transgenosis that comprises construct as claimed in claim 1 or 2 in the regenerable cell of dicotyledons; B) make that described reproducible cell regeneration is dicotyledons; C) obtain seed from described plant; D) select one or many seeds of the amino acid levels with increase, described level is relatively from the seed of the non-transgenic plant of identical plant species; And e) plant described seed, wherein, if Isoleucine exists with the level that increases, so at least a extra amino acid levels also has been increased.
38. method as claimed in claim 37, wherein, described dicotyledons is a soybean plants.
39. method as claimed in claim 37, wherein, amino acid whose level increase comprises following amino acid whose concentration increases: a) one or more among Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe and Ile; Or b) one or more among Arg, Asn, Asp, His, Met, Leu, Val, Gln, Tyr, Thr, Lys, Ala, Ser and the Phe.
40. genetically engineered soybean plant by method manufacturing as claimed in claim 37.
41. method that is used for preparing the transgenosis dicotyledons that the seed aminoacids content increases, described increase is relatively from the seed of the non-transgenic plant of same plant species, and described method comprises the steps: a) to introduce the transgenosis that comprises construct as claimed in claim 16 to the regenerable cell of dicotyledons; B) make that described reproducible cell regeneration is dicotyledons; C) obtain seed from described plant; D) select one or many seeds of the amino acid levels with increase, described level is relatively from the seed of the non-transgenic plant of identical plant species; And e) plant described seed, wherein, if Isoleucine exists with the level that increases, so at least a extra amino acid levels also has been increased.
42. method as claimed in claim 41, wherein, described dicotyledons is soybean plants or rape plant.
43. method as claimed in claim 41, wherein, amino acid whose level increase comprises following amino acid whose concentration increases: a) one or more among Arg, Asn, Asp, His, Met, Ala, Leu, Thr, Val, Gln, Tyr, Lys, Ser and the Phe and Ile; Or b) one or more among Arg, Asn, Asp, His, Met, Leu, Val, Gln, Tyr, Thr, Lys, Ala, Ser and the Phe.
44. genetically engineered soybean plant by the described method manufacturing of claim 41.
45. method that is used for preparing the transgenosis dicotyledons that the seed aminoacids content increases, described increase is relatively from the seed of the non-transgenic plant of same plant species, and described method comprises the steps: a) to introduce the transgenosis that comprises as claim 27 or 28 described constructs to the regenerable cell of dicotyledons; B) make that described reproducible cell regeneration is dicotyledons; C) obtain seed from described plant; D) select one or many seeds of the amino acid levels with increase, described level is relatively from the seed of the non-transgenic plant of identical plant species; And e) plants described seed.
46. method as claimed in claim 45, wherein, described dicotyledons is soybean plants or rape plant.
47. method as claimed in claim 45, wherein, the concentration that amino acid whose level increase comprises Ser or Val increases.
48. genetically engineered soybean plant by method manufacturing as claimed in claim 45.
49. from the break chop of producing as claim 40,44 or 48 described soybean.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US46872703P | 2003-05-07 | 2003-05-07 | |
| US60/468,727 | 2003-05-07 | ||
| US10/840,688 | 2004-05-06 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1820073A true CN1820073A (en) | 2006-08-16 |
Family
ID=36603222
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200480019478 Pending CN1820073A (en) | 2003-05-07 | 2004-05-06 | Plants with increased levels of one or more amino acids |
Country Status (3)
| Country | Link |
|---|---|
| CN (1) | CN1820073A (en) |
| AR (1) | AR047394A1 (en) |
| ZA (1) | ZA200509755B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102203243B (en) * | 2008-08-15 | 2015-05-20 | 英美烟草(投资)有限公司 | Transgenic plants |
| CN114487247A (en) * | 2020-10-26 | 2022-05-13 | 内蒙古伊利实业集团股份有限公司 | Glufosinate-ammonium and metabolite determination kit, and preparation method and determination method thereof |
-
2004
- 2004-05-06 CN CN 200480019478 patent/CN1820073A/en active Pending
- 2004-05-06 AR ARP040101544A patent/AR047394A1/en unknown
-
2005
- 2005-12-01 ZA ZA200509755A patent/ZA200509755B/en unknown
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102203243B (en) * | 2008-08-15 | 2015-05-20 | 英美烟草(投资)有限公司 | Transgenic plants |
| CN114487247A (en) * | 2020-10-26 | 2022-05-13 | 内蒙古伊利实业集团股份有限公司 | Glufosinate-ammonium and metabolite determination kit, and preparation method and determination method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| ZA200509755B (en) | 2006-12-27 |
| AR047394A1 (en) | 2006-01-18 |
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