CN1270636A - DNA sequence coding for a hydroxyphenylpyruvate dioxygenase and overproduction thereof in plants - Google Patents
DNA sequence coding for a hydroxyphenylpyruvate dioxygenase and overproduction thereof in plants Download PDFInfo
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- CN1270636A CN1270636A CN98809105A CN98809105A CN1270636A CN 1270636 A CN1270636 A CN 1270636A CN 98809105 A CN98809105 A CN 98809105A CN 98809105 A CN98809105 A CN 98809105A CN 1270636 A CN1270636 A CN 1270636A
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Abstract
The invention relates to a method for producing plants with an increased yield of vitamin E from biosynthesis by overexpression of a plant HPPD gene from barley.
Description
What the present invention relates to is a kind of by external source or endogenous HPPD gene are expressed the method for the plant that produces the content of vitamin E raising in plant or plant part.The invention further relates to the corresponding nucleic of coding HPPD gene is applied in the transgenic plant so that the latter to HPPD inhibitor tool resistance, and the dna sequence dna of using coding HPPD produces one in order to identify the checking system of HPPD inhibitor.
The genetic important goal of plant molecular is exactly to produce the plant that sugar, enzyme and aminoacids content improve.The exploitation vitamin contents improves, and the plant of improving as content of vitamin E has economic implications too.
Derivative (the Ullmann ' s Encyclopedia of Industrial Chemistry that to have the active eight kinds of natural compoundss of vitamin-E all be 6-chromanol (6-Chromanol), Vol.A 27 (1996), VCH Verlagsgesellschaft, the 4th chapter, 478-488, vitamin-E).First group (1a-d) is the derivative of tocol (tocol), then is made up of the derivative (2a-d) of tocotrienols (tocotrienol) for second group:
1a, alpha-tocopherol: R
1=R
2=R
3=CH
31b, 5,8-dimethyl tocol [148-03-08]: R
1=R
3=CH
3, R
2=H1c, Gamma-Tocopherol [54-28-4]: R
1=H, R
2=R
3=CH
31d, Delta-Tocopherol [119-13-1]: R
1=R
2=H, R
3=CH
3 
2a, alpha-tocotrienol [1721-51-3]: R
1=R
2=R
3=CH
3
2b, β-tocotrienols [490-23-3]: R
1=R
3=CH
3, R
2=H
2c, γ-tocotrienols [14101-61-2]: R
1=H, R
2=R
3=CH
3
2d, δ-tocotrienols [25612-59-3]: R
1=R
2=H, R
3=CH
3
Alpha-tocopherol has very large Economic Importance.
By the crop of tissue culture or seeds mutagenesis and the content of vitamin E raising that natural selection obtained, its development has its limitation.On the one hand, content of vitamin E must just can be monitored to when tissue culture as early as possible, on the other hand, has only those could operate by tissue culture technique from the plant that cell cultures successfully is regenerated as plant.Moreover crop may show the feature that does not conform to demand after through mutagenesis and screening, and these features must be by backcrossing, and eliminates by backcrossing repeatedly in some cases.Equally, improving content of vitamin E by hybridization also is limited within the plant with kind.
These are exactly genetic engineering method why, promptly method in the crop is separated and specifically transferred to the crucial biosynthesis gene of coding vitamin-E synthetic, have more the reason of superiority than traditional cultural method.The condition of this method is that biosynthesizing and regulation and control thereof are known and influence biosynthetic gene and identified.
The biosynthesizing mode of tocopherol in plant and algae is known, and be specific as follows:
4-hydroxyphenyl pyruvic acid homogentisic acid
↓?????????????????↓
δ-tocotrienols (2d) Delta-Tocopherol (1d)
↓?????????????????↓
β-or γ-tocotrienols → β-or Gamma-Tocopherol
(2b?or?2c)??????????(1b?or?1c)
↓?????????????????↓
Alpha-tocotrienol (2a)---→ alpha-tocopherol (1a)
The precursor of tocopherol aromatic nucleus is right-hydroxyphenyl pyruvic acid (3), the latter is at hydroxyphenyl pyruvic acid dioxygenase (hydroxyphenylpyruvate dioxygenase, HPPD) change homogentisic acid (4) under auxiliary, homogentisic acid and phytylpyrophosphoric acid reactant salt are sloughed CO
2Obtain precursor (6).The biosynthetic route of tocotrienols opens the condensation reaction that starts between homogentisic acid (4) and the geranyl geranyl pyrophosphate salt, obtains precursor (5).Precursor 5 or 6 enzyme cyclic action obtain δ-tocotrienols or Delta-Tocopherol respectively.In these biosynthetic enzymes some are separated.
When in seeking Arabidopsis (Ah cloth's genus), the mutant of carotenoid biosynthesizing defective being arranged, identify the albefaction phenotype mutant that can't produce active HPPD.If this mutant that is named as pds 2 is cultivated when having homogentisic acid to exist, it will be as wild-type and green plant, produce carotenoid people such as (, Plant Cell (1995) 7:2139-2149) Norris.This work shows that the HPPD activity is to generate to have the prerequisite of the active chloroplast(id) of photosynthesis.Do not have this kind of enzyme just can't generate plastoquinone, in carotenoid biosynthesizing (phytoene desaturation) process, need serve as the acceptor of d/d reduction equivalent by plastoquinone.HPPD brings into play in plastid metabolism has important effect, this fact to make it become interested target of weedicide.Sulcotrione suppresses the activity (people such as Schultz, FEBS Lett. (1993) 318:162-166) of this enzyme effectively.
From following organism, known the sequence of HPPD specific gene:
| Organism | The sequence title | The database access number |
| The people | ????HPPD_HUMAN | ????X?72389 |
| Pig | ????HPPD_PIG | ????D?13390 |
| Mouse | ????HPPD_RAT | ????M?18405 |
| Home mouse | ????HPPD_MOUSE | ????D?29987 |
| Streptomyces avermitilis | ????SA11864 | ????U?11864 |
| Pseudomonas Sp.Strain P.J.874 | ????HPPD_PSESP | ????P?80064 |
| Arabidopsis | ????HPPD_ARAB?1 | ????AF?900228 |
| ????HPPD_ARAB?2 | ????U?89267 |
In addition, following sequence and HPPD sequence have significant homology, and they also can find in database:
PEA3_MOUSE:Mus muscula (home mouse) PEA 3 polypeptide, AC X 63190;
MELA_SHECO:Shewanella colwelliana, mel A albumen, AC M59289;
WO96/38567 has described the HPPD dna sequence dna that derives from Arabidopsis thaliana and Daucus carota.
The dna sequence dna of knowing HPPD be produce herbicide resistant plants carry out the crop protection and increase in the plant vitamin-E synthetic, as producing the absolute prerequisite of the animal-feed that content of vitamin E improves.
A target of the present invention is the transgenic plant that a kind of content of vitamin E of exploitation improves.
Further target of the present invention is the resistant transgenic plants of a kind of antagonism HPPD inhibitor of exploitation.
We are surprised to find these targets and can realize by overexpression HPPD gene in plant.
Another one target of the present invention is checking system in order to evaluation HPPD inhibitor of exploitation.
We have found that this target can realize by the restraining effect that expression one barley HPPD gene in a plant or microorganism check chemical substance that the HPPD enzyme is lived then.
What a first aspect of the present invention related to is to clone complete barley HPPD gene by the cDNA (HvSD 36) that separates the HPPD gene specific.
In the process that leaf senesces, the content of vitamin-E significantly improves (people such as Rise, Plant Physiol. (1989) 89:1028-1030) in the leaf.The leaf of barley monocotyledons formula shows the gradient of different cell of age because its leaf has the base portion meristem, new cell by division continuously from these tissues formation.The minimus base portion that then is positioned in the tip of like this, the oldest cell seat leaf.Fig. 1 shows the diagram figure of different number of days barley primary leaf after planting.The total length of the leaf of measuring can be seen from the scale in left side.Shown what be labeled as I-IV is the blade fragment of primary leaf, and they break up different degree and are selected to gene expression analysis.Plant is that the light/half-light same period (L/D) in every day is cultivated down, senesces in order to bring out, and they is sheared off and cultivate in dark 2 days (2nD) after 6 days.To barley
NascentThe segmental RNA that leaf breaks up in various degree carries out " Northern trace " analysis, and the expression of results suggest HPPD in barley is controlled with the form that depends on growth.Therefore, the long abundant transcription product accumulation of about 1600nt is arranged in the meristem zone of primary leaf base portion (I).The content of this transcription product reduces along with the increase of organizing the age (IIa and IIb), and improves again in the cell that contains ripe chloroplast(id) (III) of differentiation fully.Finally, the content of the transcription product of 1600nt length is the highest in the cross section that senesces of primary leaf (IV).In addition, in the meristem cell of first leaf base, can detect a transcription product that about 3100nt is long.Equally, this transcription product is along with the increase of organizing maturity no longer is detected.
By means of so-called " difference shows (Differential Display) " method, at first isolate the cDNA fragment of one 207 bp, its corresponding transcription product is gathered in the primary leaf of barley in secretly inducing the situation that senesces.Subsequently this fragment (sequence solution: sequence number: 1: nucleosides position 1342-1549) isolate a cDNA clone who contains a longer insertion sequence from the cDNA library (in λ-ZAP-II) of the barley boot leaf that senesces as probe.
The diagram figure that derives from the cDNA subclone HvSD 36 in λ-ZAP-II library is:
This cDNA fragment (sequence solution: sequence number: 1: nucleosides position 771-1529) be cloned on the EcoRI shearing site of pBluescript (SK).In addition, all assemble the joint of one 14 bp at the two ends of this cDNA, this sequence is that to be connected to λ-ZAP-II necessary.There is shown the restriction site of selected carrier and cDNA itself.
Further in the experiment the long cDNA fragment of this 759bp is being used as probe to obtain the complete sequence of HvSD6.For this purpose, prepared a cDNA library that derives from barley seedlings meristem cross section RNA in five day age.(production number: 27-5011 45.5kb) is used to this cDNA library to the lambda particles phage ExCell Eco RICIP of Pharmacia (Freiburg).
Isolate one the 1565 long cDNA clone of bp, see sequence solution: sequence number: 1 and 2.
In the sequence of database, length is that 434 amino acid whose protein sequences are the highest with the homology of the HPPD sequence that derives from Arabidopsis thaliana, and 58% homology is arranged.
For the genomic clone that finds to contain complete HPPD gene order, obtain a barley λ FIXII library (production number 946104) from Stratagene (Heidelberg).This library is to use the yellow leaf DNA of winter barley Cv.Igri to prepare.DNA carries out the part enzymolysis with Sau 3AI.Before being cloned on the Xhol shearing site that cuts body, segmental end and bacteriophage arm all are full of with nucleosides.The first round has carried out 200 to the library, and the screening of 000pfu only obtains a clone who hybridizes with cDNA HvSD6.After this recombinant phage carried out limited enzymatic hydrolysis with PstI and SacI, obtain size respectively and be 5400,3800 and the fragment of 1800bp, these fragments with " Southern " blot hybridization of HvSD6 probe in can be detected.It is to be existed by the form of being cloned that these subfragments cut in the body at Bluescript.Fig. 3 shows the formation of understanding barley HPPD gene with the form of diagram figure.
The present invention is specifically related to the expression cartridge clip of its sequence encoding HPPD or its function phase jljl, and uses these and express the plant that cartridge clip produces the content of vitamin E raising.Nucleotide sequence can be, for example, and DNA or cDNA sequence.The code sequence that is suitable for being inserted in the expression cartridge clip of the present invention is shown, and for example, those coding HPPD and those tools make the sequence of the ability of host's overexpression vitamin-E.
In addition, expression cartridge clip of the present invention contains the regulatory nucleic acid sequence, its domination encoding sequence expression in host cell.According to a preferred embodiment, expression cartridge clip of the present invention comprises the upstream, it is 5 of encoding sequence ' end, promotor and downstream, be its 3 ' end, polyadenylation signal and, if suitable, other controlling element, these elements are operability with the encoding sequence of therebetween HPPD gene and are connected.Operability connect should be understood that to make promotor, encoding sequence, terminator and, if be fit to, the order of presentation of other controlling element can make all controlling elements can both finish the function of its expection when encoding sequence is expressed.The preferred sequence that is used for the operability connection has, but be not limited to, be used for guaranteeing guiding peptide (targeting sequence) and translational enhancer in the Subcellular Localization in apoplast, vacuole, plastid, plastosome, endoplasmic reticulum, nucleus, liposome or other chamber district, as 5 of tobacco mosaic virus (TMV) ' end leader sequence (people such as Gallie, Nucl.Acids Res.15 (1987) 8693-8711).
For example, can be admixed to the expression of plants cartridge clip among the tobacco conversion carrier pBinAR-Hyg.That shown in Figure 4 is the pBinAR-Hyg that has the tobacco conversion carrier pBinAR-Hyg (A) of 35S promoter and have the promotor phaseolin 796 (B) of seed specific
-HPT: hygromix phosphotransferase
-OCS: octopine synthase terminator
-PNOS: nopaline synthase promoter
-show the bright unicity restriction site that also has those carriers simultaneously.
In principle, all can be controlled alien gene expression promoter in plant and all are fit to serve as the promotor that the present invention expresses cartridge clip.Particularly, plant promoter or the promotor that derives from plant virus are preferred.Cauliflower mosaic virus CaMV 35S promoter (people such as Frank, Cell 21 (1980) 285-294) is particularly preferred.Known this promotor contains the recognition sequence that various differences are transcribed effector, and these effectors are united permanent, the constitutive expression (people such as Benfey, EMBO is (1989) 2195-2202 J.8) that causes quiding gene.
Expression cartridge clip of the present invention can contain the promotor of a chemical induction type in addition, and it can be controlled external source HPPD gene and express in plant at a specific time point.This type of promotor that can be used has, PRP1 promotor (people such as Ward for example, Plant, Mol.Biol 22 (1993), 361-366), can be by the promotor of Induced by Salicylic Acid (WO95/19443), can be by benzene sulfonamide inductive promotor (EP-A 388186), can be by tsiklomitsin inductive promotor (people such as Gatz, (1992) Plant J.Z, 397-404), can be by dormin inductive promotor (EP-A 335528) or can be by ethanol or pimelinketone inductive promotor (WO93/21334).
In addition, particularly preferred promotor is that those guarantee to express can be at those vitamin-Es or its precursor in the promotor of wherein carrying out carrying out in biosynthetic tissue or the plant organ.Noteworthy especially promotor is those promotors that can guarantee the leaf specifically expressing.The promotor of being worth mentioning is potato kytoplasm FBPase or potato ST-LSI promotor (people EMBO such as Stockhaus is (1989) 2445-2455 J.8).
Can make stably express in the seed of a foreign protein at transgene tobacco down the auxiliary of seed-specific expression promoter, its quantity can reach whole solubility seeds proteic (Fiedler and Conrad more than 0.67%, Bio/Technology 10 (1995), 1090-1094).Therefore, expression cartridge clip of the present invention can contain, for example, seed-specific expression promoter (phaseolin (US 5504200) preferably, USP (Baumlein, people Mol.Gen such as H, Genet. (1991) 225 (3), 459-467) or LEB4 promotor (Fiedler and Conrad, 1995)), the LEB4 signal peptide is waited the gene and an endoplasmic reticulum maintenance signal of being expressed.Fig. 4 shows the formation of such expression cartridge clip with the form of example.
An expression cartridge clip of the present invention is like this preparation: one contain appropriate H PPDDNA sequence and preferably one insert between promotor and the HPPD dna sequence dna and the suitable promotor of the DNA of the special transit peptides of chloroplast(id) of encoding, and a polyadenylation signal merges with the reorganization and the clone technology of routine.Above-mentioned conventional reorganization and clone technology are described in, for example, T.Maniatis, the Molecular Cloning:ALaboratory Manual that E.F.Fritsch and J.Sambrook write, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and T.J.Silhary M.L.Berman and L, the Experiments With Gene Fusions that W.Enquist writes, Cold Spring Harbor Laboratory, ColdSpring Harbor, the Current Protocols inMolecular Biology that people such as NY (1984) and Ausubel write, Greene Publishing Assoc, and Wiley-Interscience (1987).
Particularly preferred sequence is that those can guarantee that guiding enters non-protogenous matter, plastid, vacuole, plastosome, endoplasmic reticulum, perhaps by default proper control sequence, stay in the chamber district and the cytosol (Kermode of formation, Crit.Rev.Plant Sci.15,4 (1996), 285-423), be proved to be to albumen the quantity accumulation in transgenic plant in the location on the endoplasmic reticulum and be particularly advantageous (people such as Schouten, Plant Mol.Biol.30 (1996), 781-792).
The invention still further relates to its dna sequence encoding HPPD Expression of Fusion Protein cartridge clip, the part of this fusion rotein is a transit peptides, and it arranges the displacement of this polypeptide.Particularly preferably be the special transit peptides of chloroplast(id), it after the HPPD gene product has been displaced in the chloroplast(id) from the HPPD digested down.Particularly preferably be and derive from plastid transketolase (Plastidtransketolase, TK) transit peptides or its function equivalent (as ribulose-1,5-bisphosphate, the transit peptides of 5-bisphosphate hydroxylation/oxygenase (rubisco) small subunit or ferredoxin NADP oxydo-reductase).
The nucleosides insertion sequence of coding HPPD can be a synthetic, also can be natural, or the mixture of synthetic and n DNA component.Usually have prepare have plant the synthetic nucleosides sequence of preferred codon.These plants preferred codon can have high protein expression frequency and most of interested plant species, determined in the codon of expressing from those.When cartridge clip is expressed in preparation, can operate to obtain one and can and possess the nucleotide sequences that proper reading frame is arranged different dna fragmentations with correct direction reading.For dna fragmentation is interconnected, can on fragment, connect top connection.
Can advantageously dispose one or more joints for promotor of the present invention and terminator along the direction of transcribing, this joint can contain one or more restriction sites, in order to insert this sequence.On the experience, joint contains 1 to 10, is 1 to 8 under most of situation, and preferably 2 to 6, restriction site.Usually, the size that is positioned at the joint of control region is less than 100bp, and great majority are less than 60bp, but has 5bp at least.Promotor of the present invention can be host plant itself or homologous, also can be external or allogenic.Expression cartridge clip of the present invention contains promotor of the present invention along 5 '-3 ' transcriptional orientation, the dna sequence dna of any needs and a transcription termination region.Various terminator can exchange as required mutually.
Also can carry out certain operations in addition provides suitable restriction site or removes unnecessary DNA or restriction site.When being fit to insert, delete or replace, during as conversion and transversion, can use methods such as vitro mutagenesis, primer reparation, restriction or connection.
As long asCarry out suitable operation, as restriction, blunt end overlapping
RetractionOr fill, just can be used for connecting for fragment provides the complementary end.
The important part that the present invention can achieve success be to have connected specific endoplasmic reticulum maintenance signal SEKDEL (Schouten, people such as A., Plant Mol.Biol.30 (1990), 781-792), this makes expression level higher 3 to 4 times than mean value.Naturally occurring other maintenance signal that is positioned on the endoplasmic reticulum also can be used to the construction expression cartridge clip in plant animal protein.
Preferred polyadenylation signal is the plant polyadenylation signal, preferably those are in essence corresponding to agrobacterium tumefaciens T-DNA polyadenylation signal, specifically be gene 3 (octopine synthase) (people such as Gielen, EMBO is (1984) 835 pages of beginnings J.3) or its function equivalent of Ti plastid pTiACH5 T-DNA.
Expression cartridge clip of the present invention can comprise, for example, and a constitutive promoter (preferably CaMV 35S promoter), LeB
4Signal peptide, wait the gene and the endoplasmic reticulum maintenance signal of being expressed.Preferred endoplasmic reticulum maintenance signal is aminoacid sequence KDEL (Methionin, aspartic acid, L-glutamic acid, a leucine).
Coding HPPD gene Fusion expression cartridge clip preferably is cloned into a carrier, and on pBin 19, this carrier is suitable for transforming agrobacterium tumefaciens.The Agrobacterium that transforms with this carrier subsequently can by with known method in order to transform plant, crop particularly, as tobacco, for example can be by through the leaf of cutting or the leaf fragment is immersed in the Agrobacterium solution and then they are placed on to cultivate on the suitable medium transform.The method of carrying out Plant Transformation with Agrobacterium sees, for example, Transgenic Plants, the first roll, Engineering and Utilization, S.D.Kung and R.Wu write, Academic Press, the Vectors for Gene Transfer in Higher Plants of F.F.White in 1993, the 15-38 pages or leaves.Through the leaf of cutting or leaf segmental by cell transformed can by with known method in order to regeneration, transgenic plant, these plants contain one and are integrated in the gene that the present invention expresses the HPPD gene in the cartridge clip in order to express one.
For the DNA with a coding HPPD transforms host plant, the form of one expression cartridge clip of the present invention with insertion sequence is admixed in the recombinant vectors, the carrier DNA of this recombinant vectors also contains functional adjustment signal in addition, as is used to the sequence duplicating or integrate.Suitable carriers is described in, for example, and " Methods in Plant Molecular Biology and Biotechnology " (CRC Press), the 6/7th chapter, 71-119 page or leaf (1993).
Use above-mentioned reorganization and clone technology, can be cloned into the suitable carriers that allows them to duplicate to expression cartridge clip of the present invention, in intestinal bacteria.Suitable cloning vector has, for example pBR332, pUC series, M13 mp series and pACYC 184.Specially suitable is that those can not only duplicate in intestinal bacteria, and the binary vector that can duplicate in Agrobacterium.
The present invention and then relate to expression cartridge clip of the present invention is used to transform plant, vegetable cell, plant tissue or plant organ.The preferred purpose of this application is to improve the content of vitamin E of plant.
According to the difference of selected promotor, expression can specifically betide in leaf, seed or other plant organ.The present invention also relates to these transgenic plant, their reproductive material, their vegetable cell, plant tissue and plant organ simultaneously.
In addition, table bullet cartridge clip of the present invention also can be used to bacterium, blue-green algae, yeast, filamentous fungus are transformed with algae, to improve its vitamin-E output.
Alien gene transferred to call conversion in the Plant Genome.This process utilizes the above-mentioned method that plant tissue or vegetable cell were transformed and be regenerated as plant to obtain temporary transient or stable conversion.Suitable method have by the picked-up of polyoxyethylene glycol inducing DNA transform, use particle gun ballistics method-promptly so-called " particle bombardment method ", electroporation, place dried embryo the solution that contains DNA to carry out incubation, micro-injection and agriculture bacillus mediated transgenosis.Above-mentioned method is described in, for example, Transgenic Plants, the first roll, Engineering andUtilizotion, S.D.Kung and R.Wu write, the Techniques for Gene Transfer of the people such as B.Jenes in Academic Press (1993) the 128-143 pages or leaves and in Potrykus Annu.Rev.Plant Physiol.Plant Molec.Biol.42 (1991) 205-225 pages or leaves.Preferably gene to be expressed is constituted and be cloned into a carrier that is fit to transform agrobacterium tumefaciens, for example pBin 19, on people such as (, Nucl.Acids Res.12 (1984) 8711) Bevan.
The Agrobacterium of expressing the cartridge clip conversion via the present invention also can be used to transform plant with known method, crop particularly, as cereal, corn, oat, soybean, rice, cotton, sugar beet, Canola, Sunflower Receptacle, flax, hemp, potato, tobacco, tomato, oleaginous seed rape, alfalfa, lettuce and various trees, nut and grape vine kind, leaf after for example can cutting by handle or leaf fragment be impregnated in the Agrobacterium solution and then their cultivations and transform in suitable medium.
According to the present invention, though the function of coding HPPD gene equates that sequence is that those contain the sequence that different nucleotide sequences has required function.Therefore, the function equivalent comprises the natural mutation and the artificial nucleotide sequences of the sequence that those are described herein, as the artificial nucleotide sequences that vegetable codon is selected that is adapted to by chemosynthesis.
The function equivalent also can be understood that, particularly: the original natural or artificial mutant of the sequence by extracting the coding HPPD that obtains, these mutant still have required function.Sudden change comprises displacement, interpolation, deletion, transversion or inserts one or more nucleosides residues.Therefore, the present invention also comprises those nucleotide sequences by being obtained after nucleotide sequences described herein is modified.The purpose that this class is modified is, for example, the encoding sequence that is contained in is wherein further limited, and perhaps, for example inserts other Restriction Enzyme restriction enzyme site.
Function equates that body comprises that also those compare the remarkable more or more inapparent mutant of function with initial gene or gene fragment.
As long as what other was suitable also has those to make the artificial DNA sequence that content of vitamin E improves in the plant by overexpression HPPD gene in crop as mentioned above.This artificial DNA sequence to be passing through, for example, by the molecular simulation assisting building have an active proteinic reverse translation of HPPD, perhaps in-vitro screening is determined.Specially suitable is the DNA sequences encoding that the peptide sequence reverse translation is obtained by the specific codon selection according to host plant.Specific codon is selected and can easily be obtained by the computer evaluation of other known of an expert who is familiar with the plant genetic method by treating plant transformed.
Noteworthy other suitable nucleotide sequence equivalent of the present invention also has: the sequence of encoding fusion protein, a component of this fusion rotein are a plant HPPD polypeptide or its function equivalent part.The second section of fusion rotein can be that for example, a polypeptide or the antigenic polypeptide sequence (as myc-tag or his-tag) with enzymic activity is in the auxiliary expression that can detect HPPD down of above-mentioned sequence.Yet this is a modulin sequence preferably, as, a signal peptide or transit peptides, it can be directed to required action site to HPPD albumen.
Yet what the invention still further relates to that a transit peptides and produced according to the present invention has active polypeptide expression product of HPPD and a fusion product.
Improve content of vitamin E, according to purpose of the present invention, refer to by the HPPD gene in plant, carry out functional overexpression artificial obtain, with the vitamin-E biosynthesis ability that can continue the raising of generation plant without genetic engineering modified plant at least.
The biosynthetic position of vitamin-E is leaf tissue normally, is favourable so the HPPD gene carries out specifically expressing at blade.Yet it should be understood that the vitamin-E biosynthesizing might not be confined to leaf tissue, it also can be at other organ of plant, as fatty seed, in carry out with tissue-specific form.
In addition, the constitutive expression of external source HPPD gene is favourable.On the other hand, inducible expression also conforms with demand.
Render a service and for example to determine by the breeding of seedling meristem by the HPPD expression of gene of transgene expression external.In addition, the essence of HPPD genetic expression and the variation of level and to the biosynthetic influence of test plant vitamin-E can be tested by the greenhouse experiment.
The present invention and then relate to the present invention and express the cartridge clip transgenic plant transformed, and the transgenic cell of this class plant, tissue, organ and reproductive material.In the present invention, particularly preferably be genetically modified crops, as barley, wheat, rye, corn, oat, soybean, rice, cotton, sugar beet, Canola, Sunflower Receptacle, flax, hemp, Ma Lingzhu, tobacco, tomato, oleaginous seed rape, alfalfa, lettuce and various trees, nut and grape vine kind.
The plant that is used for the object of the invention can be unifacial leaf or dicotyledons, perhaps algae.
Address as top, HPPD is the suitable target of Sulcotrione type weedicide.Consider to have more effective HPPD inhibitor, the suitable pilot system that can use in conjunction with research for inhibitor/enzyme must be provided.For this purpose, for example, complete barley HPPD cDNA sequence clone to an expression vector (pQE, Qiagen) in and in intestinal bacteria overexpression.
The HPPD albumen of expressing down of assisting of expressing cartridge clip in the present invention is particularly suitable for the inhibitor of finding that HPPD is special.
For this purpose, for example, can be used for the enzyme chemical examination to HPPD, determine the active variation of HPPD when active substance to be measured exists and do not exist.By being compared, twice determined activity just can obtain some quality and quantitative evidence in the active substance inhibition behavior to be measured.
Use pilot system of the present invention to screen a large amount of chemical substances simply fast, determine its weeding characteristic.With this method can be from a large amount of materials on purpose, can repeatedly filter out material that height tires so that subsequently it is carried out more deep test, these tests all are that the expert is familiar with in the industry.
The present invention and then relate to the weedicide that to identify with above-mentioned pilot system.
The gene order of coding HPPD (sequence number: 1) in plant, carry out overexpression and can make the resistance raising of plant to the HPPD inhibitor.The present invention also relates to the transgenic plant of generation like this simultaneously.
The present invention and then also relate to :-transform the method for plant, comprise expression cartridge clip of the present invention is imported vegetable cell, callus, puts in order among strain plant or the plant protoplast.The application of-plant in producing plant HPPD.-by more the efficiently expressing of dna sequence dna of the present invention, produce HPPD inhibitor resistance enhanced plant with expression cartridge clip of the present invention.-expression cartridge clip of the present invention is by the application in the plant of expressing dna sequence dna generation content of vitamin E of the present invention raising in plant.-produce one in order to identify the test macro of HPPD inhibitor with expression cartridge clip of the present invention.
Come the present invention is made an explanation with embodiment below, but these embodiment are not limitation of the present invention: general cloning process
Clone's step of being carried out within the scope of the present invention, as restriction enzyme digestion, agarose gel electrophoresis, dna fragmentation purifying, nucleic acid transfer on cellulose nitrate and the nylon membrane, the sequential analysis of dna fragmentation connection, Bacillus coli cells conversion, microbial culture, phage propagation and recombinant DNA, all be according to people such as Sambrook (1989) Cold Spring Harbor LaboratoryPress; Method described in the ISBN 0-87969-309-6 is carried out.
From Stratagene, NP66 is then from Pharmacia for the bacterial isolates that uses below (intestinal bacteria, XL-I Blue).Agrobacterium strains (agrobacterium tumefaciens, C58C1 have plasmid pGV 2260 or pGV 3850 Kann) in order to the conversion plant is people described (Nucl.Acids Res.13 (1985) 4777) such as Deblaere.As an alternative, also can use agrobacterium strains LBA4404 (Clontech) or other suitable bacterial strain.The carrier that can be used to clone has pUC19 (Yanish-Perron, Gene 33 (1985), 103-109), pBluescript SK-(Stratagene), pGEM-T (Promega), pZerO (Invitrogen), pBin19 (people such as Bevan, Nucl.Acids Res.12 (1984), 8711-8720) and pBinAR (Hofgen and Willmitzer, Plant Science 66 (1990) 212-230).
The sequential analysis of recombinant DNA
(5463-5467) the laser fluorescence dna sequencing instrument (MWG Biotech, Ebersbach is on sale) produced of usefulness-Licor checks order to recombinant DNA molecules for people such as Sanger, Proc.Natl.Acad.Sci.USA74 (1977) according to the described method of Sanger.The preparation of expression of plants cartridge clip
Plasmid pBin19 (people such as Bevan, Nucl.Acids Res. (1984) 12,8711) insertion-35S CaMV promotor in, this-promotor is-the EcoRI-KpnI fragment, corresponding to cauliflower mosaic virus Nucleotide 6909-7437 (people such as Franck, Cell 21 (1980) 285).Ti-plasmids pTiACH5 (people such as Gielen, EMBO J.3 (1984) 835) the polyadenylation signal (nucleosides 11749-11939) of T-DNA gene 3 separated with the segmental form of PvuII-Hind III, after adding the SphI joint, be cloned on the Pvu II restriction enzyme site between the SpHI-Hind III restriction enzyme site of carrier pBmAR-Hyg.So just obtain plasmid pBinAR (Hofgon and Willmitzer, Plant Science 66 (1990) 221-230).Use the separation of the special cDNA sequence of EXAMPLE Example 1.HPPD
(Science (1992) 257 by means of the DDRT-RCR method of Liang and Pardee, 967-972), the composition of cultivating the primary leaf mRNA population of 9 days barley in the L/D photoperiod (illumination 16 hours/dark 8 hours) down (was cultivated with cultivation 11 days in 9 days, the dark place reason of carrying out subsequently 2 days senesces to induce) barley primary leaf compare (Humbeck and Krupinska, J.Photochem, Photobiol.36 (1996), 321-326).In each situation, (Gibco BRL Eggenstein) changes total RNA of 0.2 μ g into cDNA with " Superscript RT " enzyme.Except RNA, each reaction batch (20 μ l) also contains 20 μ M dNTPs, 10 μ M DTT, 1xRT damping fluid and 1 μ M (dT) 12VN primer.The required grappling " primer " (anchor " primers ") of these reactions is the data synthetic according to Liang and Pardee: 1. 5 '-TTTTTTTTTTTTAG-3 ' 2. 5 '-TTTTTTTTTTTTCA-3 ' 3. 5 '-TTTTTTTTTTTTAC-3 ' 4. 5 '-TTTTTTTTTTTTGT-3 '
After cDNA is synthesized, relevant sequence in each situation is all carried out 10 batches amplification, uses following different " primer " at random respectively: 1. 5 '-TACAACGAGG-3 ' 2. 5 '-GGAACCAATC-3 ' 3. 5 '-AAACTCCGTC-3 ' 4. 5 '-TGGTAAAGGG-3 ' 5. 5 '-CTGCTTGATG-3 ' 6. 5 '-GTTTTCGCAG-3 ' 7. 5 '-GATCTCAGAC-3 ' 8. 5 '-GATCTAACCG-3 ' 9. 5 '-GATCATGGTC-3 ' 10.5 '-GATCTAAGGC-3 '
In the 20 μ l volumes in each situation, each PCR reaction batch contains the 1xPCR damping fluid, 2 μ M dNTPs, 2.5 μ Ci (α
33P)-and dATP, 1 μ M (dT)
12VN-" primer ", the RT mixed solution of 1/10 volume (people such as Sambrook, Molecular Cloning-ALaboratory Manual, 1989), 1 Taq of unit archaeal dna polymerase (Boehringer, Mannhein) and 1 μ M 10-mer " primer " at random.Carry out the PCR reaction in-Unoblock (Biometra), program is as follows:
94 ℃ 2 minutes
94 ℃ 30 seconds
40 ℃ 2 minutes
72 ℃ 30 seconds
72 ℃ 5 minutes
6. 4 ℃ of preservations are up to further processing
Step 2,3,4 is carried out 40 times continuously.So each reaction and " primer " are in conjunction with approximately obtaining 100 cDNA.
Be different from Liang and Pardee scheme be, cDNA fragment after the amplification is to carry out isolating with non-denaturing polyacrylamide gel, gel is composed as follows: 6% (v/v) acrylamide (Long Ranger, AT Biochem), 1.2xTBE damping fluid, 0.005% (v/v) TEMED and 0.005% (w/v) APS (people such as Bauer, Nucl.Ac.Res. (1993) 21,4272-4280).
In every kind of situation, each PCR batch 3.5 μ l use the sample-loading buffer (dye II, people such as Sambrook, 1989) of 2 μ l to handle respectively, go up sample then to gel.In order to determine the repeatability of cDNA band pattern (Fig. 5), prepare two parts of independently RNA samples respectively from the barley primary leaf in Ninth Heaven age and 11 ages, equality is used for following analysis.That shown is the results of two different primers in conjunction with (A and B); As an example, emphasized the two places difference of band pattern between Ninth Heaven age and the 11 age samples with arrow.When gel analysis, only those are all occurred in two parts of samples that derive from the plant that senesces subsequently, and absent variable band takes in control sample.Electrophoresis is at 40 watts (0.8 watt/centimetre
3), carry out 2.5 hours time in the 1xTBE damping fluid.After the cDNA fragment has been separated, gel is transferred to filter paper (Schleicher ﹠amp; Schull, Dassel) on.After 50 ℃ of dryings, placed on it at gel an X-ray film.With an operation blade only come across sample 11 and 11 ' the radioautograph photograph in the cDNA band downcut from dried glue, and boil with 100 μ l 1xTE damping fluids and to boil wash-out.Through the DNA of ethanol sedimentation with the resuspended usefulness of the water of 10 μ l for further test." primer " used with this batch increases again to DNA, and then its clone, order-checking and as the probe of Northern blot hybridization.
Whether in order to test relevant cDNA really is the transcription product of special (seneszenzspezifisch) of senescing, and it is hybridized with the RNA that derives from the blade of different developmental phases: A.1. derive from the RNA that derives from the primary leaf of (2 darknesses cultivate after capable again illumination) plant in 12 day age at the RNAA.5. that the RNAA.4. that L/D cultivates 9 days plant in the photoperiod the RNAA.3. of primary leaf derives from the primary leaf of (the 10th day unglazed photograph) plant in 10 day age derives from the primary leaf of (the 10th and 11 day unglazed photograph) plant in 11 day age
In each situation, be used for sample that RNA analyzes and be at the intermediate collection of original dark phase.B. derive from the RNA (Fig. 6) of the boot leaf of collecting from the field seven different time points.These
Leaf was grown up fully May 29, at the beginning of June 21, its chlorophyll content only was
The beginning 10% a little less than.In Fig. 6, show that with arrow the starting point of understanding the process that senesces is (promptly from June
Reached in 15th after total length counted 17 days).The starting point that senesces is defined as photosystem II
The time point that render a service to descend (people such as Humbeck, Plant Cell Environment
(1996)19：337-344)。
For filter paper and above-mentioned RNA sample are hybridized, except using the HPPD probe,, also used the coding ribulose-1,5-bisphosphate, the specific probe of the rbcS gene of 5-bisphosphate carboxylase small subunit for relatively.Shown in Figure 6 is the hybridization of using cDNA HvSD 36 and rbcS gene specific probe and " Northern trace " A and B.Filter paper A carries and derives from, the RNA that grows under the photoperiod 9 days (9), cultivates 1 and 2 day (10,11) more respectively and return the barley primary leaf of illumination condition 1 day (12) subsequently in dark at L/D.Filter paper B contains the RNA that derives from the boot leaf of collecting to June 21 from the field from May 29th, 1992.Arrow has indicated the starting point June 15 of senescing.As can be seen from Figure 6, the special mRNA quantity of rbcS is many, and the special mRNA of HPPD is then less relatively.In the primary leaf of plant in 9 day age, the special mRNA of HPPD before in transferring to dark be detect less than, but in the dark interim significant accumulation that then has.When to plant recovery illumination, the quantity of this mRNA significantly reduces.In boot leaf, in total length and the leaf that do not senesce, just can detect the special mRNA of a spot of HPPD long.Before really begin as far back as senescing 4 days, expression level is higher.The maximum quantity of this mRNA comes across in the leaf that senesces.This is used the detected transcription product of cDNA probe HvSD36 (S: sequence; D: dark, No. 36 fragment in the DDRT gel) carries out big or small comparison with known RNA kind, show that its length is approximately 1.6Kb.
Adopt DDRT-PCR to obtain three separate cDNA fragments, they show that all this pattern and its sequential analysis also show their certain representatives together-transcription product.The size of long segment is 230bp.Use " Sure Clone at last
TMLigaion Kit " (Pharmacia, Freiburg), with reference to the operation instruction of manufacturers, the long PCR product cloning of 230 bp to the SmaI restriction enzyme site of carrier pUC18.Recombinant plasmid transformed in the competent cell of e.colistraindh5.Owing to because methodological reason, this fragment is only corresponding to 3 of associated retroviral product ' end, so its sequence information is not sufficient to identify the sequence that clear and definite homology is arranged with it in the database when beginning.In order to isolate bigger corresponding cDNA, with the long fragment of 230bp as probe screened the boot leaf RNA that senesces a λ ZAPII library (Stratagene, Heidelberg).When carrying out this step, (Boehringer, operation instruction Mannheim) is carried out mark with Dig-dUTP to probe with reference to " DNA Labeling and Detection Kit ".(Stratagene, scheme Heidelberg) is tested to the library with reference to " ZAP-cDNA Synthesis Kit ".
Probe described herein has been carried out 150, the check of 000pfu.Wherein there are 39 plaques to be positive, wherein 12 phage bacterium colonies are further experimentized.After the preparation phage, the fragment of insertion increases by PCR and separates by electrophoresis.Carry out the Southern blot hybridization with HvSD 36 probes and can from 12 phage bacterium colonies, pick out the phage bacterium colony that contains the longest positive " insertion sequence " through as above processing.After drawing flat board again, phage is further hybridized.Single plaque is scaled off and wash-out, with a helper phage it is carried out external shearing (Exassit with reference to the scheme of Stratagene then
TMInterference-Resistant Helper Phage With SOLR
TMStrain).Contain the cDNA that clones in pBlueskript (SK-) through so-called " phagemids " that obtains after the above-mentioned processing.
Carry out the preparation of plasmid subsequently, and then go up relevant " insertion sequence " with EcoRI from Blueskript (SK-) and shear.The cDNA that obtains with HvSD 36 contains the insertion sequence that a length is approximately 800bp.With " Sequi Therm Excel Long-Read DNA-Sequenzierangs-Kit " (Epicentre Technologies, Biozym Diagnostic, Oldendorf) and be attached to the complete sequence that the universal primer with the IRD41 mark of sequence area on the Blueskript carrier has been measured cDNA.The infrared laser of the automatic sequencer 4000L that produces with Licor is monitored dna fragmentation.After the order-checking, all have a length in each situation and just be the sequence of 759bp, the two ends of this sequence all are connected with-the long joint of 14bp.These joints are used to when the construction cDNA library cDNA fragment to be connected to phage ZAPII, and (Stratagene is on arm Heidelburg).
In the sequence of database, the sequence homology that contains more than 180 amino acid whose protein sequence HvSD 36 and people HPPD is up to 41%.Consider that the length (general 1600nt) of the detected transcription product of usefulness " Northern trace " can infer, this cDNA still lacks 850-900bp.
In order to obtain a complete cDNA, we have made up another cDNA library.At " Dynabeads " (Dynal, Hamburg) auxiliary down from five day age barley seedlings base portion meristem district extract mRNA and with " Time Saver cDNA Synthese Kit " (Pharmacia, Freiburg) it is transcribed into cDNA, connecting EcoRI/NotI connector (Pharmacia on the cDNA then, Freiburg) and its be connected to λ Excell carrier (Pharmacia, Freiburg) on.At last, (Stratagene, assisting down Heidelburg) is packaged into phage albumen to recombinant phage dna at " Gigapack II Gold Set ".HvSD 36 probes long with 759 bp have carried out 400, and the screening of 000pfu detects 5 phages.Operation instruction with reference to Pharmacia (Freiburg) is sheared from phage at bacterial isolates NP66 auxiliary following " phagemids ".From the individual bacterium colony of bacterium, isolate reorganization pExCell plasmid and it is transferred among the bacterial isolates D115 α and breed.
So separating the longest cDNA clone HvSD36 length that obtains is 1565bp, and its sequence obtains complete mensuration (seeing sequence table).Embodiment 2. genome sequence character are determined
In order to identify the genomic clone that contains the HPPD gene order, obtain the λ FIXII library of a barley from Stratagene (Heidelberg).This library is to use the albefaction leaf DNA of winter barley CV, Igri to prepare.This DNA is through Sau 3AI part enzymolysis.Before the XhoI shearing site of it being cloned into carrier, the arm of segmental two ends and phage all is full of with nucleosides.The first round 200 is carried out in the library, and the screening of 000pfu only obtains a clone who hybridizes with cDNAHvSD36.With PstI and SacI this recombinant phage is carried out limited enzymatic hydrolysis, separate to obtain length then and be 5400,3800, the fragment of 1800bp, these fragments can detect by " Southern " blot hybridization with the HvSD36 probe.These subfragments exist with the form of being cloned in Bluescript vector.
Library screening is to carry out with reference to the scheme of HybondN film.Use the Klenow enzyme, use
32P-dATP carries out " random priming " amplification so that it is carried out mark to probe, usefulness (people (1989) Molecular Cloning.A laboratory manual.Cold Spring Harbor Laboratory such as Sambrook, New York) for library screening and " Southern " blot hybridization.
Total DNA with barley (Carina) carries out genome " Southern trace " (Fig. 7).In each situation, with BamHI (B), EcoRI (E), HindIII (H) or XBAI (X) carry out enzymolysis to 15 μ gDNA to be separated it with 0.75% sepharose then.Transferring to Hybond N
+Film (Amersham, Braunschweig) afterwards, operation instruction with reference to the film manufacturer is hybridized with the long HvSD 36 incomplete cDNA probes of 759 bp, and detect following fragment: BamHI:6.0,3.9 and 3.0Kbp.EcoRI:＞10KbpHindIII:8.3,2.6,1.1 and 1.0KbpXbaI:9.0,5.2 and 4.2Kbp
Segmental length is by (Kb-Leiter, GibcoBRL Eggenstein) compare and estimate with-DNA size criteria thing.The homology of embodiment 3.HvSD protein sequence relatively
Protein sequence in HvSD 36 protein sequences and the database is compared, finds it and known following protein sequence tool homology up to the present:
10?????????20?????????30?????????40?????????50HPPD_Hv???????..........?..........?..........?........MP?PTPTTPAATGHPPD_Ath??????..........?..........?..........?...MGHQNAA?VSENQNHDDGHPPD_HUMAN????..........?..........?..........?..........?..........HPPD_RAT??????..........?..........?..........?..........?..........HPPD_PIG??????..........?..........?..........?..........?..........HPPD_MOUSE????..........?..........?..........?..........?..........HPPD_PSESP????..........?..........?..........?..........?..........MELA_SHECO????..........?..........?..........?..........?..........PEA3_MOUSE????MTKSSNHNCL?LRPENKPGLW?GPGAQAASLR?PSPATLVVSS?PGHAEHPPAA
60?????????70?????????80?????????90????????100HPPD_Hv???????AAAAVTPEHA?RPHRMVRFNP?RSDRFHTLSF?HHVEFWCADA?ASAAGRFAFAHPPD_Ath??????AASSPGFKLV?GFSKFVRKNP?KSDKFKVKRF?HHIEFWCGDA?TNVARRFSWGHPPD_HUMAN?????????????M?TTYSDKGAKP?ERGRFLH--F?HSVTFWVGNA?KQAASFYCSKHPPD_RAT???????????????????YWDKGPKP?ERGRFLH--F?HSVTFWVGNA?KQAASFYCNKHPPD_PIG???????????????M?TSYSDKGEKP?ERGRFLH--F?HSVTFWVGNA?KQAASYYCSKHPPD_MOUSE?????????????M?TTYNNKGPKP?ERGRFLH--F?HSVTFWVGNA?KQAASFYCNKHPPD_PSESP??????????????????ADLYENP?MGLMGFEFIE?LASPTPNTLEMELA_SHECO??????????????????MASEQNP?LGLLGIEFTE?FATPDLDFMHPEA3_MOUSE????PAQTPGPQVS?ASARGPGPVA?GGSGRMERRM?KGGYL---DQ?RVPYTFCSKS
110????????120????????130????????140????????150HPPD_Hv???????LGAPLAARSD?LSTGNSAHAS?QLLRSGSLAF?LPT--APYAN?G-CDAA----HPPD_Ath??????LGMRFSAKSD?LSTGNMVHAS?YLLTSGDLRF?LFT--APYSP?S-LSAGEIKPHPPD_HUMAN????MGFEPLAYRG?LETGSREVVS?HVIKQGKIVF?VLS--SA---?-------LNPHPPD_RAT??????MGFEPLAYKG?LETGSREVVS?HVIKQGKIVF?VLC--SA---?-------LNPHPPD_PIG??????IGFEPLAYKG?LETGSREVVS?HVVKQDKIVF?VFS--SA---?-------LNPHPPD_MOUSE????MGFEPLAYRG?LETGSREVVS?HVIKRGKIVF?VLC--SA---?-------LNPHPPD_PSESP????PIFEIMGFTK?VATHRSKDV-?HLYRQGAINL?ILN--NE---?----------MELA_SHECO????KVFIDFGFSK?LKKHKQKDI-?VYYKQNDINF?LLN--NE---?----------PEA3_MOUSE????PGNGSLGEAL?MVPQGKLMDP?GSLPPSDSED?LFQDLSHFQE?TWIAEAQVPD
160????????170????????180????????190????????200HPPD_Hv???????--TASLPSFS?ADAARRFSAD?HGIAVRSVAL?RVADAAEAFR?ASRRRGARPAHPPD_Ath??????TTTASIPSFD?HGSCRSFFSS?HGLGVRAVAI?EVEDAESAFS?ISVANGAIPSHPPD_HUMAN????--------WN?KEMGDHL-VK?HGDGVKDIAF?EVEDCDYIVQ?KARERGAKIMHPPD_RAT??????--------WN?KEMGDHL-VK?HGDGVKDIAF?EVEDCEHIVQ?KARERGAKIVHPPD_PIG??????--------WN?KEMGDHL-VK?HGDGVKDIAF?EVEDCDYIVQ?KARERGAIIVHPPD_MOUSE????--------WN?KEMGDHL-VK?HGDGVKDIAF?EVEDCDHIVQ?KARERGAKIVHPPD_PSESP????---------P?HSVASYFAAE?HGPSVCGMAF?RVKDSQKAYK?RALELGAQPIMELA_SHECO????---------K?QGFSAQFAKT?HGPAISSMGW?RVEDANFAFE?GAVARGAKPAPEA3_MOUSE????SDEQFVPDFE?---SENLAFH?SPTTRIKKEP?QSPRTDPALS?CSRKPPLPYH
210????????220????????230????????240????????250HPPD_Hv???????FAPV------?-----DLGRG?FAFAEVELYG?--DVVLRFVS?HP--DG--TDHPPD_Ath??????SPPI------?-----VLNEA?VTIAEVKLYG?--DVVLRYVS?YKAEDT--EKHPPD_HUMAN????REP-------?-WVEQDKFGK?VKFAVLQTYG?--DTTHTLVE?KMN-----YIHPPD_RAT??????REP-------?-WVEEDKFGK?VKFAVLQTYG?--DTTHTLVE?KIN-----YTHPPD_PIG??????REEVC-CAAD?VRGHHTPLDR?AR----QVWE?--GT---LVE?KMT-----FCHPPD_MOUSE????REP-------?-WVEQDKFGK?VKFAVLQTYG?--DTTHTLVE?KIN-----YTHPPD_PSESP????HI--------?----ETGPME?LNLPAIKGIG?--GAPLYLID?RFGEGSSIYDMELA_SHECO????AD--------?----EV--KD?LPYPAIYGIG?--DSLIYFID?TFGDDNNIYTPEA3_MOUSE????HGEQCLYSRQ?IAIKSPAPGA?PGQSPLQPFS?RAEQQQSLLR?ASSSSQSHPG
260????????270????????280????????290????????300HPPD_HV???????VPFLPGFEGV?TNPDA-----?VDYGLTRFDH?VVGNVP--EL?-APAAAYIAGHPPD_Ath??????SEFLPGFERV?EDASSF---P?LDYGIRRLDH?AVGNVP--EL?-GPALTYVAGHPPD_HUMAN????GQFLPGYEAP?AFMDPLLPKL?PKCSLEMIDH?IVGNQPDQEM?-VSASEW---HPPD_RAT??????GRFLPGFEAP?TYKDTLLPKL?PSCNLEIIDH?IVGNQPDQEM?-ESASEW---HPPD_PIG??????LDSRPQPSQT?LLHRLLLSKL?PKCGLEIIDH?IVGNQPDQEM?-ESASQW---HPPD_MOUSE????GRFLPGFEAP?TYKDTLLPKL?PRCNLEIIDH?IVGNQPDQEM?-QSASEW---HPPD_PSESP????IDFV--FLEG?VDRHPVGA--?---GLKIIDH?LTHNVYRGRM?-A---YWANFMELA_SHECO????SDF-----EA?LDEPIITQ--?-EKGFIEVDH?LTNNVHKGTM?-E---YWSNFPEA3_MOUSE????HGYLGEHSSV?FQQPVDMCHS?FTSPQGGGRE?PLPAPYQHQL?SEPCPPYPQQ
310????????320????????330????????340????????350HPPD_Hv???????FT---GFHEF?AEFTAEDVGT?TESGLNSVVL?ANNSEGVLLP?LNEPVHGTKRHPPD_Ath??????FT---GFHQF?AEFTADDVGT?AESGLNSAVL?ASNDEMVLLP?INEPVHGTKRHPPD_HUMAN????YLKNLQFHRF?WSVDDTQVHT?EYSSLRSIVV?ANYEESIKMP?INEPAPG-KKHPPD_RAT??????YLKNLQFHRF?WSVDDTQVHT?EYSSLRSIVV?ANYEESIKMP?INEPAPG-RKHPPD_PIG??????YMRNLQFHRF?WSVDDTQIHT?EYSALRSVVM?ANYEESIKMP?INEPAPG-KKHPPD_MOUSE????YLKNLQFHRF?WSVDDTQVHT?EYSSLRSIVV?TNYEESIKMP?INEPAPG-RKHPPD_PSESP????YEKLFNFREI?RYF---DIKG?EYTGLTSKAM?TAPDGMIRIP?LNE--ESSKGMELA_SHECO????YKDIFGFTEV?RYF---DIKG?SQTALISYAL?RSPDGSFCIP?INE--GKGDDPEA3_MOUSE????NFKQ-EYHDP?LYEQAGQPAS?SQGGVSGHRY?PGAGVVIKQE?RTDFAYDSDV
360????????370????????380????????390????????400HPPD_Hv???????RSQIQTFLEH?HGGPGVQH-I?AVASSDVLRT?LRKMRARSAM?GGFDFLPPPLHPPD_Ath??????KSQIQTYLEH?NEGAGLQH-L?ALMSEDIFRT?LREMRKRSSI?GGFDFMPSPPHPPD_HUMAN????KSQIQEYVDY?NGGAGVQH-I?ALKTEDIITA?IRHLRER---?-GLEFLSVP-HPPD_RAT??????KSQIQEYVDY?NGGAGVQH-I?ALRTEDIITT?IRHLRER---?-GMEFLAVP-HPPD_PIG??????KSQIQEYVDY?NGGAGVQH-I?ALKTEDIITA?IRSLRER---?-GVEFLAVP-HPPD_MOUSE????KSQIQEYVDY?NGGAGVQH-I?ALKTEDIITA?IRHLRER---?-GTEFLAAP-HPPD_PSESP????AGQIEEFLMQ?FNGEGIQH-V?AFLSDDLIKT?WDHLKSI---?-GMRFMTAPPMELA_SHECO????RNQIDEYLKE?YDGPGVQH-L?AFRSRDIVAS?LDAMEGS---?-SIQTLDIIPPEA3_MOUSE????PGCASMYLHP?EGFSGPSPGD?GVMGYGYEKS?LRPFPDDVCI?VPKKFEGDIK
410????????420????????430????????440????????450HPPD_Hv???????PKYYEGVRRL?AGD--VLSEA?QIKECQELGV?LVDRDDQG--?--VLL-----HPPD_Ath??????PTYYQNLKKR?VGD--VLSDD?QIKECEELGI?LVDRDDQG--?--TLL-----HPPD_HUMAN????STYYKQLREK?LKTAKIKVKE?NIDALEELKI?LVDYDEKG--?--YLL-----HPPD_RAT??????SSYYRLLREN?LKTSKIQVKE?NMDVLEELKI?LVDYDEKG--?--YLL-----HPPD_PIG??????FTYYKQLQEK?LKSAKIRVKE?SIDVLEELKI?LVDYDEKG--?--YLL-----HPPD_MOUSE????SSYYKLLREN?LKSAKIQVKE?SMDVLEELHI?LVDYDEKG--?--YLL-----HPPD_PSESP????DTYYEMLEGR?LPN----HGE?PVGELQARGI?LLDGSSESGD?KRLLL-----MELA_SHECO????E-YYDTIFEK?LPQ----VTE?DRDRIKHHQI?LVDGDEDG--?--YLL-----PEA3_MOUSE????QEGIGAFREG?PPYQR-----?-RGALQLWQF?LVALLDDPTN?AHFIAWTGRG
460????????470????????480????????490????????500HPPD_Hv???????QIFTKPVGDR?PTLFLEMIQR?IGCMEKDERG?EE----YQKG?GCGGFGKGNFHPPD_Ath??????QIFTKPLGDR?PTIFIEIIOR?VGCMMKDEEG?KA----YOSG?GCGGFGKGNFHPPD_HUMAN????QIFTKPVQDR?PTLFLEVIQR?HNHQ------?----------?---GFGAGNFHPPD_RAT??????QIFTKPMQDR?PTLFLEVIQR?HNHQ------?----------?---GFGAGNFHPPD_PIG??????QIFTKPMQDR?PTVFLEVIQR?NNHQ------?----------?---GFGAGNFHPPD_MOUSE????QIFTKPMQDR?PTLFLEVIQR?HNHQ------?----------?---GFGAGNFHPPD_PSESP????QIFSETLMGP?--VFFEFIQR?-----KGDD-?----------?---GFGEGNFMELA_SHECO????QIFTKNLFGP?--IFIEIIQR?-----KNNL-?----------?---GFGEGNFPEA3_MOUSE????MEFKLIEPEE?VARLWGIQKN?RPAMNYDKLS?RSLRYYYEKG?IMQKVAGERY
510????????520????????530????????540????????550HPPD_Hv???????--------SE?LFK-SIE-DY?--EKS--LEA?KQSAAV-QGSHPPD?Ath??????--------SE?LFK-SIE-EY?--EKT--LEA?KQLVGHPPD?HUMAN????--------NS?LFK-AFEEEQ?--NLRGNLTN?METNGVVPGMHPPD?RAT??????--------NS?LFK-AFEEEQ?--ALRGHPPD_PIG??????--------NS?LFK-AFEEEQ?--ELRGNLTD?TDPNGVPFRLHPPD_MOUSE????--------NS?LFK-AFEEEQ?--ALRGNLTD?LEPNGVRSGMHPPD_PSESP????--------KA?LFE-SIERDQ?--VRRGVLST?-DMELA_SHECO????--------KA?LFE-SIERDQ?--VRRGVLPEA3_MOUSE????VYKFVCEPEA?LFSLAFPDNQ?RPALKAEFDR?PVSEEDTVPL?SHLDESPAYL
560 570HPPD_HvHPPD_AthHPPD_HUMANHPPD_RATHPPD_PIGHPPD_MOUSE HPPD_PSESPMELA_SHECOPEA3_MOUSE PELTGPAPPF GHRGGYSY nouns: HPPD-Hv:Hordeum vulgare 4-hydroxyphenyl pyruvic acid dioxygenase (HvSD 36) HPPD-Ath:Arabidopsis thaliana 4-hydroxyphenyl pyruvic acid dioxygenase HPPD-HUMAN:H.Sapiens 4-hydroxyphenyl pyruvic acid dioxygenase HPPD-PIG: pig 4-hydroxyphenyl pyruvic acid dioxygenase HPPD-RAT: mouse F alloantigen HPPD-MOUSE: home mouse 4-hydroxyphenyl pyruvic acid dioxygenase MELA-SHECO:S.colwelliana melA albumen HPPD-PSESP:Pseudomonas sp. (P.J.874 bacterial strain) 4-hydroxyphenyl pyruvic acid
Dioxygenase PEA3-MOUSE:Mus musculus (home mouse) PEA3 polypeptide
What homology was the highest is the Arabidopsis sequence, the homology of whole sequence reaches 58% (412 amino acid whose homologys are 62%), next be HPPD_RAT, be 35% (365 amino acid whose comparisons), HPPD-HUMAN, be 34% (365 amino acid whose comparisons), and HPPD-MOUSE, be 34% (371 amino acid whose comparisons).The cultivation of embodiment 4 barleys (Hordeum vulgare)
Casing an ambient controlled, be among the so-called Mitscherlich pots, condition with control, contain 4 gram Osmocote 5M (Urania at every liter, Hamburg cultivates barley seedlings (Hordeum Vulgare L.CV.Carina, AckermannSaatzucht in soil Germany), Irbach, Germany) 15 days.For growth is evenly carried out, seed is placed on rudiment on the moistening filter paper, 4 ℃ 2 days, 21 ℃ 1 day, only plant the identical seedling of those primary root longitudinal growths.After transferring in the soil, cover the thick soil of 1.5cm to this seedling through screening to them.After this, plant culturing 9 days, 16 hours (120 μ mm of illumination
-2S
-1), dark 8 hours, in conjunction with temperature inversion (21 ℃ of daytimes, 16 ℃ of evenings).After 9 days, in above-mentioned temperature plant is preserved 2 days (the 10th and 11 day) in the dark and senesce to induce it.The cultivation of embodiment 5 tobaccos
Tobacco is cultivated with reference to known method.Used tobacco cultivation kind is Nicotiana tabacum cv.Xanthi.The conversion of embodiment 6 tobaccos
The expression cartridge clip of the present invention that contains HPPD gene order 1 is cloned into (Fig. 4) among the carrier pBinAR-Hyg.Tobacco plant described in the embodiment 5 is transformed with this carrier with known method subsequently.Embodiment 7 improves the biosynthesis ability of tocopherol in tobacco
For HPPD cDNA provides a CaMV 35S promoter and makes its overexpression in tobacco with this 35S promoter.Simultaneously, the Yunnan lentil protein gene promotor of use seed specific specifically improves the content of tocopherol in the tobacco seed.Place the tobacco plant that transforms through relevant construction the greenhouse to cultivate.In all situations, to compare with the plant of unconverted, the concentration of alpha-tocopherol has all improved.Sequence solution (1) general information
(i) applicant
(A) title: BASF AG
(B) street: Carl Bosch
(C) city: Ludwigshafen
(D) federal state: Germany
(F) postcode: 67056
(G) phone: 0621-60-52698
(ii) apply for exercise question: barley HPPD sequence
(iii) sequence number: 2 covers
(iv) the computer-readable lattice show:
(A) recording medium: floppy disk
(B) computer: IBM PC can compatible type
(C) operating system: PC-DOS/MS-DOS
(D) software: PatertIn release #1.0, Version #1.25 (EPA) (2) sequence number: 1 information
(i) sequence signature:
(A) length: 1565 base pairs
(B) type: nucleic acid
(C) chain feature: two strands
(D) topology: linearity
(ii) molecule type: cDNA
(iii) suppose: do not have
(iii) antisense: do not have
(vi) primary source
(A) organism: barley hppd
(D) etap: phylogerontic
(vii) direct sources
(A) library: barley λ FIXII library
(B) clone: pHvSD 36 sequences
(ix) feature:
(A) title: CDS
(B) position: 9..1313
(x) deliver details:
(A) author: Krupinska, Karin
(B) exercise question: Overexpression of HPPD
(C) publication: Overexpression of HPPD
(G) date: 1998
(K) at sequence number: the relevant residue in 1: 1 to 1565
(xi) sequence description: sequence number: 1:CGCACACC ATG CCG CCC ACC CCC ACC ACC CCC GCG GCT ACC GGC GCC GCC 50
Met?Pro?Pro?Thr?Pro?Thr?Thr?Pro?Ala?Ala?Thr?Gly?Ala?Ala
1???????????????5??????????????????10GCC?GCG?GTG?ACG?CCG?GAG?CAC?GCG?CGA?CCG?CAC?CGA?ATG?GTC?CGC?TTC?????98Ala?Ala?Val?Thr?Pro?Glu?His?Ala?Arg?Pro?His?Arg?Met?Val?Arg?Phe?15??????????????????20??????????????????25??????????????????30AAC?CCG?CGC?AGC?GAC?CGC?TTC?CAC?ACG?CTC?TCC?TTC?CAC?CAC?GTC?GAG????146Asn?Pro?Arg?Ser?Asp?Arg?Phe?His?Thr?Leu?Ser?Phe?His?His?Val?Glu
35??????????????????40??????????????????45TTC?TGG?TGC?GCG?GAC?GCC?GCC?TCC?GCC?GCC?GGC?CGC?TTC?GCG?TTC?GCG????194Phe?Trp?Cys?Ala?Asp?Ala?Ala?Ser?Ala?Ala?Gly?Arg?Phe?Ala?Phe?Ala
50??????????????????55??????????????????60CTC?GGC?GCG?CCG?CTC?GCC?GCC?AGG?TCC?GAC?CTC?TCC?ACG?GGG?AAC?TCC????242Leu?Gly?Ala?Pro?Leu?Ala?Ala?Arg?Ser?Asp?Leu?Ser?Thr?Gly?Asn?Ser
65??????????????????70??????????????????75GCG?CAC?GCC?TCC?CAG?CTG?CTC?CGC?TCG?GGC?TCC?CTC?GCC?TTC?CTC?TTC????290Ala?His?Ala?Ser?Gln?Leu?Leu?Arg?Ser?Gly?Ser?Leu?Ala?Phe?Leu?Phe
80??????????????????85??????????????????90ACC?GCG?CCC?TAC?GCC?AAC?GGC?TGC?GAC?GCC?GCC?ACC?GCC?TCC?CTG?CCC????338Thr?Ala?Pro?Tyr?Ala?Asn?Gly?Cys?Asp?Ala?Ala?Thr?Ala?Ser?Leu?Pro?95?????????????????100?????????????????105?????????????????110TCC?TTC?TCC?GCC?GAC?GCC?GCG?CGC?CGG?TTC?TCC?GCC?GAC?CAC?GGG?ATC????386Ser?Phe?Ser?Ala?Asp?Ala?Ala?Arg?Arg?Phe?Ser?Ala?Asp?His?Gly?Ile
115?????????????????120?????????????????125GCG?GTG?CGC?TCC?GTA?GCG?CTG?CGC?GTC?GCA?GAC?GCC?GCC?GAG?GCC?TTC????434Ala?Val?Arg?Ser?Val?Ala?Leu?Arg?Val?Ala?Asp?Ala?Ala?Glu?Ala?Phe
130?????????????????135?????????????????140CGC?GCC?AGT?CGT?CGA?CGG?GGC?GCG?CGC?CCG?GCC?TTC?GCC?CCC?GTG?GAC?????482Arg?Ala?Ser?Arg?Arg?Arg?Gly?Ala?Arg?Pro?Ala?Phe?Ala?Pro?Val?Asp
145?????????????????150?????????????????155CTC?GGC?CGC?GGC?TTC?GCG?TTC?GCG?GAG?GTC?GAG?CTC?TAC?GGC?GAC?GTC?????530Leu?Gly?Arg?Gly?Phe?Ala?Phe?Ala?Glu?Val?Glu?Leu?Tyr?Gly?Asp?Val
160?????????????????165?????????????????170GTG?CTC?CGC?TTC?GTC?AGC?CAC?CCG?GAC?GGC?ACG?GAC?GTG?CCC?TTC?TTG?????578Val?Leu?Arg?Phe?Val?Ser?His?Pro?Asp?Gly?Thr?Asp?Val?Pro?Phe?Leu175?????????????????180?????????????????185?????????????????190CCG?GGG?TTC?GAG?GGC?GTA?ACC?AAC?CCG?GAC?GCC?GTG?GAC?TAC?GGC?CTG?????626Pro?Gly?Phe?Glu?Gly?Val?Thr?Asn?Pro?Asp?Ala?Val?Asp?Tyr?Gly?Leu
195?????????????????200?????????????????205ACG?CGG?TTC?GAC?CAC?GTC?GTC?GGC?AAC?GTC?CCG?GAG?CTT?GCC?CCC?GCC?????674Thr?Arg?Phe?Asp?His?Val?Val?Gly?Asn?Val?Pro?Glu?Leu?Ala?Pro?Ala
210?????????????????215?????????????????220GCA?GCC?TAC?ATC?GCC?GGG?TTC?ACG?GGG?TTC?CAC?GAG?TTC?GCC?GAG?TTC?????722Ala?Ala?Tyr?Ile?Ala?Gly?Phe?Thr?Gly?Phe?His?Glu?Phe?Ala?Glu?Phe
225????????????????230?????????????????235ACG?GCG?GAG?GAC?GTG?GGC?ACG?ACC?GAG?AGC?GGG?CTC?AAC?TCG?GTG?GTG?????770Thr?Ala?Glu?Asp?Val?Gly?Thr?Thr?Glu?Ser?Gly?Leu?Asn?Ser?Val?Val
240?????????????????245?????????????????250CTC?GCC?AAC?AAC?TCG?GAG?GGC?GTG?CTG?CTG?CCG?CTC?AAC?GAG?CCG?GTG?????818Leu?Ala?Asn?Asn?Ser?Glu?Gly?Val?Leu?Leu?Pro?Leu?Asn?Glu?Pro?Val255?????????????????260?????????????????265?????????????????270CAC?GGC?ACC?AAG?CGC?CGG?AGC?CAG?ATA?CAG?ACG?TTC?CTG?GAA?CAC?CAC?????866His?Gly?Thr?Lys?Arg?Arg?Ser?Gln?Ile?Gln?Thr?Phe?Leu?Glu?His?His
275?????????????????280?????????????????285GGC?GGC?CCG?GGC?GTG?CAG?CAC?ATC?GCG?GTG?GCC?AGC?AGT?GAC?GTG?CTC?????914Gly?Gly?Pro?Gly?Val?Gln?His?Ile?Ala?Val?Ala?Ser?Ser?Asp?Val?Leu
290?????????????????295?????????????????300AGG?ACG?CTC?AGG?AAG?ATG?CGT?GCG?CGC?TCC?GCC?ATG?GGC?GGC?TTC?GAC?????962Arg?Thr?Leu?Arg?Lys?Met?Arg?Ala?Arg?Ser?Ala?Met?Gly?Gly?Phe?Asp
305?????????????????310?????????????????315TTC?CTG?CCA?CCC?CCG?CTG?CCG?AAG?TAC?TAC?GAA?GGC?GTG?CGA?CGC?CTT????1010Phe?Leu?Pro?Pro?Pro?Leu?Pro?Lys?Tyr?Tyr?Glu?Gly?Val?Arg?Arg?Leu
320?????????????????325?????????????????330GCC?GGG?GAT?GTC?CTC?TCG?GAG?GCG?CAG?ATC?AAG?GAA?TGC?CAG?GAG?CTG????1058Ala?Gly?Asp?Val?Leu?Ser?Glu?Ala?Gln?Ile?Lys?Glu?Cys?Gln?Glu?Leu335?????????????????340?????????????????345?????????????????350GGT?GTG?CTC?GTC?GAT?AGG?GAC?GAC?CAA?GGG?GTG?TTG?CTC?CAA?ATC?TTC????1106Gly?Val?Leu?Val?Asp?Arg?Asp?Asp?Gln?Gly?Val?Leu?Leu?Gln?Ile?Phe
355?????????????????360?????????????????365ACC?AAG?CCA?GTA?GGG?GAC?AGG?CCG?ACC?TTG?TTC?CTG?GAG?ATG?ATC?CAG????1154Thr?Lys?Pro?Val?Gly?Asp?Arg?Pro?Thr?Leu?Phe?Leu?Glu?Met?Ile?Gln
370?????????????????375?????????????????380AGG?ATC?GGG?TGC?ATG?GAG?AAG?GAC?GAG?AGA?GGG?GAA?GAG?TAC?CAG?AAG????1202Arg?Ile?Gly?Cys?Met?Glu?Lys?Asp?Glu?Arg?Gly?Glu?Glu?Tyr?Gln?Lys
385?????????????????390?????????????????395GGT?GGC?TGC?GGC?GGG?TTC?GGC?AAA?GGC?AAC?TTC?TCC?GAG?CTG?TTC?AAG????1250Gly?Gly?Cys?Gly?Gly?Phe?Gly?Lys?Gly?Asn?Phe?Ser?Glu?Leu?Phe?Lys
400?????????????????405?????????????????410TCC?ATT?GAA?GAT?TAC?GAG?AAG?TCC?CTT?GAA?GCC?AAG?CAA?TCT?GCT?GCA????1298Ser?Ile?Glu?Asp?Tyr?Glu?Lys?Ser?Leu?Glu?Ala?Lys?Gln?Ser?Ala?Ala415?????????????????420?????????????????425?????????????????430GTT?CAG?GGA?TCA?TAGGATAGAA?GCTGGTCCTT?GTATCATGGT?CTCATGGAGC????????1350Val?Gln?Gly?Ser
435AAAAGAAAAC AATGTTGTTT GTAATATGCG TCGCACAATT ATATCAATGT TATAATTGGT 14l0GAAGCTGAAG ACAGATGTAT CCTATGTATG ATGGGTGTAA TGGATGGTAG AGGGGCTCAC 1470ACATGAAGAA AATGTAGCGT TGACATTGTT GTACAATCTT GCTTGCAAGT AAAATAAAGA 1530ACAGATTTTG AGTTCTGCAA AAAAAAAAAA AAAAA 1565 (2) sequence numbers: 2 information
(i) sequence signature:
(A) length: 434 amino acid
(B) type: amino acid
(D) topology: linearity
(ii) molecule type: protein
(xi) sequence description: sequence number: 2:Met Pro Pro Thr Pro Thr Thr Pro Ala Ala Thr Gly Ala Ala Ala Ala 15 10 15Val Thr Pro Glu His Ala Arg Pro His Arg Met Val Arg Phe Asn Pro
20??????????????????25??????????????????30Arg?Ser?Asp?Arg?Phe?His?Thr?Leu?Ser?Phe?His?His?Val?Glu?Phe?Trp
35??????????????????40??????????????????45Cys?Ala?Asp?Ala?Ala?Ser?Ala?Ala?Gly?Arg?Phe?Ala?Phe?Ala?Leu?Gly
50??????????????????55??????????????????60Ala?Pro?Leu?Ala?Ala?Arg?Ser?Asp?Leu?Ser?Thr?Gly?Asn?Ser?Ala?His?65??????????????????70??????????????????75??????????????????80Ala?Ser?Gln?Leu?Leu?Arg?Ser?Gly?Ser?Leu?Ala?Phe?Leu?Phe?Thr?Ala
85??????????????????90??????????????????95Pro?Tyr?Ala?Asn?Gly?Cys?Asp?Ala?Ala?Thr?Ala?Ser?Leu?Pro?Ser?Phe
100?????????????????105?????????????????110Ser?Ala?Asp?Ala?Ala?Arg?Arg?Phe?Ser?Ala?Asp?His?Gly?Ile?Ala?Val
115?????????????????120?????????????????125Arg?Ser?Val?Ala?Leu?Arg?Val?Ala?Asp?Ala?Ala?Glu?Ala?Phe?Arg?Ala
130?????????????????135?????????????????140Ser?Arg?Arg?Arg?Gly?Ala?Arg?Pro?Ala?Phe?Ala?Pro?Val?Asp?Leu?Gly145?????????????????150?????????????????155?????????????????160Arg?Gly?Phe?Ala?Phe?Ala?Glu?Val?Glu?Leu?Tyr?Gly?Asp?Val?Val?Leu
165?????????????????170?????????????????175Arg?Phe?Val?Ser?His?Pro?Asp?Gly?Thr?Asp?Val?Pro?Phe?Leu?Pro?Gly
180?????????????????185?????????????????190Phe?Glu?Gly?Val?Thr?Asn?Pro?Asp?Ala?Val?Asp?Tyr?Gly?Leu?Thr?Arg
195?????????????????200?????????????????205Phe?Asp?His?Val?Val?Gly?Asn?Val?Pro?Glu?Leu?Ala?Pro?Ala?Ala?Ala
210?????????????????215?????????????????220Tyr?Ile?Ala?Gly?Phe?Thr?Gly?Phe?His?Glu?Phe?Ala?Glu?Phe?Thr?Ala225?????????????????230?????????????????235?????????????????240Glu?Asp?Val?Gly?Thr?Thr?Glu?Ser?Gly?Leu?Asn?Ser?Val?Val?Leu?Ala
245?????????????????250?????????????????255Asn?Asn?Ser?Glu?Gly?Val?Leu?Leu?Pro?Leu?Asn?Glu?Pro?Val?His?Gly
260?????????????????265?????????????????270Thr?Lys?Arg?Arg?Ser?Gln?Ile?Gln?Thr?Phe?Leu?Glu?His?His?Gly?Gly
275?????????????????280?????????????????285Pro?Gly?Val?Gln?His?Ile?Ala?Val?Ala?Ser?Ser?Asp?Val?Leu?Arg?Thr
290?????????????????295?????????????????300Leu?Arg?Lys?Met?Arg?Ala?Arg?Ser?Ala?Met?Gly?Gly?Phe?Asp?Phe?Leu305?????????????????3l0?????????????????315?????????????????320Pro?Pro?Pro?Leu?Pro?Lys?Tyr?Tyr?Glu?Gly?Val?Arg?Arg?Leu?Ala?Gly
325?????????????????330?????????????????335Asp?Val?Leu?Ser?Glu?Ala?Gln?Ile?Lys?Glu?Cys?Gln?Glu?Leu?Gly?Val
340?????????????????345?????????????????350Leu?Val?Asp?Arg?Asp?Asp?Gln?Gly?Val?Leu?Leu?Gln?Ile?Phe?Thr?Lys
355?????????????????360?????????????????365Pro?Val?Gly?Asp?Arg?Pro?Thr?Leu?Phe?Leu?Glu?Met?Ile?Gln?Arg?Ile
370?????????????????375?????????????????380Gly?Cys?Met?Glu?Lys?Asp?Glu?Arg?Gly?Glu?Glu?Tyr?Gln?Lys?Gly?Gly385?????????????????390?????????????????395?????????????????400Cys?Gly?Gly?Phe?Gly?Lys?Gly?Asn?Phe?Ser?Glu?Leu?Phe?Lys?Ser?Ile
405?????????????????410?????????????????415Glu?Asp?Tyr?Glu?Lys?Ser?Leu?Glu?Ala?Lys?Gln?Ser?Ala?Ala?Val?Gln
420?????????????????425?????????????????430Gly?Ser
Claims (24)
1. the sequence number of coding HPPD: 1 dna sequence dna and with the dna sequence dna of its hybridization.
2. the expression cartridge clip that contains the dna sequence dna described in a promotor and the claim 1.
3. the expression cartridge clip described in claim 2 comprises the CaMV 35S promoter.
4. the expression cartridge clip described in claim 2 comprises the common bean promotor of seed specific.
5. the expression cartridge clip described in claim 2, wherein the dna sequence dna described in claim 1 and another albumen are functional and are connected and form an associating translation product.
6. the application of the expression cartridge clip described in claim 2 in transforming plant.
7. transform the method for plant, comprise that the expression cartridge clip of handle described in claim 2 imports among vegetable cell, callus, whole strain plant or the plant protoplast.
8. transform the method for plant, comprising:
1) the expression cartridge clip described in claim 2 is transferred in the agrobacterium strains,
2) recombinant clone that forms is separated, and
3) above-mentioned recombinant clone is used to transform plant.
9. the method described in claim 8, wherein transforming is to finish down the auxiliary of agrobacterium tumefaciens bacterial strain.
10. the method for the conversion plant described in claim 7, wherein transforming is to finish down the auxiliary of electroporation.
11. the method for the conversion plant described in claim 7 wherein transforms by means of the particle bombardment method and finishes.
12. the plant that content of vitamin E improves contains just like arbitrary described expression cartridge clip in the claim 2 to 5.
13. the plant described in claim 12 is selected from soybean, barley, oat, wheat, oleaginous seed rape, corn or Sunflower Receptacle.
14. produce the method for the plant of its content of vitamin E raising, it is characterized in that the dna sequence dna described in claim 1 is expressed in plant.
15. the method described in claim 14, wherein dna sequence dna is expressed in tobacco.
16., wherein express to be in the blade of plant or seed, to carry out as claim 14 or 15 arbitrary described methods.
17., produce the plant that its content of vitamin E improves with arbitrary described expression cartridge clip in the claim 2 to 5 by in plant, expressing the dna sequence dna described in claim 1.
18. be used to identify the test macro of HPPD inhibitor with the generation of the expression cartridge clip described in claim 2 one.
19. the test macro that is used to identify the HPPD inhibitor that is expressed as the basis with the expression cartridge clip described in claim 2.
20. the herbicidal activity material that can identify with the test macro described in claim 19.
21. the application of the plant described in claim 12 in producing plant HPPD.
22., produce HPPD inhibitor resistance enhanced plant with the expression cartridge clip described in claim 2 by expressing the dna sequence dna described in claim 1 more efficiently.
23. produce method to HPPD inhibitor resistance enhanced plant by expressing dna sequence dna described in claim 1 more efficiently.
24., contain just like arbitrary described expression cartridge clip in the claim 2 to 5 to HPPD inhibitor resistance enhanced plant.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19730066A DE19730066A1 (en) | 1997-07-14 | 1997-07-14 | DNA sequence coding for a hydroxyphenylpyruvate dioxygenase and its overproduction in plants |
| DE19730066.9 | 1997-07-14 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1270636A true CN1270636A (en) | 2000-10-18 |
Family
ID=7835618
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN98809105A Pending CN1270636A (en) | 1997-07-14 | 1998-06-23 | DNA sequence coding for a hydroxyphenylpyruvate dioxygenase and overproduction thereof in plants |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP1009841A1 (en) |
| CN (1) | CN1270636A (en) |
| AR (1) | AR014893A1 (en) |
| AU (1) | AU8216998A (en) |
| BR (1) | BR9811006A (en) |
| CA (1) | CA2296840A1 (en) |
| DE (1) | DE19730066A1 (en) |
| WO (1) | WO1999004021A1 (en) |
| ZA (1) | ZA986188B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1317388C (en) * | 2003-11-28 | 2007-05-23 | 中国科学院微生物研究所 | 2-amino phenol 1,6-dioxygenase, its gene and use thereof |
| CN100335641C (en) * | 2002-09-11 | 2007-09-05 | 拜尔农科股份有限公司 | Transformed plants with enhanced prenylquinone biosynthesis |
| CN102088994B (en) * | 2008-04-17 | 2014-06-25 | 海莱乌医院 | Indoleamine 2, 3-dioxygenase based immunotherapy |
| WO2014177992A3 (en) * | 2013-04-30 | 2015-06-25 | Basf Se | Plants having increased tolerance to herbicides |
| CN105543245A (en) * | 2015-12-28 | 2016-05-04 | 北京未名凯拓作物设计中心有限公司 | HPPD (hydroxyphenylpyruvate dioxygenase) inhibitor-resistant gene derived from pseudomonas aeruginosa, as well as application thereof |
| CN105567705A (en) * | 2015-12-28 | 2016-05-11 | 北京未名凯拓作物设计中心有限公司 | HPPD inhibitor-resistant gene derived from ochrobactrum anthropi and application thereof |
| CN105567707A (en) * | 2015-12-28 | 2016-05-11 | 北京未名凯拓作物设计中心有限公司 | HPPD inhibitor-resistant gene and application thereof |
| CN105567706A (en) * | 2015-12-28 | 2016-05-11 | 北京未名凯拓作物设计中心有限公司 | HPPD inhibitor-resistant gene derived from pseudomonas putida and application thereof |
Families Citing this family (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2778527A1 (en) * | 1998-05-18 | 1999-11-19 | Rhone Poulenc Agrochimie | Producing tomato, cereal or oilseed plants, especially maize, oilseed rape, soya or sunflower plants, from which tocopherols can be extracted and purified |
| DE19903493A1 (en) * | 1999-01-29 | 2000-08-03 | Basf Ag | Overexpression of a DNA sequence coding for a transketolase in plants |
| ATE316578T1 (en) | 1999-04-15 | 2006-02-15 | Calgene Llc | NUCLEIC ACID SEQUENCES FOR PROTEINS INVOLVED IN TOCOPHEROL BIOSYNTHESIS |
| FR2796954B1 (en) * | 1999-07-30 | 2003-10-31 | Aventis Cropscience Sa | HYDROXY-PHENYL PYRUVATE DIOXYGENASE FUSED TO A SIGNAL PEPTIDE, DNA SEQUENCE AND THE PRODUCTION OF PLANTS CONTAINING SUCH A GENE, HERBICIDE TOLERANT |
| DE19937957A1 (en) * | 1999-08-11 | 2001-02-15 | Sungene Gmbh & Co Kgaa | Homogenate dioxygenase |
| CN1301841A (en) * | 1999-12-27 | 2001-07-04 | 上海博德基因开发有限公司 | New polypeptide-human dioxygenase 13 and polynucleotide codign such polypeptide |
| US6872815B1 (en) | 2000-10-14 | 2005-03-29 | Calgene Llc | Nucleic acid sequences to proteins involved in tocopherol synthesis |
| EP1356033B1 (en) | 2000-08-07 | 2009-10-28 | Monsanto Technology LLC | Methyl-d-erythritol phosphate pathway genes |
| DE10046462A1 (en) * | 2000-09-19 | 2002-05-29 | Sungene Gmbh & Co Kgaa | Improved procedures for vitamin E biosynthesis |
| CA2427787C (en) | 2000-12-07 | 2012-07-17 | Syngenta Limited | Herbicide resistant plants |
| US7161061B2 (en) | 2001-05-09 | 2007-01-09 | Monsanto Technology Llc | Metabolite transporters |
| EP1392106B1 (en) | 2001-05-09 | 2008-12-31 | Monsanto Technology LLC | Tyra genes and uses thereof |
| WO2003016482A2 (en) | 2001-08-17 | 2003-02-27 | Monsanto Technology Llc | Methyltransferase genes and uses thereof |
| CN100400661C (en) | 2001-10-25 | 2008-07-09 | 孟山都技术有限公司 | Aromatic methyltransferases and uses thereof |
| CA2478957C (en) | 2002-03-19 | 2013-07-02 | Monsanto Technology, Llc | Homogentisate prenyl transferase ("hpt") nucleic acids and polypeptides, and uses thereof |
| CN1681928A (en) | 2002-08-05 | 2005-10-12 | 孟山都技术公司 | Tocopherol biosynthesis related genes and uses thereof |
| US9175305B2 (en) | 2009-01-22 | 2015-11-03 | Syngenta Participations Ag | Mutant hydroxyphenylpyruvate dioxygenase polypeptides and methods of use |
| EP2573183B1 (en) | 2009-01-22 | 2017-01-04 | Syngenta Participations AG | Mutant hydroxyphenylpyruvate dioxgenase polypeptids and methods of use |
| US9347046B2 (en) | 2009-01-22 | 2016-05-24 | Syngenta Participations Ag | Hydroxyphenylpyruvate dioxygenase polypeptides and methods of use |
| WO2014177991A2 (en) * | 2013-04-30 | 2014-11-06 | Basf Se | Plants having increased tolerance to herbicides |
| CN116064430B (en) * | 2021-09-14 | 2023-08-11 | 山东舜丰生物科技有限公司 | Mutant HPPD polypeptides and uses thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2734842B1 (en) * | 1995-06-02 | 1998-02-27 | Rhone Poulenc Agrochimie | DNA SEQUENCE OF A HYDROXY-PHENYL PYRUVATE DIOXYGENASE GENE AND OBTAINING PLANTS CONTAINING A HYDROXY-PHENYL PYRUVATE DIOXYGENASE GENE, TOLERANT TO CERTAIN HERBICIDES |
| US6087563A (en) * | 1996-01-29 | 2000-07-11 | Arizona Board Of Regents On Behalf Of The University Of Arizona | Cloned arabidopsis p-hydroxyphenyl pyruvic acid dioxygenase DNA |
| WO1997049816A1 (en) * | 1996-06-27 | 1997-12-31 | E.I. Du Pont De Nemours And Company | Plant gene for p-hydroxyphenylpyruvate dioxygenase |
-
1997
- 1997-07-14 DE DE19730066A patent/DE19730066A1/en not_active Withdrawn
-
1998
- 1998-06-23 EP EP98932179A patent/EP1009841A1/en not_active Withdrawn
- 1998-06-23 AU AU82169/98A patent/AU8216998A/en not_active Abandoned
- 1998-06-23 WO PCT/EP1998/003832 patent/WO1999004021A1/en not_active Application Discontinuation
- 1998-06-23 CN CN98809105A patent/CN1270636A/en active Pending
- 1998-06-23 BR BR9811006-3A patent/BR9811006A/en not_active IP Right Cessation
- 1998-06-23 CA CA002296840A patent/CA2296840A1/en not_active Abandoned
- 1998-07-13 ZA ZA9806188A patent/ZA986188B/en unknown
- 1998-07-14 AR ARP980103428A patent/AR014893A1/en not_active Application Discontinuation
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100335641C (en) * | 2002-09-11 | 2007-09-05 | 拜尔农科股份有限公司 | Transformed plants with enhanced prenylquinone biosynthesis |
| CN1317388C (en) * | 2003-11-28 | 2007-05-23 | 中国科学院微生物研究所 | 2-amino phenol 1,6-dioxygenase, its gene and use thereof |
| CN102088994B (en) * | 2008-04-17 | 2014-06-25 | 海莱乌医院 | Indoleamine 2, 3-dioxygenase based immunotherapy |
| WO2014177992A3 (en) * | 2013-04-30 | 2015-06-25 | Basf Se | Plants having increased tolerance to herbicides |
| US10829778B2 (en) | 2013-04-30 | 2020-11-10 | Basf Se | Plants having increased tolerance to herbicides |
| CN105543245A (en) * | 2015-12-28 | 2016-05-04 | 北京未名凯拓作物设计中心有限公司 | HPPD (hydroxyphenylpyruvate dioxygenase) inhibitor-resistant gene derived from pseudomonas aeruginosa, as well as application thereof |
| CN105567705A (en) * | 2015-12-28 | 2016-05-11 | 北京未名凯拓作物设计中心有限公司 | HPPD inhibitor-resistant gene derived from ochrobactrum anthropi and application thereof |
| CN105567707A (en) * | 2015-12-28 | 2016-05-11 | 北京未名凯拓作物设计中心有限公司 | HPPD inhibitor-resistant gene and application thereof |
| CN105567706A (en) * | 2015-12-28 | 2016-05-11 | 北京未名凯拓作物设计中心有限公司 | HPPD inhibitor-resistant gene derived from pseudomonas putida and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2296840A1 (en) | 1999-01-28 |
| EP1009841A1 (en) | 2000-06-21 |
| WO1999004021A1 (en) | 1999-01-28 |
| AU8216998A (en) | 1999-02-10 |
| BR9811006A (en) | 2000-08-22 |
| DE19730066A1 (en) | 1999-01-21 |
| AR014893A1 (en) | 2001-04-11 |
| ZA986188B (en) | 2000-01-13 |
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