CN101798577A - Jatropha curcas phytoene synthetase gene sequence and application thereof in plants - Google Patents

Abstract

The invention relates to the technical fields of molecular biology and genetic engineering and relates to a JcPSY protein for expressing the Jatropha curcas phytoene synthetase and a nucleotide sequence thereof. Due to the use of the Jatropha curcas PSY protein, substances interacting with the Jatropha curcas PSY protein, or receptors, inhibitors or antagonists, and the like can be screened by various conventional screening methods. The Jatropha curcas PSY protein genes can be used for improving the beta-carotene content in the plants and improving the economic values of the plants through the genetic engineering technology, and have obvious market application prospects.

Description

Jatropha curcas phytoene synthetase gene sequence and the application in plant

Technical field

The present invention relates to molecular biology, gene engineering technology field.Particularly, the present invention relates to a kind of in Cortex jatrophae, express JcPSY albumen (jatropha curcas phytoene synthetase, Jatropha curcas phytoenesynthase, JcPSY) and nucleotide sequence.

Background technology

Known, (biosynthesizing of β-Carotene) mainly betides in higher plant, algae, fungi and the bacterial body β-Hu Luobusu, can not the de novo synthesis β-Hu Luobusu in the animal body.β-Hu Luobusu is the precursor of vitamin A (Vitamin A), can be converted into vitamin A as required in human body.It has nutrition, painted dual function, is the outstanding nourishing food additive of category-A (food additives) that the Food and Argriculture OrganizationFAO (FAO) and the foodstuff additive joint specialist council of The World Health Organization (WHO) are assert.In recent years, increasing medical research shows that β-Hu Luobusu can resist multiple cancer, particularly can reduce the lung cancer morbidity rate.β-Hu Luobusu strengthens body immunity at the cancellation free radical, and protection human health aspects such as preventing cardiovascular disease play an important role.Lyeopene is the main pigment of structure in the carotenoid, experiment showed, that the absorption of Lyeopene helps reducing the incidence probability of prostate cancer, protect cardiovascularly, reduces ultraviolet to multiple uses such as human skin injuries.Phytoene synthetase is the biosynthetic specific enzymes of catalysis carotenoid, and it is with synthetic first the carotenoid-phytoene of bimolecular geranyl geranyl tetra-sodium (GGPP) catalysis.Studies show that phytoene synthase gene is the rate-limiting enzyme in the carotenoid biosynthetic pathway, this is proved in tamato fruit.Fray etc. import the phytoene synthetase in the tomato (PSY) gene antisense in the tomato, in the transfer-gen plant that the result obtains carotenoid content significantly reduce (Fray RG, etc.Constitutive?expression?of?a?fruit?phytoene?synthasegene?in?transgenic?tomatoes?causes?dwarfism?by?redirecting?metabolitesfrom?the?gibberellin?pathway.Plant?J，1995，8(5)：693-701)。Overexpression phytoene synthetase in tomato, rape, Arabidopis thaliana and potato, the level of accumulation carotenoid significantly improves in the respective organization, β-Hu Luobusu has improved 1.9 times in tamato fruit, average raising is 43 times in the Arabidopis thaliana seed, improve 50 times in the Semen Brassicae campestris, improve 8 times in the potato tuber.The phytoene synthetase PSY of catalysis carotenoid pathways metabolism the first step reaction is the crucial regulatory enzyme of this pathways metabolism, and generates " bottleneck " that this step of phytoene has become the carotenoid pathways metabolism by GGPP.Therefore, the PSY gene has now become the first-selected goal gene (Zhu Changfu in the genetic engineering of plant carotenoid, Deng. the application in genetically engineered of biosynthesizing of plant carotenoid and genes involved thereof. Mol.Biol., 2004,30 (6): 609-618).Before the present invention comes forth, any cDNA sequence that discloses or reported the coding jatropha curcas phytoene synthetase of mentioning in the present patent application is not arranged as yet.The clone of relevant jatropha curcas phytoene gene and functional study are subjected to state natural sciences fund (30771745) subsidy among the present invention.

Summary of the invention

First purpose of the present invention provides a kind of new Cortex jatrophae PSY gene, and this gene is a Cortex jatrophae PSY protein gene.

Second purpose of the present invention provides a kind of new Cortex jatrophae PSY albumen.

The 3rd purpose of the present invention provides a kind of recombinant technology that utilizes and prepares the above-mentioned new Cortex jatrophae PSY albumen and the method for nucleotide sequence.

In one aspect of the invention, a kind of isolated dna molecular is provided, this molecule comprises: coding has the nucleotide sequence of the polypeptide of Cortex jatrophae PSY protein active, shows at least 70% homology from the nucleotides sequence of Nucleotide 78-1364 position dna molecular among described nucleotide sequence and the SEQ ID NO.1; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.1 in from the nucleotide sequence hybridization of Nucleotide 78-1364 position.Preferably, described sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO.2.More preferably, described sequence has among the SEQ ID NO.1 nucleotide sequence from Nucleotide 78-1364 position.

In another aspect of this invention, provide a kind of isolated Cortex jatrophae PSY protein and peptide, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.2 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.2 polypeptide of sequence.

In another aspect of this invention, also provide a kind of carrier, it comprises above-mentioned dna molecular.

In another aspect of this invention, also provide a kind of usefulness above-mentioned carrier transformed host cells.This host cell is yeast cell, tobacco and other vegetable cell in example.

In another aspect of this invention, also provide a kind of generation to have the method for the polypeptide of Cortex jatrophae PSY protein active, its step is as follows:

(1) nucleotide sequence that coding is had a purifying of Cortex jatrophae PSY protein active polypeptide operationally is connected in expression regulation sequence, form Cortex jatrophae PSY protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 78-1364 position among described nucleotide sequence and the SEQ ID NO.1;

(2) change the expression vector in the step (1) over to prokaryotic host cell, form the proteic reconstitution cell of Cortex jatrophae PSY;

(3) be fit to express under the condition of Cortex jatrophae PSY protein polypeptide the reconstitution cell in the culturing step (2);

(4) isolate pure substantially polypeptide with Cortex jatrophae PSY protein-active.

Preferably, the nucleotide sequence that uses in the method has the sequence of 78-1364 position among the SEQ ID NO.1.

In another aspect of this invention, also provide a kind of transgenic technology of utilizing that the nucleotide sequence that coding has Cortex jatrophae PSY protein active polypeptide is transformed into plant to improve the method for β-Hu Luobusu in the plant, its step is as follows:

(1) nucleotide sequence of purifying that coding is had a polypeptide of Cortex jatrophae PSY protein-active operationally is connected in the expression of plants regulating and controlling sequence, formation contains the plant expression vector of Cortex jatrophae PSY protein gene, shows at least 70% homology from the nucleotides sequence of Nucleotide 78-1364 position among described nucleotide sequence and the SEQ ID NO.1;

(2) change the expression vector in the step (1) over to Agrobacterium, the Agrobacterium that will contain expression vector is cultivated altogether with eukaryotic host cell, under 22-28 ℃ of condition, the dark cultivation after 1-2 days, by screening as antibiotic-screening, acquisition contains transformant and the final regeneration of transgenic plant and the offspring thereof of Cortex jatrophae PSY protein gene, comprises plant seed and plant tissue.Contain that β-Hu Luobusu significantly improves in the transfer-gen plant of Cortex jatrophae PSY protein gene.

Preferably, the nucleotide sequence that uses in the method has the sequence of 78-1364 position among the SEQ ID No.1.The present invention also provides and PSY protein polypeptide specificity bonded antibody, and it comprises polyclonal antibody and monoclonal antibody.In the present invention, " isolating ", " purifying " or " sill is pure " DNA are meant, this DNA or fragment have been arranged in the sequence of its both sides and have separated under native state, refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.

In the present invention, refer to encode has the nucleotide sequence of the active polypeptide of jatropha curcas phytoene synthetase to term " jatropha curcas phytoene synthetase (or polypeptide) encoding sequence ", as 78-1364 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1.This degenerate sequence is meant, is arranged in the encoder block 78-1364 position Nucleotide of SEQ ID NO.1 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.1 in 78-1364 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.2 of also encoding out.This term also comprises can be under the moderate stringent condition, better under the height stringent condition with SEQ IDNO.1 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 78-1364 position.This term also comprise with SEQ ID NO.1 in from the homology of nucleotide sequence at least 70% of Nucleotide 78-1364 position, preferably at least 80%, more preferably at least 90%, at least 95% nucleotide sequence best.

This term also comprises encoding to have the variant form of open reading frame sequence among the proteic SEQ IDNO.1 with natural Cortex jatrophae PSY albumen identical function.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.

In the present invention, " pure substantially " protein or polypeptide are meant that it accounts at least 20% of the total material of sample at least, preferably at least 50%, more preferably at least 80%, and at least 90% (by dry weight or weight in wet base) best.Purity can be measured with any suitable method, as measure the purity of polypeptide with column chromatography, PAGE or HPLC method.Substantially pure polypeptide is substantially free of the component of following it under the native state.

In the present invention, term " Cortex jatrophae PSY albumen or polypeptide " refers to have the SEQ ID NO.1 polypeptide of sequence of Cortex jatrophae PSY protein-active.This term also comprises the variant form that has with the SEQ ID NO.2 sequence of natural Cortex jatrophae PSY albumen identical function.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of Cortex jatrophae PSY and reactive derivative.

The variant form of Cortex jatrophae PSY protein polypeptide of the present invention comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low stringent condition can with the coded albumen of the DNA of Cortex jatrophae PSY protein D NA hybridization and the polypeptide or the albumen that utilize the serum of Cortex jatrophae PSY protein polypeptide to obtain.

In the present invention, " Cortex jatrophae PSY albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO.1, has 10 at the most, and preferably at the most 8, more preferably 5 amino acid similar performances or close amino acid are replaced and formed polypeptide at the most.These conservative property variation polypeptide are preferably replaced according to table 1 and are produced.

Table 1

Initial residue	Representational replacement	The preferred replacement
			??Ala(A)	?Val；Leu；Ile	??Val
??Arg(R)	?Lys；Gln；Asn	??Lys

Initial residue	Representational replacement	The preferred replacement
			??Asn(N)	?Gln；His；Lys；Arg	??Gln
??Asp(D)	?Glu	??Glu
			??Cys(C)	?Ser	??Ser
??Gln(Q)	?Asn	??Asn
			??Glu(E)	?Asp	??Asp
??Gly(G)	?Pro；Ala	??Ala
			??His(H)	?Asn；Gln；Lys；Arg	??Arg
??Ile(I)	?Leu；Val；Met；Ala；Phe	??Leu
			??Leu(L)	?Ile；Val；Met；Ala；Phe	??Ile
??Lys(K)	?Arg；Gln；Asn	??Arg
			??Met(M)	?Leu；Phe；Ile	??Leu
??Phe(F)	?Leu；Val；Ile；Ala；Tyr	??Leu
			??Pro(P)	?Ala	??Ala
??Ser(S)	?Thr	??Thr
			??Thr(T)	?Ser	??Ser
??Trp(W)	?Tyr；Phe	??Tyr
			??Tyr(Y)	?Trp；Phe；Thr；Ser	??Phe
??Val(V)	?Ile；Leu；Met；Phe；Ala	??Leu

The present invention also comprises the analogue of Cortex jatrophae PSY albumen or polypeptide.The difference of these analogues and natural PS Y protein polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

Described modification (not changing primary structure usually) form comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its proteolysis performance or optimized solubility property by modifying.

In the present invention, can select various carrier known in the art for use, the carrier as commercially available comprises plasmid, clay etc.When producing Cortex jatrophae PSY protein polypeptide of the present invention, Cortex jatrophae PSY albumen coded sequence operationally can be connected in expression regulation sequence, thereby form Cortex jatrophae PSY protein expression vector.

As used herein, " operationally being connected in " refer to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjacent, then means in reading frame adjacent for the secretion leader sequence.

In the present invention, term " host cell " is an eukaryotic cell.Eukaryotic host cell commonly used comprises yeast cell, tobacco cell and other vegetable cell.

Whether and quantity the expression of also available Northern blotting technical Analysis Cortex jatrophae PSY protein gene product promptly analyzes the existence of the proteic rna transcription thing of Cortex jatrophae PSY in cell.

The Western engram analysis of the Northern engram analysis of Cortex jatrophae PSY RNA and Cortex jatrophae PSY specific antibody can be united use, to confirm the expression of Cortex jatrophae PSY in biological specimen.

In addition, can be used as the nucleic acid molecule of probe among the present invention, this molecule has 8-100 continuous nucleotide of Cortex jatrophae PSY albumen nucleotide coding sequence usually, preferably has 15-50 continuous nucleotide.This probe can be used for whether existing in the test sample the proteic nucleic acid molecule of coding Cortex jatrophae PSY.

The present invention relates to whether exist in the test sample method of Cortex jatrophae PSY pyrenoids nucleotide sequence, it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to Cortex jatrophae PSY albumen nucleotide coding sequence, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 15-50 Nucleotide.

In addition, according to Cortex jatrophae PSY pyrenoids nucleotide sequence of the present invention and aminoacid sequence, can be on the homology basis of nucleic acid homology or marking protein, screening Cortex jatrophae PSY albumen homology gene or homologous protein.

In order to obtain the dot matrix of the Cortex jatrophae cDNAs relevant with Cortex jatrophae PSY protein gene, can screen Cortex jatrophae cDNA library with dna probe, these probes are under low stringent condition, use ³²P to Cortex jatrophae PSY proteic all or part of do the radioactivity mark and.The cDNA library that most is suitable for screening is the library from Cortex jatrophae.Structure is that biology field is well-known from the method in the cDNA library of interested cell or tissue.In addition, many such cDNA libraries also can buy, for example available from Clontech, and Stratagene, Palo Alto, Cal..This screening method can be discerned the nucleotide sequence with the proteic gene family of Cortex jatrophae PSY.

Cortex jatrophae PSY pyrenoids thuja acid full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

In addition, also the method for available artificial chemosynthesis is synthesized relevant sequence.Before the application, prior art fully can be by first synthetic a plurality of polynucleotide small segments, and then connect and obtain the proteic nucleotide sequence of code book invention Cortex jatrophae PSY.Then, can be with in various existing dna moleculars (as carrier) and the cell in this nucleotide sequence introducing this area.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.

Except producing with recombination method, the also available solid phase technique of the proteic fragment of the present invention is produced (people such as Stewart, (1969) Solid-Phase Peptide Synthesis, WHFreeman Co., San Francisco by direct peptide synthesis; Merrifield J. (1963) J.Am Chem.Soc 85:2149-2154).Can carry out by hand or automatically at external synthetic protein.For example, can (Foster City CA) synthesizes peptide automatically with the 431A type peptide synthesizer of AppliedBiosystems.Can distinguish proteic each fragment of chemosynthesis the present invention, be connected to produce the molecule of total length with chemical process then.

Utilize Cortex jatrophae PSY albumen of the present invention,, can filter out with Cortex jatrophae PSY albumen interactional material takes place, perhaps acceptor, inhibitor or short of money dose etc. by various conventional screening methods.Cortex jatrophae PSY protein gene of the present invention can be used for improving β-Hu Luobusu in the plant by genetic engineering technique, and in order to improve the economic worth of plant, the present invention has great application prospect.

Table 2 is that the homology of the nucleotide sequence of Cortex jatrophae PSY of the present invention and sweet orange (Citrus sinensis) PSY compares (GAP) table.

Table 3 is that the homology of the aminoacid sequence of proteic aminoacid sequence of Cortex jatrophae PSY of the present invention and papaya (Carica papaya) PSY compares (FASTA) table.Wherein, identical amino acid marks with the amino acid monocase between two sequences, and similar amino acid marks with "+".

Table 2

gb|DQ235260.1|???Citrus?sinensis?phytoene?synthase?mRNA，complete?cds

Length＝1311

Score＝989bits(535)，Expect＝0.0

Identities＝905/1083(83％)，Gaps＝27/1083(2％)，Strand＝Plus/Plus

Query?253?TTA-ATATAGATTTGAGGAATCCTTGTATAGGTAGTGGAAGCGAATTCCCTGTATTAGCA?311

|||?|||?||||||||||?|||||||??|???|??|||||?|||?||?||||?|?||?||

Sbjct?202?TTAGATACAGATTTGAGGCATCCTTG-CT-CAT-CTGGAATCGACTTGCCTGAAATATCA?258

Query?312?AGTATGATTGCCAGTC-CAGCTGGAGAAATGGCTATCTCGTCAGAGGAGAA-GGTATACA?369

|||||?||||?||?|?|?|||||||||?||||?||?||?|||||?||?||?|||?||||

Sbjct?259?TGTATGGTTGCTAG-CACTGCTGGAGAAGTGGCCATGTCTTCAGAAGA-AATGGTTTACA?316

Query?370?ATGTGGTGATGAAGCAGGCAGCTTTGGTTAAAAAGCAATTAA----GGTCTAATCAAGAT?425

||||?|||?|?|||||||||||?||||||||?||||||??||????|||?||?||??|||

Sbjct?317?ATGTTGTGCTCAAGCAGGCAGCCTTGGTTAATAAGCAACCAAGTGGGGT-TACTCGTGAT?375

Query?426?CTTGATGTGAAACCAGATATTG-TTCTTCCAGGAAATCTGAGTTTGTTGAGTGAAGCTTA?484

|||||||||||?||||||||||?||?|?||?||||?|?|?|||?||?|?|||||||||||

Sbjct?376?CTTGATGTGAACCCAGATATTGCTT-TACCCGGAACTTTAAGTCTGCTCAGTGAAGCTTA?434

Query?485?CGATAGATGTGGAGAAGTTTGTGCTGAGTATGCAAAAACATTTTACTTGGGAACTTTGCT?544

|||?|?||||||||||||||?||?||||||||?||?|||||||||||||||||||||||

Sbjct?435??TGATCGTTGTGGAGAAGTTTGCGCCGAGTATGCTAAGACATTTTACTTGGGAACTTTGCT?494

Query?545??GATGACTTCTGAAAGAAGAAGAGCTATCTGGGCAATATATGTGTGGTGTCGCAGGACTGA?604

||||||?||||||||?|||||?|||||?||||||?||||||||||||||?|?|||||?||

Sbjct?495??GATGACCTCTGAAAGGCGAAGGGCTATATGGGCTATATATGTGTGGTGTAGGAGGACAGA?554

Query?605??TGAGCTTGTTGATGGGCCTAATGCTTCACACATAACGCCAACAGCTTTAGATAGGTGGGA?664

||||||?|||||||||||||||||||||||||||||?||||||||||||||?||||||||

Sbjct?555??TGAGCTCGTTGATGGGCCTAATGCTTCACACATAACTCCAACAGCTTTAGACAGGTGGGA?614

Query?665??GGCAAGGTTGGAAGACCTTTTCCGAGGTCGTCCATTTGATATGTTTGATGCTGCTTTATC?724

|?|?||||||||||||||||||||?||||||||||||||||||?||||||||||?|||||

Sbjct?615??GTCCAGGTTGGAAGACCTTTTCCGGGGTCGTCCATTTGATATGCTTGATGCTGCATTATC?674

Query?725??AGACACGGTTACTAAATTTCCTGTTGACATTCAGCCATTCAAAGATGTGATTGAAGGAAT?784

|||?||?||?||?|||||||||||?||||||||||||||||?||||?||||?||||||||

Sbjct?675??AGATACAGTAACCAAATTTCCTGTCGACATTCAGCCATTCAGAGATATGATAGAAGGAAT?734

Query?785??GAGGCTGGACCTGAAGAAAACAAGATATAAGAACTTTGATGAGCTT-TATCTT-TACTGT?842

||||?|||||||?|?|||??|||||||?||?|||||||||||??||?||?|||?||?|||

Sbjct?735??GAGGATGGACCTTAGGAAGTCAAGATACAAAAACTTTGATGAA-TTATA-CTTGTATTGT?792

Query?843??TATTATGTTGCTGGGACGGTTGGATTGATGAGTGTTCCGGTCATGGGCATTGCACCTGAA?902

|||||||||||||||||?||?||??|?|||||||||||?||?||||||||?|||||||||

Sbjct?793??TATTATGTTGCTGGGACCGTAGGGCTAATGAGTGTTCCAGTTATGGGCATAGCACCTGAC?852

Query?903??TCACAGGCACCAACTGAGAGCGTCTATAATGCTGCCTTGGCATTAGGAATAGCAAATCAG?962

|||||||||?||||?|||||||||||?|||||?||?||||||?||||?||?||?||||||

Sbjct?853??TCACAGGCAACAACAGAGAGCGTCTACAATGCAGCATTGGCACTAGGGATTGCTAATCAG?912

Query?963??CTTACCAACATACTCAGGGATGTGGGAGAGGATGCAAGAAGAGGAAGGATTTATTTACCA?1022

||?||?|||||||||||?|||||?|||||||||||??|||||||||||?|||||?|||||

Sbjct?913??CTCACTAACATACTCAGAGATGTTGGAGAGGATGCCCAAGAGGAAGGGTTTATCTACCA??972

Query?1023?CAAGATGAGCTCGTGCAGGCTGGACTTTCAGATGATGACATATTTGCTGGGAAAG-TGAC?1081

|||||||||?|?|??|||||?||?||||||||||||||||||||||||||?|?||?||||

Sbjct?973??CAAGATGAGTTGGCACAGGCAGGGCTTTCAGATGATGACATATTTGCTGG-AGAGGTGAC?1031

Query?1082?AGAT-AAATGGAGAAATTTTATGAAGAGTCAAATTAAGAGGGCAAGGATGTTCTTCAATG?1140

||?|||||||||||?||?|||||||??||||||||||||||||||||||||||??||

Sbjct?1032?C-ATTAAATGGAGAAACTTCATGAAGAACCAAATTAAGAGGGCAAGGATGTTCTTTGAT-?1089

Query?1141?A-GGCAGAGAAAGGAGTGACCGAGTTGAGTGCCGCAAGTAGATGGCCGGTATGGGCATCT?1199

|?|||?|||||?||?|||||||||?||||||??||?||||||||||||||||||||?||

Sbjct?1090?ATGGCTGAGAACGGTGTGACCGAGCTGAGTGAAGCTAGTCGATGGCCGGTATGGGCTTCA?1149

Query?1200?TTAGAA-TTATATCAGAAAATTCTAGATGAGATAGAAGCCAATGATTACAATAACTTCAC?1258

||?|???||?||?|?|?||||?||?||||||||?||?||||||||||||||?||||||||

Sbjct?1150?TT-GCTGTTGTACCGGCAAATACTGGATGAGATTGAGGCCAATGATTACAACAACTTCAC??1208

Query?1259?AAAGAGAGCATATGTAAGCAAAGCCAAGAAGTTAGCTTCTTTACCAATTGCATATGCAAG?1318

|||||||||?|||||?||?||||||||||||?|||||?|??||||||||||||||||||

Sbjct?1209?AAAGAGAGCTTATGTGAGTAAAGCCAAGAAGATAGCTGCACTACCAATTGCATATGCAAA??1268

Query?1319?ATC?1321

|||

Sbjct?1269ATC?1271

Query: the proteic nucleotide sequence of Cortex jatrophae JcPSY

Sbjct: the proteic nucleotide sequence of sweet orange CsPSY (GenBank Accession No.DQ235260)

Table 3

gb|ABG72805.1|phytoene?synthase?protein(Carica?papaya)

Length＝438

Score＝715bits(1846)，Expect＝0.0，Method：Compositional?matrix?adjust.

Identities＝349/435(80％)，Positives＝384/435(88％)，Gaps＝7/435(1％)

Query?1???MTVALLWVATPSTEASNSFGFLHSVR----VLDSSKVGSLDRNLTFKGRAKKGRSQKWKS?56

M+VAL?WVA+?S+E?SNSFGF??S+R????+?DSS++GS?DR+L?F??RAKKGRSQ+

Sbjct?1???MSVALFWVASTSSELSNSFGFFDSLRDGNRLFDSSRLGSRDRSLFFSSRAKKGRSQRLNP?60

Query?57??SSVNIDLRNPC--IGSGSEFPVLASMIASPAGEMAISSEEKVYNVVMKQAALVKKQLRSN?114

S+??DLR??C??????S??P+?+SM+?SPAGEMAISSEEKVYNVV++QAALVK+QLRS+

Sbjct?61??GSICTDLRLGCSNFDGQSNMPLTSSMVVSPAGEMAISSEEKVYNVVLRQAALVKRQLRSS?120

Query?115?Q-DLDVKPDIVLPGNLSLLSEAYDRCGEVCAEYAKTFYLGTLLMTSERRRAIWAIYVWCR?173

+LDVKPDIVLPG?LSL++EAYDRCGEVCAEYAKTFYLGTLLMT?ERR+AIWAIYVW?R

Sbjct?121?SGELDVKPDIVLPGTLSLMTEAYDRCGEVCAEYAKTFYLGTLLMTPERRKAIWAIYVWWR?180

Query?174?RTDELVDGPNASHITPTALDRWEARLEDLFRGRPFDMFDAALSDTVTKFPVDIQPFKDVI??233

RT+ELVDGPNASHITPTALDRWEARLEDLF+GRPFDM?DAALSDTV?KFPVDIQPF?D+I

Sbjct?181?RTEELVDGPNASHITPTALDRWEARLEDLFQGRPFDMLDAALSDTVAKFPVDIQPFIDMI?240

Query?234?EGMRLDLKKTRYKNFDELYLYCYYVAGTVGLMSVPVMGIAPESQAPTESVYNAALALGIA?293

EGMR+DL+K+RYKNFDELYLYCYYVAGTVGLMSVP+MGIAPESQA?TESVYNAALALGIA

Sbjct?241?EGMRMDLRKSRYKNFDELYLYCYYVAGTVGLMSVPIMGIAPESQATTESVYNAALALGIA??300

Query?294?NQLTNILRDVGEDARRGRIYLPQDELVQAGLSDDDIFAGKVTDKWRNFMKSQIKRARMFF?353

NQLTNILRDVGEDARRGRIYLPQDEL?QAGLSDDDIFAG?VTDKWRNFMK?QIKRARMFF

Sbjct?301?NQLTNILRDVGEDARRGRIYLPQDELAQAGLSDDDIFAGTVTDKWRNFMKKQIKRARMFF?360

Query?354?NEAEKGVTELSAASRWPVWASLELYQKILDEIEANDYNNFTKRAYVSKAKKLASLPIAYA?413

+EAEKGVTELSAASRWPVWASL?LY+KILDEIEANDYNNFTKRAYVSKAKK+?+LPI+YA

Sbjct?361?DEAEKGVTELSAASRWPVWASLLLYRKILDEIEANDYNNFTKRAYVSKAKKILALPISYA?420

Query?414?RSFVGPSRISTPLRK?428

RS?+GPSR?S+???K

Sbjct?421RSLIGPSRTSSSANK?435

Query: the proteic aminoacid sequence of Cortex jatrophae JcPSY

Sbjct: the proteic aminoacid sequence of papaya CpPSY (GenBank Accession No.ABG72805)

Description of drawings

Fig. 1 is the proteic hydrophobicity plot of Cortex jatrophae PSY.Wherein, the hydrophobicity plot amino acid whose hydrophobic value that is based on Kyte and Doolittle report is set up.

Fig. 2 is the proteic transmembrane amino acid analysis chart of Cortex jatrophae PSY.

Fig. 3 is the domain analyses figure of the proteic aminoacid sequence of Cortex jatrophae PSY.

Embodiment

Below in conjunction with the concrete testing data in laboratory and in conjunction with specific embodiments, further set forth the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The clone of Cortex jatrophae PSY protein gene

1. separate tissue (isolation)

Jatropha curcas seed derives from Sichuan Province's Panzhihua Region, after jatropha curcas seed is gathered, is placed on the laboratory and preserves.

2.RNA separation (RNA isolation)

The kind skin of jatropha curcas seed is peeled off, taken out kind of a benevolence, grind, behind the adding liquid nitrogen, after pulverizing, get 100mg and move in the 1.5mL EP pipe, extracted total RNA (two step cracking processs) with mortar.Identify total RNA quality with the denaturing formaldehyde gel electrophoresis, on spectrophotometer, measure rna content then.

3. the full-length clone of gene (Cloning of Full-length cDNA)

According to the amino acid conserved sequence of some plant PSY, the design degenerate primer utilizes homologous genes clone principle, SMART ^TMRACE cDNA amplification method (Clonetech test kit) carries out the cDNA full-length clone, divides four-stage to carry out:

(1) clone of core sequence

PCR (JcPSYF+JcPSYR) obtains PSY-1 (422bp), reclaim, be connected on the pMD-18T carrier, with M13F or M13R as universal primer, adopt thing fluorescent mark (Big-Dye, Perkin-Elmer, method USA) of stopping, (Perkin-Elmer checks order on USA) at ABI 377 sequenators.Sequencing result GCG software package (Wisconsin group, USA) BLAST in and the existing database of FASTA software search (Genebank+EMBL), the homology of PSY gene of knowing its nucleotide sequence and proteins encoded and known model plant papaya etc. is very high, so think that tentatively it is a PSY gene.

(2)3’-RACE

Result according to the amplification of core sequence designs two forward special primers (JcPSYF1 and JcPSYF2).Adopt the secondary PCR amplification method to carry out.For the first time PCR (JcPSYF1+AP) obtains the PCR product, dilute 100 times after, do template with it, carry out PCR second time (JcPSYF2+AP), obtain JcPSY-3 (850bp), reclaim connection, order-checking process same (1)).

(3)5’-RACE

Result according to the amplification of core sequence designs two reverse special primers (JcPSYR1 and JcPSYR2).Adopt the secondary PCR amplification method to carry out.For the first time PCR (JcPSYR1+UPM) obtains the PCR product, dilute 100 times after, do template with it, carry out PCR second time (JcPSYR2+NUP), obtain JcPSY-5 (678bp), reclaim, connect check order (process is (1) together).

(4) clone of encoding sequence

With the comparison of 5 ' RACE sequencing result and 3 ' RACE sequencing result and splice, obtain the full length fragment sequence information, and design a pair of special primer and carry out JcPSY coding region (JcPSYfull-F+JcPSYfull-R) pcr amplification, obtain JcPSY coding region (1287bp) (process is with (1)).

By being used in combination above-mentioned 4 kinds of methods, obtained candidate's the proteic complete encoding sequence of Cortex jatrophae PSY.Obtain on the total length basis of (comprising complete open reading frame at least) in splicing, further design primer JcPSYfull-F:5-ATGACCGTCGCA TTACTATGGGTTGCA-3 (SEQ ID NO.3) is a forward primer, oligonucleotide JcPSYful l-R:5-TTATGCCTTCCTCAA AGGAGTTGAGATTC-3 (SEQ ID NO.4) is a reverse primer, with total RNA is template, carry out the RT-PCR amplification, the PCR condition be 94 ℃ 5 minutes, carried out 35 circulations in 2 minutes with 94 ℃ 1 minute, 58 ℃ 1 minute and 72 ℃ thereupon, extended 10 minutes with 72 ℃ at last.The electrophoresis detection pcr amplification product, the acquisition expanding fragment length is 1290bp.Clone, check order with pcr amplification product according to a conventional method then, obtain the sequence shown in SEQ ID NO.3 and the SEQ ID NO.4.

Embodiment 2

The sequence information and the homology analysis of Cortex jatrophae PSY protein gene

The length of the Cortex jatrophae PSY albumen full-length cDNA that the present invention is new is 1755bp, and detailed sequence is seen SEQ IDNO.1, and wherein open reading frame is positioned at 78-1364 position Nucleotide.Derive the proteic aminoacid sequence of Cortex jatrophae PSY according to full-length cDNA, totally 429 amino-acid residues, molecular weight 48068.52, pI are 8.96.Detailed sequence is seen SEQ ID NO.2.

Proteic full length cDNA sequence of Cortex jatrophae PSY and coded protein thereof are carried out Nucleotide and protein homology retrieval with blast program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR database, found that the nucleotide sequence of the nucleotide sequence of Cortex jatrophae PSY gene and other plant such as sweet orange (C.sinensis) (DQ235260), oranges and tangerines (C.unshiu) (AF220218), shaddock (C.maxima) (EU375852), natsudaidai (C.paradisi) (AF152892), calamondin (Citrofortunella mitis) (EU375851), papaya (C.papaya) etc. has certain homology.But the aminoacid sequence of Cortex jatrophae PSY and the aminoacid sequence of other plant have more extensive and higher similarity, as similarity widely being arranged with the aminoacid sequence of Cortex jatrophae PSY and the aminoacid sequence of other plant, as with papaya (C.papaya) (ABG72805), Radix Dauci Sativae (D.carota) (ABB52068), coffee (Coffea canephora) (ABA43898), tomato (S.lycopersicum) (ABU40771), oranges and tangerines (C.unshiu) (AF220218), plum blossom (Prunus mume) consistence (BAF49052) is respectively 81%, 76%, 77%, 78%, 78% and 77%, similarity is respectively 90%, 88%, 88%, 87%, 86% and 88%.

The proteic structure characteristic analysis of embodiment 3 Cortex jatrophae PSY

1. the proteic domain analyses of Cortex jatrophae PSY

(network address is: index structure territory http://www.ncbi.nlm.nih.gov/Blast.cgi) obtains the results are shown in Figure 3 at ncbi database with the proteic aminoacid sequence of Cortex jatrophae PSY.

In aminoacid sequence, exist the isopentenyl pyrophosphate synthetic enzyme (Trans-Isoprenyl DiphosphateSynthases, catalytic site Trans_IPPS) (be in the sequence table SEQ ID.1 from aminoterminal (N end) 137-404 amino acids residue.

2. Cortex jatrophae PSY protein signal peptide analysis

Plant carotenoid synthase class is encoded by cell nucleus gene, the coding translation forms the precursor (precursors) that there is transit peptide sequence in the N end, transit peptides has the function that plant carotenoid synthase class is transported to plastid, then be sheared after transport process or transhipment, plant carotenoid synthase class enters plastid with maturation protein (mature proteins) form.The aminoacid sequence of JcPSY is analyzed with ChloroP software, finding has the translocation peptide (being cTP) that is made of 28 amino acid in the total length JcPSY aminoacid sequence, its shearing site is between the 28th and 29 amino acid, and this also has report in plants such as Arabidopis thaliana, tomato and Sunflower Receptacle.

3. the proteic hydrophobicity analysis of Cortex jatrophae PSY

Ripe PSY after the transit peptides shearing is analyzed, find that it contains a plurality of water repellent regions, especially 224-245 amino acids hydrophobicity is very strong, has significantly to stride film trend (Fig. 1).

4. the proteic transmembrane amino acid analysis of Cortex jatrophae PSY

Find that by the TMHMM prediction 223-245 amino acids of ripe PSY is a transmembrane helix structure, show the ripe PSY of Cortex jatrophae be a N end inwardly, the C end outside stride embrane-associated protein, the 223-245 amino acids is the site (Fig. 2) of itself and film secure bond.

Embodiment 4

The activation analysis of jatropha curcas phytoene synthetase cDNA

In this embodiment, Cortex jatrophae JcPSY encoding sequence or the fragment with total length is built into commercial albumen

Among the matter fusion expression vector, to identify the activity of JcPSY.

Cortex jatrophae JcPSY construction of prokaryotic expression vector, and transformed into escherichia coli

According to the aminoacid sequence of Cortex jatrophae JcPSY, the primer of design protein-coding region, and on positive anti-primer, introduce restriction endonuclease sites (this decides according to the pBluescript II KS-vector carrier of selecting for use) respectively, so that construction of expression vector.Amplified production with acquisition among the embodiment 1 is a template, behind pcr amplification, Cortex jatrophae JcPSY gene is being guaranteed to be cloned into pBluescript II KS-vector (Stratahene) under the correct prerequisite of reading frame.Identify that good expression vector utilizes CaCl ₂Method changes intestinal bacteria DH10B over to, and (there is pACCAR25 Δ crtB plasmid in this cingula, have the ctr gene cluster that derives from the synthetic carotenoid of uredo erwinia phage (Erwiniauredovora) on the plasmid, but the ctrB that lacks in this kind is the PSY gene), Screening and Identification obtains containing the engineering bacteria DH10B-JcPSY of JcPSY expression vector.

The result observes

On the colibacillary flat board of growth, do not change the color that the intestinal bacteria DH10B of JcPSY gene grows over to and be white on flat board, changed color that the intestinal bacteria DH10B of JcPSY gene grows over to for yellow on flat board, explanation has the generation of carotenoid on the conversion flat board that has changed the JcPSY gene over to, further specify JcPSY and can replace ctrB gene in the uredo erwinia phage (E.uredovora), promote the generation of carotenoid.Use derives from the PSY gene of mouse and Arabidopis thaliana and does contrast, and the color of bacterium that changes Cortex jatrophae JcPSY gene over to is darker than the color of the bacterium of the psy gene that changes mouse and Arabidopis thaliana over to, illustrates that Cortex jatrophae JcPSY gene has higher activity.

Embodiment 5

The functional analysis of jatropha curcas phytoene synthetase cDNA

In this step, the cDNA sequence construct of coding jatropha curcas phytoene synthetase is gone among the plant expression vector, and transformation of tobacco, to identify its function.

1. the structure of plant expression vector

According to jatropha curcas phytoene synthetase full length cDNA sequence (table 1), design amplifies the primer of entire reading frame, introduces restriction endonuclease sites respectively on positive anti-primer.Amplified production with acquisition in embodiment 1 step 3 is a template, behind pcr amplification, guarantees that the reading frame of jatropha curcas phytoene synthetase is entirely true.Use endonuclease digestion, carrier p2300 reclaims the purpose fragment respectively with also using identical endonuclease digestion.The purpose fragment of the jatropha curcas phytoene synthetase cDNA that will cut through enzyme is connected with the p2300 carrier of cutting through corresponding restriction endonuclease, and transformed into escherichia coli DH5 α cultivates 20h for 37 ℃, and the PCR that carries out recon identifies and enzyme is cut evaluation.

2. plant expression vector transforms Agrobacterium

(1) gets a competence Agrobacterium EHA105, add the about 1pg of plant expression vector that contains jatropha curcas phytoene synthetase cDNA, gently mixing;

(2) quick-frozen 2 minutes in liquid nitrogen, 37 ℃ of incubations 5 minutes;

(3) add 500 μ L YEB liquid nutrient mediums, 28 ℃ jog 2-4 hour, eliminate competence;

(4) get 50-200 μ L bacterium liquid respectively, separate application is selected on the flat board in containing suitable antibiotic YEB, is inverted for 28 ℃ and cultivates two days.

(5) identify the single bacterium colony of the Agrobacterium EHA105 that is positive, be inoculated into and contain the 50mg/L Rifampin, in the 20ml liquid YEB substratum of that mycin of 100mg/L, to logarithmic phase, get an amount of Agrobacterium doubly with liquid MS medium dilution 20-30 in 28 ℃ of constant temperature shaking table shaking culture 30 hours.

3. genetic transformation of tobacco and regeneration

(1) gets aseptic tobacco leaf, excision blade edge and Zhong Mai, 28 ℃ of pre-cultivations 2 days in Ms+1.0mg/L NAA solid medium;

(2) retrieve material, put into the Agrobacterium that has goal gene that aseptic MS liquid nutrient medium diluted, soaked 15 minutes, low speed shook 15 minutes in 28 ℃ of shaking tables are arranged then;

(3) take out vanelets, inhale with aseptic filter paper and remove unnecessary bacterium liquid, 28 ℃ of dark cultivations two days in the MS solid medium;

(4) vanelets, with the aseptic washing twice that adds 100mg/LCb, after aseptic filter paper is inhaled and removed unnecessary bacterium liquid, change over to and contain among 100mg/L Km and the 100mg/L Cb division culture medium M1, be cultured under 28 ℃ of light and differentiate callus, until growing bud, per therebetween 15 days subcultures once;

(5) bud that will grow to 3-5cm changes root media 1/2MS over to and goes up root induction.

(6) after being accredited as positive transgene tobacco, continue to cultivate into seedling.

4. the extraction of transgene tobacco blade carotenoid

(1) get each transgene tobacco blade, lyophilized powder is made in lyophilize rapidly, puts in the vacuum drying oven and keeps in Dark Place, and measure to the greatest extent and as early as possible.

(2) accurately take by weighing lyophilized powder 1.000g in tool plug triangular flask, and the adding extracting solution (acetone: 45mL ethanol=3: 2), shake up, cover bottle stopper, use ultrasonic extraction 20 minutes, filter in the 50mL volumetric flask and constant volume.Getting 20mL filtrate puts in the separating funnel, add 10mL sherwood oil mixing, adding 10mL distilled water distributes, getting sherwood oil puts in the rotary evaporation bottle mutually, surplus water adds the 10mL sherwood oil reallocates, and merges 2 sherwood oils and puts on the Rotary Evaporators 40-45 ℃ of evaporate to dryness mutually, with the dissolving of 2.0mL Virahol, filter the back and go up machine mensuration.

5. high effective liquid chromatography for measuring transgene tobacco blade carotenoid

(1) preparation of B-carotene standard specimen: B-carotene standard substance (Sigma company product) 1.0mg, with a small amount of trichloromethane dissolving, use petroleum ether dissolution again, solution changes in the 25.0ml volumetric flask, uses the sherwood oil constant volume, and concentration is 0.04mg/ml, and refrigerator is preserved.Face the time spent, draw 1.0ml in the 10.0ml volumetric flask, add moving phase to scale, this moment, concentration was 0.004mg/ml.

(2) preparation of Lyeopene standard specimen: Pure Lycopene (Sigma company product) 1.0mg, with a small amount of trichloromethane dissolving, use petroleum ether dissolution again, solution changes in the 25.0ml volumetric flask, uses the sherwood oil constant volume, and concentration is 0.04mg/ml, and refrigerator is preserved.Face the time spent, draw 1.0ml in the 10.0ml volumetric flask, add moving phase to scale, this moment, concentration was 0.004mg/ml.

(3) measuring method: moving phase: second eyeball: trichloromethane (92: 8).Detect wavelength: dual wavelength is measured, 470nm (0-min) and 450nm (8-13min).Flow velocity: 1.0mL/min.Column temperature: 35 ℃.Observing samples has the size variation of absorption peak at 470nm and 450nm place.

Sequence that the present invention relates to and mark apportion are as follows:

＜110〉Fudan University

＜120〉jatropha curcas phytoene synthetase gene sequence and the application in plant

<160>2

<170>PatentIn?version?3.1

<210>1

<211>1755

<212>mRNA

＜213〉Cortex jatrophae (Jatropha carcas L.)

<220>

<221>CDS

<222>(78)..(1364)

<223>

<400>1

GGAAATTTGC?ATAGACAAGG?TAAACAAAGA?AAGAAAGAAA?AAAATAAAAA?AATAAAAATA

AAAATCTGGT?TTTGAACATG?ACC?GTC?GCA?TTA?CTA?TGG?GTT?GCA?ACA?CCA?AGT

Met?Thr?Val?Ala?Leu?Leu?Trp?Val?Ala?Thr?Pro?Ser

1????????????????5??????????????????10

ACA?GAG?GCT?TCC?AAT?TCA?TTT?GGG?TTT?CTC?CAT?TCG?GTT?CGG?GTC?TTA?GAT

Thr?Glu?Ala?Ser?Asn?Ser?Phe?Gly?Phe?Leu?His?Ser?Val?Arg?Val?Leu?Asp

15???????????????????20??????????????????25

TCG?TCG?AAA?GTT?GGT?TCT?CTA?GAT?CGG?AAT?TTG?ACG?TTT?AAG?GGA?AGA?GCA

Ser?Ser?Lys?Val?Gly?Ser?Leu?Asp?Arg?Asn?Leu?Thr?Phe?Lys?Gly?Arg?Ala

30???????????????????35??????????????????40??????????????????45

AAA?AAG?GGT?AGG?AGT?CAA?AAA?TGG?AAG?TCA?AGC?TCA?GTT?AAT?ATA?GAT?TTG

Lys?Lys?Gly?Arg?Ser?Gln?Lys?Trp?Lys?Ser?Ser?Ser?Val?Asn?Ile?Asp?Leu

50???????????????????55??????????????????60

AGG?AAT?CCT?TGT?ATA?GGT?AGT?GGA?AGC?GAA?TTC?CCT?GTA?TTA?GCA?AGT?ATG

Arg?Asn?Pro?Cys?Ile?Gly?Ser?Gly?Ser?Glu?Phe?Pro?Val?Leu?Ala?Ser?Met

65???????????????????70??????????????????75?????????????????80

ATT?GCC?AGT?CCA?GCT?GGA?GAA?ATG?GCT?ATC?TCG?TCA?GAG?GAG?AAG?GTA?TAC

Ile?Ala?Ser?Pro?Ala?Gly?Glu?Met?Ala?Ile?Ser?Ser?Glu?Glu?Lys?Val?Tyr

85???????????????????90??????????????????95

AAT?GTG?GTG?ATG?AAG?CAG?GCA?GCT?TTG?GTT?AAA?AAG?CAA?TTA?AGG?TCT?AAT

Asn?Val?Val?Met?Lys?Gln?Ala?Ala?Leu?Val?Lys?Lys?Gln?Leu?Arg?Ser?Asn

100??????????????????105????????????????110

CAA?GAT?CTT?GAT?GTG?AAA?CCA?GAT?ATT?GTT?CTT?CCA?GGA?AAT?CTG?AGT?TTG

Gln?Asp?Leu?Asp?Val?Lys?Pro?Asp?Ile?Val?Leu?Pro?Gly?Asn?Leu?Ser?Leu

115?????????????????120??????????????????125?????????????????130

TTG?AGT?GAA?GCT?TAC?GAT?AGA?TGT?GGA?GAA?GTT?TGT?GCT?GAG?TAT?GCA?AAA

Leu?Ser?Glu?Ala?Tyr?Asp?Arg?Cys?Gly?Glu?Val?Cys?Ala?Glu?Tyr?Ala?Lys

135??????????????????140?????????????????145

ACA?TTT?TAC?TTG?GGA?ACT?TTG?CTG?ATG?ACT?TCT?GAA?AGA?AGA?AGA?GCT?ATC

Thr?Phe?Tyr?Leu?Gly?Thr?Leu?Leu?Met?Thr?Ser?Glu?Arg?Arg?Arg?Ala?Ile

150?????????????????155?????????????????160

TGG?GCA?ATA?TAT?GTG?TGG?TGT?CGC?AGG?ACT?GAT?GAG?CTT?GTT?GAT?GGG?CCT

Trp?Ala?Ile?Tyr?Val?Trp?Cys?Arg?Arg?Thr?Asp?Glu?Leu?Val?Asp?Gly?Pro

165?????????????????170?????????????175?????????????????180

AAT?GCT?TCA?CAC?ATA?ACG?CCA?ACA?GCT?TTA?GAT?AGG?TGG?GAG?GCA?AGG?TTG

Asn?Ala?Ser?His?Ile?Thr?Pro?Thr?Ala?Leu?Asp?Arg?Trp?Glu?Ala?Arg?Leu

185????????????????190?????????????????195

GAA?GAC?CTT?TTC?CGA?GGT?CGT?CCA?TTT?GAT?ATG?TTT?GAT?GCT?GCT?TTA?TCA

Glu?Asp?Leu?Phe?Arg?Gly?Arg?Pro?Phe?Asp?Met?Phe?Asp?Ala?Ala?Leu?Ser

200?????????????????205?????????????????210?????????????????215

GAC?ACG?GTT?ACT?AAA?TTT?CCT?GTT?GAC?ATT?CAG?CCA?TTC?AAA?GAT?GTG?ATT

Asp?Thr?Val?Thr?Lys?Phe?Pro?Val?Asp?Ile?Gln?Pro?Phe?Lys?Asp?Val?Ile

220??????????????????225?????????????????230

GAA?GGA?ATG?AGG?CTG?GAC?CTG?AAG?AAA?ACA?AGA?TAT?AAG?AAC?TTT?GAT?GAG

Glu?Gly?Met?Arg?Leu?Asp?Leu?Lys?Lys?Thr?Arg?Tyr?Lys?Asn?Phe?Asp?Glu

235?????????????????240?????????????????245?????????????????250

CTT?TAT?CTT?TAC?TGT?TAT?TAT?GTT?GCT?GGG?ACG?GTT?GGA?TTG?ATG?AGT?GTT

Leu?Tyr?Leu?Tyr?Cys?Tyr?Tyr?Val?Ala?Gly?Thr?Val?Gly?Leu?Met?Ser?Val

255?????????????????260??????????????????265

CCG?GTC?ATG?GGC?ATT?GCA?CCT?GAA?TCA?CAG?GCA?CCA?ACT?GAG?AGC?GTC?TAT

Pro?Val?Met?Gly?Ile?Ala?Pro?Glu?Ser?Gln?Ala?Pro?Thr?Glu?Ser?Val?Tyr

270?????????????????275?????????????????280

AAT?GCT?GCC?TTG?GCA?TTA?GGA?ATA?GCA?AAT?CAG?CTT?ACC?AAC?ATA?CTC?AGG

Asn?Ala?Ala?Leu?Ala?Leu?Gly?Ile?Ala?Asn?Gln?Leu?Thr?Asn?Ile?Leu?Arg

285?????????????????290?????????????????295?????????????????300

GAT?GTG?GGA?GAG?GAT?GCA?AGA?AGA?GGA?AGG?ATT?TAT?TTA?CCA?CAA?GAT?GAG

Asp?Val?Gly?Glu?Asp?Ala?Arg?Arg?Gly?Arg?Ile?Tyr?Leu?Pro?Gln?Asp?Glu

305?????????????????310??????????????????315

CTC?GTG?CAG?GCT?GGA?CTT?TCA?GAT?GAT?GAC?ATA?TTT?GCT?GGG?AAA?GTG?ACA

Leu?Val?Gln?Ala?Gly?Leu?Ser?Asp?Asp?Asp?Ile?Phe?Ala?Gly?Lys?Val?Thr

320?????????????????325??????????????????330????????????????335

GAT?AAA?TGG?AGA?AAT?TTT?ATG?AAG?AGT?CAA?ATT?AAG?AGG?GCA?AGG?ATG?TTC

Asp?Lys?Trp?Arg?Asn?Phe?Met?Lys?Ser?Gln?Ile?Lys?Arg?Ala?Arg?Met?Phe

340?????????????????345??????????????????350

TTC?AAT?GAG?GCA?GAG?AAA?GGA?GTG?ACC?GAG?TTG?AGT?GCC?GCA?AGT?AGA?TGG

Phe?Asn?Glu?Ala?Glu?Lys?Gly?Val?Thr?Glu?Leu?Ser?Ala?Ala?Ser?Arg?Trp

355?????????????????360??????????????????365

CCG?GTA?TGG?GCA?TCT?TTA?GAA?TTA?TAT?CAG?AAA?ATT?CTA?GAT?GAG?ATA?GAA

Pro?Val?Trp?Ala?Ser?Leu?Glu?Leu?Tyr?Gln?Lys?Ile?Leu?Asp?Glu?Ile?Glu

370??????????????????375??????????????????380????????????????385

GCC?AAT?GAT?TAC?AAT?AAC?TTC?ACA?AAG?AGA?GCA?TAT?GTA?AGC?AAA?GCC?AAG

Ala?Asn?Asp?Tyr?Asn?Asn?Phe?Thr?Lys?Arg?Ala?Tyr?Val?Ser?Lys?Ala?Lys

390?????????????????395?????????????????400

AAG?TTA?GCT?TCT?TTA?CCA?ATT?GCA?TAT?GCA?AGA?TCA?TTT?GTT?GGG?CCA?TCA

Lys?Leu?Ala?Ser?Leu?Pro?Ile?Ala?Tyr?Ala?Arg?Ser?Phe?Val?Gly?Pro?Ser

405?????????????????410??????????????????415????????????????420

AGA?ATC?TCA?ACT?CCT?TTG?AGG?AAG?GCA?TAAATGTCAA?ATCATGAACA?TCATATATAG

Arg?Ile?Ser?Thr?Pro?Leu?Arg?Lys?Ala

425

AAGAAAAGGG?AAAAATACCA?TAATTAATTT?GTATATTAGC?AACTTTTGTA?ATGCAGCAAG

TGGAAGCAAG?AACAGTCAAT?TCAAATTTTA?TTTGAAGGGA?TGAACTGTAT?ATATTCTGCT

AGGAACTTAA?AGCTTCTTGG?TTTAGAGAGT?GTTTAGTGTT?TTAAGAGAAG?ACTCATCTAA

GAGTCTGCTT?GTGCTTTTCT?ACCCTCACGT?AGACATGGCT?GGTTAACCAA?ACCGGTGGTT

CATGGCCTTT?AAAAGTAGGA?ACTGAGAACG?GTCTTAATCA?GGCTGGTTTA?ATTCAATTTC

GATTCAATCG?GGCTGGTTTA?ATTCAATTTC?GATTCTGTAA?AATGAAAAAA?AAAAAAAAAAA

<210>2

<211>429

<212>PRT

＜213〉Cortex jatrophae (Jatropha carcas L.)

<400>2

Met?Thr?Val?Ala?Leu?Leu?Trp?Val?Ala?Thr?Pro?Ser?Thr?Glu?Ala?Ser

1????????????????5??????????????????10??????????????????15

Asn?Ser?Phe?Gly?Phe?Leu?His?Ser?Val?Arg?Val?Leu?Asp?Ser?Ser?Lys

20??????????????????25???????????????????30

Val?Gly?Ser?Leu?Asp?Arg?Asn?Leu?Thr?Phe?Lys?Gly?Arg?Ala?Lys?Lys

35??????????????????40??????????????????45

Gly?Arg?Ser?Gln?Lys?Trp?Lys?Ser?Ser?Ser?Val?Asn?Ile?Asp?Leu?Arg

50???????????????????55??????????????????60

Asn?Pro?Cys?Ile?Gly?Ser?Gly?Ser?Glu?Phe?Pro?Val?Leu?Ala?Ser?Met

65???????????????????70??????????????????75?????????????????80

Ile?Ala?Ser?Pro?Ala?Gly?Glu?Met?Ala?Ile?Ser?Ser?Glu?Glu?Lys?Val

85???????????????????90?????????????????95

Tyr?Asn?Val?Val?Met?Lys?Gln?Ala?Ala?Leu?Val?Lys?Lys?Gln?Leu?Arg

100??????????????????105????????????????110

Ser?Asn?Gln?Asp?Leu?Asp?Val?Lys?Pro?Asp?Ile?Val?Leu?Pro?Gly?Asn

115?????????????????120??????????????????125

Leu?Ser?Leu?Leu?Ser?Glu?Ala?Tyr?Asp?Arg?Cys?Gly?Glu?Val?Cys?Ala

130?????????????????135??????????????????140

Glu?Tyr?Ala?Lys?Thr?Phe?Tyr?Leu?Gly?Thr?Leu?Leu?Met?Thr?Ser?Glu

145?????????????????150?????????????????155?????????????????160

Arg?Arg?Arg?Ala?Ile?Trp?Ala?Ile?Tyr?Val?Trp?Cys?Arg?Arg?Thr?Asp

165?????????????????170????????????????175

Glu?Leu?Val?Asp?Gly?Pro?Asn?Ala?Ser?His?Ile?Thr?Pro?Thr?Ala?Leu

180?????????????????185?????????????????190

Asp?Arg?Trp?Glu?Ala?Arg?Leu?Glu?Asp?Leu?Phe?Arg?Gly?Arg?Pro?Phe

195??????????????????200?????????????????205

Asp?Met?Phe?Asp?Ala?Ala?Leu?Ser?Asp?Thr?Val?Thr?Lys?Phe?Pro?Val

210?????????????????215?????????????????220

Asp?Ile?Gln?Pro?Phe?Lys?Asp?Val?Ile?Glu?Gly?Met?Arg?Leu?Asp?Leu

225?????????????????230??????????????????235????????????????240

Lys?Lys?Thr?Arg?Tyr?Lys?Asn?Phe?Asp?Glu?Leu?Tyr?Leu?Tyr?Cys?Tyr

245?????????????????250????????????????255

Tyr?Val?Ala?Gly?Thr?Val?Gly?Leu?Met?Ser?Val?Pro?Val?Met?Gly?Ile

260?????????????????265?????????????????270

Ala?Pro?Glu?Ser?Gln?Ala?Pro?Thr?Glu?Ser?Val?Tyr?Asn?Ala?Ala?Leu

275?????????????????280?????????????????285

Ala?Leu?Gly?Ile?Ala?Asn?Gln?Leu?Thr?Asn?Ile?Leu?Arg?Asp?Val?Gly

290?????????????????295?????????????????300

Glu?Asp?Ala?Arg?Arg?Gly?Arg?Ile?Tyr?Leu?Pro?Gln?Asp?Glu?Leu?Val

305?????????????????310??????????????????315????????????????320

Gln?Ala?Gly?Leu?Ser?Asp?Asp?Asp?Ile?Phe?Ala?Gly?Lys?Val?Thr?Asp

325??????????????????330????????????????335

Lys?Trp?Arg?Asn?Phe?Met?Lys?Ser?Gln?Ile?Lys?Arg?Ala?Arg?Met?Phe

340?????????????????345??????????????????350

Phe?Asn?Glu?Ala?Glu?Lys?Gly?Val?Thr?Glu?Leu?Ser?Ala?Ala?Ser?Arg

355?????????????????360??????????????????365

Trp?Pro?Val?Trp?Ala?Ser?Leu?Glu?Leu?Tyr?Gln?Lys?Ile?Leu?Asp?Glu

370??????????????????375?????????????????380

Ile?Glu?Ala?Asn?Asp?Tyr?Asn?Asn?Phe?Thr?Lys?Arg?Ala?Tyr?Val?Ser

385?????????????????390?????????????????395?????????????????400

Lys?Ala?Lys?Lys?Leu?Ala?Ser?Leu?Pro?Ile?Ala?Tyr?Ala?Arg?Ser?Phe

405?????????????????410?????????????????415

Val?Gly?Pro?Ser?Arg?Ile?Ser?Thr?Pro?Leu?Arg?Lys?Ala

420??????????????????425

<210>3

<211>27

<212>DNA

＜213〉Cortex jatrophae (Jartropha curcas L.)

<400>3

ATGACCGTCGCATTACTATGGGTTGCA

<210>4

<211>29

<212>DNA

＜213〉Cortex jatrophae (Jatropha curcas L.)

<400>4

TTATGCCTTCCTCAAAGGAGTTGAGATTC

Claims (14)

1. isolate dna molecular for one kind, it is characterized in that, it comprises: coding has the nucleotide sequence of the polypeptide of Cortex jatrophae JcPSY protein active, and shows at least 70% homology from the nucleotides sequence of Nucleotide 78-1364 position among described nucleotide sequence and the SEQ ID NO.1; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.1 in from the nucleotide sequence hybridization of Nucleotide 78-1364 position.

2. dna molecular as claimed in claim 1 is characterized in that described sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQID NO.2.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has among the SEQ ID NO.1 nucleotide sequence from Nucleotide 78-1364 position.

4. isolating Cortex jatrophae JcPSY protein polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ IDNO.2 aminoacid sequence, or its reactive derivative.

5. polypeptide as claimed in claim 4 is characterized in that, this polypeptide is to have SEQ ID NO.2 polypeptide of sequence.

6. a carrier is characterized in that, it comprises the described DNA of claim 1.

7. carrier as claimed in claim 6 is characterized in that, is used to make up the carrier that sets out of described plant expression vector, and the described carrier that sets out is selected from p3301-BI121, pBll21, pBin19, pCAMBIA2301, pCAMBIA1301 or pCAMBIA1300.

8. one kind with the described carrier transformed host cells of claim 6, it is characterized in that it is protokaryon or eukaryotic cell.

9. a generation has the method for the polypeptide of Cortex jatrophae JcPSY protein active, is characterised in that its step is as follows:

(1) nucleotide sequence of purifying that coding is had a polypeptide of Cortex jatrophae JcPSY protein-active operationally is connected in expression regulation sequence, form Cortex jatrophae JcPSY protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 78-1364 position among described nucleotide sequence and the SEQID NO.1;

(2) change the expression vector in the step (1) over to host cell, form the proteic reconstitution cell of Cortex jatrophae JcPSY;

(3) be fit to express under the condition of Cortex jatrophae JcPSY protein polypeptide the reconstitution cell in the culturing step (2);

(4) isolate pure substantially polypeptide with Cortex jatrophae JcPSY protein-active.

10. one kind is utilized transgenic technology that the nucleotide sequence that coding has Cortex jatrophae JcPSY protein active polypeptide is transformed into plant to improve the method for β-Hu Luobusu in the plant, it is characterized in that comprising the steps:

(1) nucleotide sequence of purifying that coding is had a polypeptide of Cortex jatrophae JcPSY protein-active operationally is connected in the expression of plants regulating and controlling sequence, formation contains the proteic plant expression vector of Cortex jatrophae JcPSY, shows at least 70% homology from the nucleotides sequence of Nucleotide 78-1364 position among described nucleotide sequence and the SEQ ID NO.1;

(2) change the expression vector in the step (1) over to plant host cell.It is characterized in that: described plant host is paddy rice, wheat, soybean, corn, tobacco, rape, Chinese sorghum, cotton, clover, Cortex jatrophae or Arabidopis thaliana.

(3) by screening as antibiotic-screening, obtain transformant and final regeneration of transgenic plant and offspring thereof, comprise plant seed and plant tissue.Transgenic plant and offspring thereof have the enhanced effect to the plant drought tolerance.

11. one kind and many skins of the described Cortex jatrophae JcPSY of claim 4 protein polypeptide specificity bonded antibody is characterized in that it comprises polyclonal antibody and monoclonal antibody.

12. a nucleic acid molecular probe is characterized in that, it comprises 8-100 continuous nucleotide in the described dna molecular of claim 1.

13. one kind is used for the method whether test sample exists Cortex jatrophae JcPSY nucleotide sequence, it is characterized in that it comprises with described probe of claim 12 and sample to hybridize, whether detection probes combination has taken place then.

14. the described detection method of claim 13, it is characterized in that described sample is the product behind the pcr amplification, wherein the pcr amplification primer is corresponding to Cortex jatrophae JcPSY nucleotide coding sequence, and can be positioned at the both sides or the centre of this encoding sequence, and primer length is a 15-50 Nucleotide.

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