CN101798579A - Jatropha curcas farnesyl pyrophosphate synthase protein encoding sequence and application thereof in plants - Google Patents
Jatropha curcas farnesyl pyrophosphate synthase protein encoding sequence and application thereof in plants Download PDFInfo
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Abstract
The invention belongs to the technical fields of molecular biology and genetic engineering, and provides an encoding sequence for encoding cDNA of Jatropha curcas farnesyl pyrophosphate synthase. The farnesyl pyrophosphate synthase catalyzes isopentenyl pyrophosphate and dimethylallyl pyrophosphate to carry out continuous condensation reaction to form farnesyl pyrophosphate, is key enzyme for metabolic pathway of Jatropha curcas isoprene, and contributes to reducing the content of phorbol esters and precursor thereof in the Jatropha curcas. Due to the use of the Jatropha curcas protein, substances interacting with the Jatropha curcas protein, or receptors, inhibitors or antagonists, and the like can be screened by various conventional screening methods. The encoding sequence for encoding the cDNA of the Jatropha curcas farnesyl pyrophosphate synthase has great application prospects.
Description
Technical field
The invention belongs to molecular biology, gene engineering technology field.Particularly, the present invention relates to a kind of jc-fps albumen of in Cortex jatrophae, expressing (jatropha curcas farnesyl pyrophosphate synthase protein, Jatropha curcas farnesyl pyrophosphate synthase, JCFPS) and nucleotide sequence.
Background technology
Cortex jatrophae has another name called the little seeds of a tung oil tree, and euphorbia plant is distributed in ground such as Sichuan, Yunnan, Guangxi in China.In recent years, Cortex jatrophae by extensive exploitation, in addition, Cortex jatrophae also is used to a plurality of fields such as pharmacy, biological pesticide, wasteland improvement, pharmacy, feed, fertilizer as a kind of novel bioenergy.Contain multiple toxin composition in the Cortex jatrophae, the composition that toxicity is stronger is curcin, phorbol ester.(12-deoxidation-16-hydroxyl phorbol 12-deoxy-16-hydroxyphorbol) is a kind of diterpene-kind compound to phorbol ester, mainly is present in jatropha curcas seed, bark, leaf, root and the milk.Phorbol ester has severe toxicity, is a kind of tumor initiator.Except that the oil length height, the phase protein content in its seed dregs of rice is up to 60% in the jatropha curcas seed, and the amino acid Compositional balance is a kind of very fine vegetable-protein source, can be used as animal-feed and uses.But for a long time because the existence of Cortex jatrophae toxin such as phorbol ester, barbadosnut pulp can not directly be used, and can only abandon or as fertilizer sources field also, and this makes this a pair of domestic animal have the resource of great potential utility value to be wasted greatly.In addition, toxin such as phorbol ester have certain residual in jatropha curcas seed oil, have also limited the application of biofuel to a certain extent.The develop rapidly of genetic engineering technique reduces the Cortex jatrophae toxin for utilizing biotechnology, improve the utilization of Cortex jatrophae and opened up a new thinking, can partly reduce the key gene of Cortex jatrophae phorbol ester route of synthesis expresses by sense-rna or RNAi technology, thereby the acquisition regeneration plant obtains the low toxicity high-quality strain of Cortex jatrophae.
In metabolic pathway of Jatropha, farnesyl pyrophosphate synthase catalysis isopentenyl pyrophosphate (IPP) and the reaction of dimethyl allene tetra-sodium (DMAPP) continuous condensating form farnesyl pyrophosphate (FPP), are key enzymes of this approach.FPP is the precursor of terpene derivant matter such as steroidal in the plant materials, saponin(e, sesquiterpene, also is the synthetic precursor of phorbol ester in the Cortex jatrophae.Significant for reducing phorbol ester content by genetic manipulation.
Before the present invention comes forth, any cDNA sequence that discloses or reported the coding jatropha curcas farnesyl pyrophosphate synthase of mentioning in the present patent application is not arranged as yet.
The clone of relevant jatropha curcas farnesyl pyrophosphate synthase gene and functional study are subjected to state natural sciences fund (30771745) subsidy among the present invention.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of Cortex jatrophae JcFPS albumen coded sequence is provided.Make it comprise said gene Fusion gene construct, carry the new recombinant expression vector of this construct, by said expression vector transformed plant cells, and the transgenic plant and the offspring thereof of the said gene that produces by transformant, comprise plant seed and plant tissue, the transgenic plant that obtain will have the phorbol ester content of reduction.
The present invention is achieved through the following technical solutions,
The present invention isolated dna molecular comprise: coding has the nucleotide sequence of polypeptide of Cortex jatrophae JcFPS protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 221-1246 position among described nucleotide sequence and the SEQ ID NO.1; Perhaps described nucleotide sequence can be under 40-55 ℃ of condition with SEQ ID NO.1 in from the nucleotide sequence hybridization of Nucleotide 221-1246 position.
Described coding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO.2.
Described sequence has among the SEQ ID NO.1 nucleotide sequence from Nucleotide 104-1246 position.
The isolated Cortex jatrophae JcFPS of the present invention protein polypeptide, 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.
Described protein polypeptide is to have SEQ ID NO.2 polypeptide of sequence.
Vector dna molecule transformed host cells provided by the present invention, it is an eukaryotic cell.This host cell comprises 8-100 continuous nucleotide in the described dna molecular.
In the present invention, " isolating ", " purifying " 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, term " Cortex jatrophae JcFPS albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with Cortex jatrophae JcFPS protein-active is as 104-1246 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1.This degenerate sequence is meant, is arranged in the encoder block 104-1246 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 221-1246 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 ID NO.1 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 221-1246 position.This term also comprise with SEQ ID NO.1 in from the homology of nucleotide sequence at least 70% of Nucleotide 221-1246 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 SEQID NO.1 with natural Cortex jatrophae JcFPS 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, term " Cortex jatrophae JcFPS albumen or polypeptide " refers to have the SEQ ID NO.1 polypeptide of sequence of Cortex jatrophae JcFPS protein-active.This term also comprises the variant form that has with the SEQ ID NO.2 sequence of natural Cortex jatrophae JcFPS 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 JcFPS and reactive derivative.
The variant form of Cortex jatrophae JcFPS 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 JcFPS protein D NA hybridization and the polypeptide or the albumen that utilize the serum of Cortex jatrophae JcFPS protein polypeptide to obtain.
In the present invention, " Cortex jatrophae JcFPS albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO.1, have 10 at the most, preferably at the most 8, more preferably 5 amino acid similar performances or close amino acid are replaced and are 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 |
| ??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 |
Table 2 is that the homology of the nucleotide sequence of Cortex jatrophae JcFPS of the present invention and Para rubber tree (Hevea brasiliensis) HbFPS compares (GAP) table.
Table 2
87％identity?in?1289nt?overlap
Query?205??TCTCCTTTTTGAAGCTATGGCGGATCTGAAATCAACATTCTTGGAGGTCTATTCTGTTCT??264
|||||??||||||?|?||||||||||||||?||||?|||||||?|||||||?|||||?||
Sbjct?47???TCTCC-GTTTGAATCCATGGCGGATCTGAAGTCAACTTTCTTGAAGGTCTACTCTGTCCT??105
Query?265??CAAGAAGGAGCTACTTCAAGACCCGGCTTTCGAATGGACACCAGATTCTCGTGAATGGGT??324
||||?|||||||?|||?|?||?||||||||||||||||||||||||||?|||?|||||||
Sbjct?106??CAAGCAGGAGCTCCTTGAGGATCCGGCTTTCGAATGGACACCAGATTCCCGTCAATGGGT??165
Query?325??CGAAAGGATGCTGGACTACAATGTGCCTGGAGGGAAGCTGAATAGGGGGCTCTCTGTGAT??384
||???|||||?||||||||||||||||||||||||||||||||||||||||?|||||?||
Sbjct?166??TGAGCGGATGTTGGACTACAATGTGCCTGGAGGGAAGCTGAATAGGGGGCTTTCTGTAAT??225
Query?385??TGACAGCTACAAATTGTTGAAAGATGGACAGGAATTAACAGAAGAAGAAATCTTTCTCGC??444
||||||||||||||||||||||||?|||||||||||||||||||||||?||||||||?||
Sbjct?226??TGACAGCTACAAATTGTTGAAAGAAGGACAGGAATTAACAGAAGAAGAGATCTTTCTTGC?285
Query?445??AAGCGCTCTTGGTTGGTGTATTGAATGGCTCCAAGCCTATTTCCTTGTCCTTGATGATAT?504
|||?||||||||||||||||||||||||||?|||||||||||?|||||?||||||||?||
Sbjct?286??AAGTGCTCTTGGTTGGTGTATTGAATGGCTTCAAGCCTATTTTCTTGTACTTGATGACAT?345
Query?505??TATGGATAGCTCTCATACACGACGTGGTC-GACCATGTTGGTTTATGGTGCCCAAGGTTG?563
|||||||||||||||||||||||||||||?|?||?||?|||||||?||||||||||||||
Sbjct?346??TATGGATAGCTCTCATACACGACGTGGTCAG-CCTTGCTGGTTTAGGGTGCCCAAGGTTG?404
Query?564??GTCTTATTGCAGCAAATGATGGGATTTTGCTTCGAAATCACATTCCCAGGATTCTTAAAA?623
||||?|||||||||||||||||||||||||||||?|||||||||||||||||||||||||
Sbjct?405??GTCTGATTGCAGCAAATGATGGGATTTTGCTTCGCAATCACATTCCCAGGATTCTTAAAA?464
Query?624??AGCACTTCCGAGGGAAAGCATACTATGTAGATCTTCTAGATTTATTTAATGAGGTGGAGT?683
||||||||||||||||?||||||||||||||||||||||||||?||||||||||||||||
Sbjct?465??AGCACTTCCGAGGGAAGGCATACTATGTAGATCTTCTAGATTTGTTTAATGAGGTGGAGT?524
Query?684??TTCAAACAGCCTCAGGACAGATGATAGATCTGATTACAACACTTGAAGGAGAAAAGGATT?743
|||||||||||||||||||||||||||||||||?||||||||||||||?|||||||||||
Sbjct?525??TTCAAACAGCCTCAGGACAGATGATAGATCTAATTACAACACTTGAAGAAGAAAAGGATT?584
Query?744??TATCGAAGTACAATTTATCGCTTCACCGGCGAATTGTTCAGTACAAAACTGCCTACTACT?803
||||?||?||||?|||?||?||?||||||?||||||||||||||||||||||||||||||
Sbjct?585??TATCCAAATACACTTTGTCACTCCACCGGAGAATTGTTCAGTACAAAACTGCCTACTACT?644
Query?804??CATTTTACCTTCCTGTTGCTTGTGCATTGCTCATGGCTGGTGAGAATCTGGACAGCCATA?863
||||||||||||||||||||||||||||||||||?||?||?|||||||||||||??||||
Sbjct?645??CATTTTACCTTCCTGTTGCTTGTGCATTGCTCATAGCGGGCGAGAATCTGGACAATCATA?704
Query?864??TTGATGTACA-GAATATTCTTGTCCAGATGGGAATCTACTTCCAAGTACAGGATGATTAT?922
|||?||||?|?||??||||||||?||||||||||||||||||||||||||||||||||||
Sbjct?705??TTGTTGTAAAAGAC-ATTCTTGTTCAGATGGGAATCTACTTCCAAGTACAGGATGATTAT?763
Query?923??TTGGATTGCTTTGGTGATCCCAAGACAATTGGCAAGATAGGGACAGATATTGAAGATTTT?982
|||||||||||||||||||||?||||||||||?||||||||?||||||||?|||||||||
Sbjct?764??TTGGATTGCTTTGGTGATCCCGAGACAATTGGTAAGATAGGAACAGATATAGAAGATTTT?823
Query?983??AAGTGCTCTTGGTTGGTCGTGAAGGCTTTGGAGCGATGCAATGAAGAACAAAAGAAAGT-1041
|||||?||?||||||||||||||||||||?||?|??||||||||||||||||?||||||
Sbjct?824??AAGTGTTCATGGTTGGTCGTGAAGGCTTTAGAACTTTGCAATGAAGAACAAAGGAAAGTG?883
Query?1042?TCTACATGAGCATTATGGGAAACCTGACCCAGCCAGTGT-GTCAAAGGTGAAAGTCCTCT?1100
|?||?|||||||?|||||||||?||||||||||||||||?|?||||||||||?|||||?|
Sbjct??884??T-TATATGAGCACTATGGGAAAGCTGACCCAGCCAGTGTAG-CAAAGGTGAAGGTCCTTT?941
Query??1101?ATGATGAGCTGGACCTTCAGGGGGTATTTATGGAGTATGAGAACCAAAGCTATGATAAAC?1160
||?||||||||?|?||||||||||||||||?||||||||||||??||||||||?|?||||
Sbjct??942??ATAATGAGCTGAAGCTTCAGGGGGTATTTACGGAGTATGAGAATGAAAGCTATAAGAAAC?1001
Query??1161?TAGTAACCTCCATTGAGGCTCACCCTAGCAAGGCAGTGCAAGCAGTGTTGAAGTC-TTTC?1219
||||||||||?|||||?|||||?|||||||||?|?||||||||||||||||||||?|||
Sbjct??1002?TAGTAACCTCTATTGAAGCTCATCCTAGCAAGCCGGTGCAAGCAGTGTTGAAGTCCTTT-?1060
Query??1220?CTTG-CCAAAATTTACAAGAGACAGAAATAAAAGAAAAGAAAATAAGAATATGCAGA-AG?1277
|||?||||||||||||||||||||||||||||?|?||?|||||???|||??|||||??|
Sbjct??1061?-TTGGCCAAAATTTACAAGAGACAGAAATAAAA-AGAA-AAAATGGG--TATCCAGATA-?1114
Query??1278?CACAGTCAATGATTGGATGATGCCGAATAATGC-GATTTTA-TTTGGGAATTGTATCATT?1335
||??||||?||?|||||||?||?|||||||||??||||??||||||??||||||||??||
Sbjct??1115?CATGGACAAAGACTGGATGACGCTGAATAATGCAGGTTT-AGTTTGG-AGTTGTATCTTT?1172
Query??1336?AATTTCCTAATT-AAGGAT-G---ATT--GCA---A-CTCTTTGTTGAACATTTTGATAT?1384
|?||||||||||?|||||??|???|||??||????|?|?|?|?||?||||||||||||||
Sbjct??1173?AGTTTCCTAATTGAAG-ATTGCTTATTCTGCTTGTAGCACATGGTTAAACATTTTGATAT?1231
Query??1385?TGGTAG-TCTTTGCTTATTT-GAAATTTGATTGAATGATTCTCATCTTTAATAAGATTTT?1442
|?|?||?||?||?||?|||??|||||??||?|||?||?||?||||?|||||||||||||
Sbjct??1232?TTGAAGGTCCTTAATTGTTTTGGAATT-GAATGAGTGGTTTTCATCATTAATAAGATTT-?1289
Query??1443?AATCCATTTGA-Taaaaaaaaaaaaaaaa??1470
||?||||||?|?||||||||||||||||
Sbjct??1290AAGCCATTTTAATCAAAAAAAAAAAAAAA?1318
Query: the nucleotide sequence of Cortex jatrophae JcFPS
Sbjct: the nucleotide sequence of Para rubber tree HbFPS (AB294712)
Table 3 is that the homology of the aminoacid sequence of proteic aminoacid sequence of Cortex jatrophae JcFPS of the present invention and Para rubber tree HbFPS compares (FASTA) table.Wherein, identical amino acid marks with the amino acid monocase between two sequences.
Table 3
93％identity?in?342aa?overlap，97％similarity?in?342aa?overlap
Query?1?MADLKSTFLEVYSVLKKELLQDPAFEWTPDSREWVERMLDYNVPGGKLNRGLSVIDSYKL?60
MADLKSTFL+VYSVLK+ELL+DPAFEWTPDSR+WVERMLDYNVPGGKLNRGLSVIDSYKL
Sbjct???1?MADLKSTFLKVYSVLKQELLEDPAFEWTPDSRQWVERMLDYNVPGGKLNRGLSVIDSYKL??60
Query??61?LKDGQELTEEEIFLASALGWCIEWLQAYFLVLDDIMDSSHTRRGQPCWFMVPKVGLIAAN?120
LK+GQELTEEEIFLASALGWCIEWLQAYFLVLDDIMDSSHTRRGQPCWF?VPKVGLIAAN
Sbjct??61?LKEGQELTEEEIFLASALGWCIEWLQAYFLVLDDIMDSSHTRRGQPCWFRVPKVGLIAAN?120
Query?121?DGILLRNHIPRILKKHFRGKAYYVDLLDLFNEVEFQTASGQMIDLITTLEGEKDLSKYNL?180
DGILLRNHIPRILKKHFRGKAYYVDLLDLFNEVEFQTASGQMIDLITTLEGEKDLSKY?L
Sbjct?121?DGILLRNHIPRILKKHFRGKAYYVDLLDLFNEVEFQTASGQMIDLITTLEGEKDLSKYTL?180
Query?181?SLHRRIVQYKTAYYSFYLPVACALLMAGENLDSHIDVQNILVQMGIYFQVQDDYLDCFGD?240
SLHRRIVQYKTAYYSFYLPVACALL+AGENLD+HI?V++ILVQMGIYFQVQDDYLDCFGD
Sbjct?181?SLHRRIVQYKTAYYSFYLPVACALLIAGENLDNHIVVKDILVQMGIYFQVQDDYLDCFGD?240
Query?241?PKTIGKIGTDIEDFKCSWLVVKALERCNEEQKKVLHEHYGKPDPASVSKVKVLYDELDLQ?300
P+TIGKIGTDIEDFKCSWLVVKALE?CNEEQKKVL+EHYGK?DPASV+KVKVLY+EL?LQ
Sbjct?241?PETIGKIGTDIEDFKCSWLVVKALELCNEEQKKVLYEHYGKADPASVAKVKVLYNELKLQ?300
Query?301?GVFMEYENQSYDKLVTSIEAHPSKAVQAVLKSFLAKIYKRQK?342
GVF?EYEN+SY?KLVTSIEAHPSK?VQAVLKSFLAKIYKRQK
Sbjct?301?GVFTEYENESYKKLVTSIEAHPSKPVQAVLKSFLAKIYKRQK?342
Query: Cortex jatrophae JcFPS aminoacid sequence
Sbjct: Para rubber tree HbFPS aminoacid sequence (GenBank Accession No.AAM98379)
The present invention also comprises the analogue of Cortex jatrophae JcFPS albumen or polypeptide.The difference of these analogues and natural farnesyl pyrophosphate synthase 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 of enumerating.
Described modification (not changing primary structure usually) form comprises: in the body or the chemically derived form such as the acetylize or carboxylated of external polypeptide.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in polypeptide synthesizes and processes 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 amino-acid residue (as Tyrosine O-phosphate, phosphoserine, the phosphothreonine) sequence with phosphorylation.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 JcFPS protein polypeptide of the present invention, Cortex jatrophae JcFPS albumen coded sequence operationally can be connected in expression regulation sequence, thereby form Cortex jatrophae JcFPS 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, Cortex jatrophae cell and other vegetable cell.
Whether and quantity the expression of also available Northern blotting technical Analysis Cortex jatrophae JcFPS protein gene product promptly analyzes the existence of the proteic rna transcription thing of Cortex jatrophae jc-fps in cell.
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 JcFPS 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 JcFPS.
The present invention relates to whether exist in the test sample method of Cortex jatrophae JcFPS 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 JcFPS 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 JcFPS 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 JcFPS albumen homology gene or homologous protein.
In order to obtain the dot matrix of the Cortex jatrophae cDNAs relevant with Cortex jatrophae JcFPS protein gene, the present invention can screen Cortex jatrophae cDNA library with dna probe, and these probes are under low stringent condition, use
32P to Cortex jatrophae JcFPS 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.This screening method can be discerned the nucleotide sequence with the proteic gene family of Cortex jatrophae JcFPS.
Cortex jatrophae JcFPS 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 obtain relevant sequence.When sequence is longer, often need carries 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 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, 1969Solid-Phase Peptide Synthesis, WHFreeman Co., San Francisco by direct peptide synthesis; Merrifield is Chem.Soc 85:2149-2154 J.1963J.Am).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 JcFPS albumen of the present invention,, can filter out with Cortex jatrophae JcFPS albumen interactional material takes place, perhaps acceptor, inhibitor or antagonist etc. by various conventional screening methods.Cortex jatrophae JcFPS protein gene of the present invention can be used for reducing the content of phorbol ester in the Cortex jatrophae or its precursor by genetic engineering technique, and phorbol ester is a kind of plant poison.Thereby the present invention has great application prospect.
Description of drawings
Fig. 1 is the proteic domain analyses figure of Cortex jatrophae FPS.
Fig. 2 is the proteic hydrophobicity analysis result of Cortex jatrophae FPS.
Fig. 3 is proteic transit peptides of Cortex jatrophae FPS and transmembrane amino acid analytical results.
Show that wherein ripe FPS does not have the film of striding district, is non-transmembrane protein.
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.
The clone of Cortex jatrophae JcFPS 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 FPS, the design degenerate primer utilizes homologous genes clone principle, adopts SMARTTMRACE cDNA amplification method (Clonetech test kit) to carry out the cDNA full-length clone, divides four-stage to carry out:
(1) clone of core sequence
PCR (JcFPSF+JcFPSR) obtains JcFPS-1 (434bp), 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 FPS gene of knowing its nucleotide sequence and proteins encoded and known xylophyta such as Para rubber tree (Hevea brasiliensis) etc. is very high, so think that tentatively it is a FPS gene.
(2)3’-RACE
Result according to the amplification of core sequence designs two forward special primers (JcFPSF1 and JcFPSF2).Adopt the secondary PCR amplification method to carry out.For the first time PCR (JcFPSF1+AP) obtains the PCR product, dilute 100 times after, do template with it, carry out PCR second time (JcFPSF2+AP), obtain JcFPS-3 (657bp), reclaim connection, order-checking process same (1)).
(3)5’-RACE
Result according to the amplification of core sequence designs two reverse special primers (JcFPSR1 and JcFPSR2).Adopt the secondary PCR amplification method to carry out.For the first time PCR (JcFPSR1+UPM) obtains the PCR product, dilute 100 times after, do template with it, carry out PCR second time (JcFPSR2+NUP), obtain JcFPS-3 (700bp), reclaim, connect check order (process is (1) together).
(4) clone of encoding sequence
With core sequence, 5 ' RACE and 3 ' RACE sequencing result sequence alignment and splice, obtain the full length fragment sequence information.Obtain on the total length basis of (comprising complete open reading frame at least) in splicing, further design oligonucleotides sequence JcFPSF1:5 '-ATGGCGGATCTGAAATCAACATTCTTG-3 ' (SEQ ID NO.3) is a forward primer, oligonucleotide sequence JcFPSR1:5 '-TTATTTCTGTCTCTTGTAAATTTTGGC-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 1029bp.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 IDNO.4.
The sequence information and the homology analysis of Cortex jatrophae JcFPS protein gene
The length of the Cortex jatrophae JcFPS albumen full-length cDNA that the present invention is new is 1029bp, and detailed sequence is seen SEQ IDNO.1, and wherein open reading frame is positioned at 221-1246 position Nucleotide.Derive the proteic aminoacid sequence of Cortex jatrophae JcFPS according to full-length cDNA, totally 381 amino-acid residues, molecular weight 43784.45, pI are 5.54.Detailed sequence is seen SEQ ID NO.2.
Proteic full length cDNA sequence of Cortex jatrophae JcFPS 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 it and Para rubber tree HbFPS gene (AB294712) have 87% homology (subordinate list 2) on nucleotide level; On amino acid levels, it and Para rubber tree HbFPS (GenBankAccession No.AAM98379) have 93% homogeny and 97% similarity (seeing Table 3).This shows that there are higher homology in Cortex jatrophae JcFPS albumen and Para rubber tree HbFPS on nucleic acid still is protein level, so can think that Cortex jatrophae JcFPS albumen is also similar on function.
The proteic structure characteristic analysis of embodiment 3 Cortex jatrophae FPS
1. the proteic domain analyses of Cortex jatrophae FPS
With the proteic aminoacid sequence of Cortex jatrophae FPS ncbi database (network address is: index structure territory http://www.ncbi.nlm.nih.gov/Blast.cgi), the result shows:
In aminoacid sequence, exist isopentenyl pyrophosphate synthetic enzyme (Trans-Isoprenyl Diphosphate Synthases, Trans IPPS) catalytic site (be in the sequence table SEQ ID.1 from aminoterminal (N end) 69-303 amino acids residue.
2. the proteic hydrophobicity analysis of Cortex jatrophae FPS
Ripe FPS after the transit peptides shearing is analyzed, and as a complete unit, hydrophilic amino acid is evenly distributed in the whole peptide chain, and whole polypeptide chain shows as wetting ability, does not have tangible hydrophobic region.Hydrophobicity plot of the present invention is based on the amino acid whose hydrophobic value of Kyte and Doolittle report and sets up.
3. proteic transit peptides of Cortex jatrophae FPS and transmembrane amino acid analysis
Find that by the TMHMM prediction ripe FPS does not have the film of striding district, be non-transmembrane protein.FPS whole piece chain all is in outside the film, does not have membrane spaning domain.The analysis revealed of TargetP 1.1Server, the score value of chloroplast transit peptides, plastosome target peptide and Secretory Pathway signal peptide is all lower, and no amino-acid residue division site thinks not have transit peptides.Both think by binding analysis, do not enter any subcellular organelle after the FPS maturation, but stay in the tenuigenin.
Embodiment 4
Cortex jatrophae JcFPS albumen carries out RNA in Cortex jatrophae disturbs
The structure that contains the rna interference vector of goal gene (Cortex jatrophae JcFPS protein gene).According to the proteic full length sequence of Cortex jatrophae JcFPS (SEQ ID NO.1) (belonging to just fragment), design a pair of primer, obtain complementary antisense fragment behind the pcr amplification, just segment and antisense segment introduced restriction endonuclease sites (this is decided by the carrier of selecting for use) respectively and connect carrier pHANNIBAL form loop-stem structure, by double digestion the segment of loop-stem structure is connected into the pCAMBIA1301 carrier then, be built into rna interference vector, again it is changed in the Agrobacterium, utilize Agrobacterium-mediated Transformation technology or gene gun technology to transform Cortex jatrophae.
Utilize Agrobacterium-mediated Transformation technical transform Cortex jatrophae
1. with the positive bacterium colony on the aseptic toothpick picking YEB selection flat board, be inoculated in 2ml YEB liquid (Sm
+, Kan
+), 28 ℃, 200rpm shaking culture 24-36 hour;
2. under the room temperature 4, the centrifugal 10min of 000g;
3. abandon supernatant, thalline suspends with the 1/2MS liquid nutrient medium, is diluted to 5-20 times of original volume, makes the OD of bacterium liquid
600About=0.5;
4. get the aseptic explant of the Cortex jatrophae of growth about month, it is cut into about 1 square centimeter of square small pieces or segment;
5. the small pieces or the segment of step (4) are put into the bacterium liquid for preparing, soak 2-5min, on aseptic filter paper, blot bacterium liquid;
6. small pieces or segment through contaminating are put on the MS substratum 28 ℃ of dark cultivations 48 hours;
7. small pieces or segment are forwarded on the callus substratum (MS+6-BA 1.0mg/L+NAA 0.1mg/L+Kan50mg/L+cb 250mg/L), the formation of 7-15 days visible callus is cultivated in 25-28 ℃ of illumination down;
8. visible differentiation bud grows after about 40 days, treat that bud is grown up after, downcut, place on the root media (1/2MS+NAA 0.5mg/L+Kan 25mg/L) and carry out root culture, take root about 10 days;
9. after waiting well developed root system, plant is taken out, clean the solid medium that adheres to, move in the soil, just begun to treat to take off lens again behind the robust plant, cultivate in the greenhouse with lens cover several days with sterilized water.
Utilize Northern blotting to detect the expression of JcFPS albumen in transgenosis Cortex jatrophae plant
1.RNA extraction: treat the long RNA that extracts the leprosy leaf during to 2-3 sheet leaf of transgenosis Cortex jatrophae blade.With the plant of normal growth (condition is the same) in contrast, (GIBCO BRL USA) extracts and with reference to the preparation chapters and sections (Sambrook etc., 1989) of " molecular cloning " relevant RNA to utilize the TRIzol test kit.
2.RNA quantitatively: with reference to " molecular cloning " (Sambrook etc., 1989), spectrophotometric instrumentation OD
260Rna content calculates: 1OD
260=40 μ g/ml.
3 total RNA agarose gel electrophoresis separate: 1. get 6ml 25 (doubly) electrophoretic buffer, add the 117ml sterilized water, mixing.2. take by weighing the 1.5g agarose, join in the above-mentioned solution, heating and melting in microwave oven changes in 55 ℃ of water-baths.3. in stink cupboard, get 26.8ml formaldehyde, join in 55 ℃ the gelating soln mixing.4. pour into rapidly in the glue plate, room temperature water placing flat 30 minutes treats that gelling is solid.5. the RNA (20 μ g) that extracts is dissolved in the RNA denaturing soln, heated 10 minutes down, be placed on ice immediately then at 65 ℃.6. in sample, add 2ul 10 * sample-loading buffer, mixing.7. do not cover point sample under the condition of glue in electrophoresis liquid, 5V/cm voltage electrophoresis is about 5 hours.
4.RNA shift on the nylon membrane: before 1. shifting, nylon membrane is soaked with 10 * SSC.2. moistening film is covered exactly on film, two filter paper identical with film size are put in 2 * SSC solution moistening, cover on film, get rid of bubble.3. put one on the filter paper and fold and the identical thieving paper of film size, put a sheet glass and a weight on thieving paper, horizontal positioned shifted 12-20 hour.4. after shifting, film was toasted 2 hours in 80 ℃.
5. the detecting of hybridization signal on the film: 1. film is immersed in 5 * Dendart ' s, 0.1%SDS, 0.1mg/ml salmon sperm dna, 65 ℃ of following prehybridizations 2 hours.2. will use Gene Images
TMContents CDP-Star
TMThe sex change 5 minutes in boiling water of the probe of labelling module mark directly adds in the hybridization solution of (1), in 65 ℃ of hybridization 16-24 hour.3. take out film, place film washing liquid I (1 * SSC, 1%SDS) in, in 65 ℃ of rinsings 3 times, each 15 minutes.Change over to film washing liquid II (0.1 * SSC, 1%SDS) in 65 ℃ of rinsings 3 times, each 15 minutes.4. use X-ray sheet compressing tablet 60-90 minute, then development, photographic fixing (method is with reference to RocheDIG labeled test kit specification sheets).Northern hybridization shows; The jc-FPS transcriptional level of transgenosis Cortex jatrophae is obviously more much lower than the expression level of genetically modified control material not.
Sequence that the present invention relates to and mark apportion are as follows:
＜110〉Fudan University
＜120〉jatropha curcas farnesyl pyrophosphate synthase protein encoding sequence and the application in plant thereof
<160>2
<170>PatentIn?version?3.1
<210>1
<211>1471
<212>mRNA
＜213〉Cortex jatrophae (Jatropha carcas L.)
<220>
<221>CDS
<222>(221)..(1246)
<223>
<400>1
AAGCAGTGGT?ATCAACGCAG?AGTACGCGGG?GGCGCAAGCG?AAAGACAGTG?ACTCCAAGTC
TCCAACGCCA?AGAACAGGAA?TATTACATAG?TCAACCAAAA?GCTGTGGTCT?CTCAGTATAA
AATTGCCACT?TTGCAACATC?AATCTCTCCT?CACTACTGCC?CTCCCTTTTC?ACTGTAGCTC
TCTTTCATTC?CCTTGGTCTC?TGAGTCTCCT?TTTTGAAGCT?ATG?GCG?GAT?CTG?AAA
Met?Ala?Asp?Leu?Lys
1???????????????5
TCA?ACA?TTC?TTG?GAG?GTC?TAT?TCT?GTT?CTC?AAG?AAG?GAG?CTA?CTT?CAA
Ser?Thr?Phe?Leu?Glu?Val?Tyr?Ser?Val?Leu?Lys?Lys?Glu?Leu?Leu?Gln
10???????????????????15??????????????????20
GAC?CCG?GCT?TTC?GAA?TGG?ACA?CCA?GAT?TCT?CGT?GAA?TGG?GTC?GAA?AGG
Asp?Pro?Ala?Phe?Glu?Trp?Thr?Pro?Asp?Ser?Arg?Glu?Trp?Val?Glu?Arg
25?????????????????30???????????????????35
ATG?CTG?GAC?TAC?AAT?GTG?CCT?GGA?GGG?AAG?CTG?AAT?AGG?GGG?CTC?TCT
Met?Leu?Asp?Tyr?Asn?Val?Pro?Gly?Gly?Lys?Leu?Asn?Arg?Gly?Leu?Ser
40???????????????????45??????????????????50
GTG?ATT?GAC?AGC?TAC?AAA?TTG?TTG?AAA?GAT?GGA?CAG?GAA?TTA?ACA?GAA
Val?Ile?Asp?Ser?Tyr?Lys?Leu?Leu?Lys?Asp?Gly?Gln?Glu?Leu?Thr?Glu
55???????????????????60??????????????????65
GAA?GAA?ATC?TTT?CTC?GCA?AGC?GCT?CTT?GGT?TGG?TGT?ATT?GAA?TGG?CTC
Glu?Glu?Ile?Phe?Leu?Ala?Ser?Ala?Leu?Gly?Trp?Cys?Ile?Glu?Trp?Leu
70??????????????????75???????????????????80?????????????????85
CAA?GCC?TAT?TTC?CTT?GTC?CTT?GAT?GAT?ATT?ATG?GAT?AGC?TCT?CAT?ACA7
Gln?Ala?Tyr?Phe?Leu?Val?Leu?Asp?Asp?Ile?Met?Asp?Ser?Ser?His?Thr
90??????????????????95?????????????????100
CGA?CGT?GGT?CAA?CCA?TGT?TGG?TTT?ATG?GTG?CCC?AAG?GTT?GGT?CTT?ATT
Arg?Arg?Gly?Gln?Pro?Cys?Trp?Phe?Met?Val?Pro?Lys?Val?Gly?Leu?Ile
105?????????????????110?????????????????115
GCA?GCA?AAT?GAT?GGG?ATT?TTG?CTT?CGA?AAT?CAC?ATT?CCC?AGG?ATT?CTT
Ala?Ala?Asn?Asp?Gly?Ile?Leu?Leu?Arg?Asn?His?Ile?Pro?Arg?Ile?Leu
120?????????????????125?????????????????130
AAA?AAG?CAC?TTC?CGA?GGG?AAA?GCA?TAC?TAT?GTA?GAT?CTT?CTC?GAT?TTA
Lys?Lys?His?Phe?Arg?Gly?Lys?Ala?Tyr?Tyr?Val?Asp?Leu?Leu?Asp?Leu
135?????????????????140?????????????????145
TTT?AAT?GAG?GTG?GAG?TTT?CAA?ACA?GCC?TCA?GGA?CAG?ATG?ATA?GAT?CTG
Phe?Asn?Glu?Val?Glu?Phe?Gln?Thr?Ala?Ser?Gly?Gln?Met?Ile?Asp?Leu
150?????????????????155?????????????????160?????????????????165
ATT?ACA?ACA?CTT?GAA?GGA?GAA?AAG?GAT?TTA?TCG?AAG?TAC?AAT?TTA?TCG
Ile?Thr?Thr?Leu?Glu?Gly?Glu?Lys?Asp?Leu?Ser?Lys?Tyr?Asn?Leu?Ser
170?????????????????175?????????????????180
CTT?CAC?CGG?CGA?ATT?GTT?CAG?TAC?AAA?ACT?GCC?TAC?TAC?TCA?TTT?TAC
Leu?His?Arg?Arg?Ile?Val?Gln?Tyr?Lys?Thr?Ala?Tyr?Tyr?Ser?Phe?Tyr
185?????????????????190?????????????????195
CTT?CCT?GTT?GCT?TGT?GCA?TTG?CTC?ATG?GCT?GGT?GAG?AAT?CTG?GAC?AGC
Leu?Pro?Val?Ala?Cys?Ala?Leu?Leu?Met?Ala?Gly?Glu?Asn?Leu?Asp?Ser
200?????????????????205?????????????????210
CAT?ATT?GAT?GTA?CAG?AAT?ATT?CTT?GTC?CAG?ATG?GGA?ATC?TAC?TTC?CAA
His?Ile?Asp?Val?Gln?Asn?Ile?Leu?Val?Gln?Met?Gly?Ile?Tyr?Phe?Gln
215?????????????????220?????????????????225
GTA?CAG?GAT?GAT?TAT?TTG?GAT?TGC?TTT?GGT?GAT?CCC?AAG?ACA?ATT?GGC
Val?Gln?Asp?Asp?Tyr?Leu?Asp?Cys?Phe?Gly?Asp?Pro?Lys?Thr?Ile?Gly
230?????????????????235?????????????????240?????????????????245
AAG?ATA?GGG?ACA?GAT?ATT?GAA?GAT?TTT?AAG?TGC?TCT?TGG?TTG?GTC?GTG
Lys?Ile?Gly?Thr?Asp?Ile?Glu?Asp?Phe?Lys?Cys?Ser?Trp?Leu?Val?Val
250?????????????????255?????????????????260
AAG?GCT?TTG?GAG?CGA?TGC?AAT?GAA?GAG?CAA?AAG?AAA?GTT?CTA?CAT?GAG
Lys?Ala?Leu?Glu?Arg?Cys?Asn?Glu?Glu?Gln?Lys?Lys?Val?Leu?His?Glu
265?????????????????270?????????????????275
CAT?TAT?GGG?AAA?CCT?GAC?CCA?GCC?AGT?GTG?TCA?AAG?GTG?AAA?GTC?CTC
His?Tyr?Gly?Lys?Pro?Asp?Pro?Ala?Ser?Val?Ser?Lys?Val?Lys?Val?Leu
280?????????????????285?????????????????290
TAT?GAT?GAG?CTG?GAC?CTT?CAG?GGG?GTA?TTT?ATG?GAG?TAT?GAG?AAC?CAA
Tyr?Asp?Glu?Leu?Asp?Leu?Gln?Gly?Val?Phe?Met?Glu?Tyr?Glu?Asn?Gln
295?????????????????300?????????????????305
AGC?TAT?GAT?AAA?CTA?GTA?ACC?TCC?ATT?GAG?GCT?CAC?CCT?AGC?AAG?GCA
Ser?Tyr?Asp?Lys?Leu?Val?Thr?Ser?Ile?Glu?Ala?His?Pro?Ser?Lys?Ala
310?????????????????315?????????????????320?????????????????325
GTG?CAA?GCA?GTG?TTG?AAG?TCT?TTC?CTT?GCC?AAA?ATT?TAC?AAG?AGA?CAG
Val?Gln?Ala?Val?Leu?Lys?Ser?Phe?Leu?Ala?Lys?Ile?Tyr?Lys?Arg?Gln
330?????????????????335?????????????????340
AAA?TAAAAGAAAA?GAAAATAAGA?ATATGCAGAA?GCACAGTCAA?TGATTGGATG?ATGCC
Lys
GAATAATGCG?ATTTTATTTG?GGAATTGTAT?CATTAATTTC?CTAATTAAGG?ATGATTGCAA
CTCTTTGTTG?AACATTTTGA?TATTGGTAGT?CTTTGCTTAT?TTGAAATTTG?ATTGAATGAT
TCTCATCTTT?AATAAGATTT?TAATCCATTT?GATAAAAAAA?AAAAAAAAAA
<210>2
<211>342
<212>PRT
＜213〉Cortex jatrophae (Jatropha carcas L.)
<400>2
Met?Ala?Asp?Leu?Lys?Ser?Thr?Phe?Leu?Glu?Val?Tyr?Ser?Val?Leu?Lys
1???????????????5???????????????????10??????????????????15
Lys?Glu?Leu?Leu?Gln?Asp?Pro?Ala?Phe?Glu?Trp?Thr?Pro?Asp?Ser?Arg
20??????????????????25??????????????????30
Glu?Trp?Val?Glu?Arg?Met?Leu?Asp?Tyr?Asn?Val?Pro?Gly?Gly?Lys?Leu
35??????????????????40??????????????????45
Asn?Arg?Gly?Leu?Ser?Val?Ile?Asp?Ser?Tyr?Lys?Leu?Leu?Lys?Asp?Gly
50??????????????????55??????????????????60
Gln?Glu?Leu?Thr?Glu?Glu?Glu?Ile?Phe?Leu?Ala?Ser?Ala?Leu?Gly?Trp
65??????????????????70??????????????????75??????????????????80
Cys?Ile?Glu?Trp?Leu?Gln?Ala?Tyr?Phe?Leu?Val?Leu?Asp?Asp?Ile?Met
85??????????????????90??????????????????95
Asp?Ser?Ser?His?Thr?Arg?Arg?Gly?Gln?Pro?Cys?Trp?Phe?Met?Val?Pro
100?????????????????105?????????????????110
Lys?Val?Gly?Leu?Ile?Ala?Ala?Asn?Asp?Gly?Ile?Leu?Leu?Arg?Asn?His
115?????????????????120?????????????????125
Ile?Pro?Arg?Ile?Leu?Lys?Lys?His?Phe?Arg?Gly?Lys?Ala?Tyr?Tyr?Val
130?????????????????135?????????????????140
Asp?Leu?Leu?Asp?Leu?Phe?Asn?Glu?Val?Glu?Phe?Gln?Thr?Ala?Ser?Gly
145?????????????????150?????????????????155?????????????????160
Gln?Met?Ile?Asp?Leu?Ile?Thr?Thr?Leu?Glu?Gly?Glu?Lys?Asp?Leu?Ser
165?????????????????170?????????????????175
Lys?Tyr?Asn?Leu?Ser?Leu?His?Arg?Arg?Ile?Val?Gln?Tyr?Lys?Thr?Ala
180?????????????????185?????????????????190
Tyr?Tyr?Ser?Phe?Tyr?Leu?Pro?Val?Ala?Cys?Ala?Leu?Leu?Met?Ala?Gly
195?????????????????200?????????????????205
Glu?Asn?Leu?Asp?Ser?His?Ile?Asp?Val?Gln?Asn?Ile?Leu?Val?Gln?Met
210?????????????????215?????????????????220
Gly?Ile?Tyr?Phe?Gln?Val?Gln?Asp?Asp?Tyr?Leu?Asp?Cys?Phe?Gly?Asp
225?????????????????230?????????????????235?????????????????240
Pro?Lys?Thr?Ile?Gly?Lys?Ile?Gly?Thr?Asp?Ile?Glu?Asp?Phe?Lys?Cys
245?????????????????250?????????????????255
Ser?Trp?Leu?Val?Val?Lys?Ala?Leu?Glu?Arg?Cys?Asn?Glu?Glu?Gln?Lys
260?????????????????265?????????????????270
Lys?Val?Leu?His?Glu?His?Tyr?Gly?Lys?Pro?Asp?Pro?Ala?Ser?Val?Ser
275?????????????????280?????????????????285
Lys?Val?Lys?Val?Leu?Tyr?Asp?Glu?Leu?Asp?Leu?Gln?Gly?Val?Phe?Met
290?????????????????295?????????????????300
Glu?Tyr?Glu?Asn?Gln?Ser?Tyr?Asp?Lys?Leu?Val?Thr?Ser?Ile?Glu?Ala
305?????????????????310?????????????????315?????????????????320
His?Pro?Ser?Lys?Ala?Val?Gln?Ala?Val?Leu?Lys?Ser?Phe?Leu?Ala?Lys
325?????????????????330?????????????????335
Ile?Tyr?Lys?Arg?Gln?Lys
340
<210>3
<211>27
<212>DNA
＜213〉Cortex jatrophae (Jartropha curcas L.)
<400>3
ATGGCGGATCTGAAATCAACATTCTTG
<210>4
<211>27
<212>DNA
＜213〉Cortex jatrophae (Jatropha curcas L.)
<400>4
TTATTTCTGTCTCTTGTAAATTTTGGC
Claims (14)
1. isolated dna molecular, it is characterized in that, it comprises: coding has the nucleotide sequence of Cortex jatrophae JcFPS protein active polypeptide, shows at least 70% homology from the nucleotides sequence of Nucleotide 221-1246 position among described nucleotide sequence and the SEQ ID N0.1; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.1 in from the nucleotide sequence hybridization of Nucleotide 221-1246 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 221-1246 position.
4. isolated Cortex jatrophae JcFPS protein polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQIDNO.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: the carrier that sets out that is used to make up described plant expression vector is p3301-BI121, pBll21, pBin19, pCAMBIA2301, pCAMBIA1301 or pCAMBIA1300.
8. the described carrier transformed host cells of claim 6 is characterized in that it is protokaryon or eukaryotic cell.
9. a generation has the method for the polypeptide of Cortex jatrophae JcFPS protein active, is characterised in that it comprises the steps:
(1) nucleotide sequence of purifying that coding is had a polypeptide of Cortex jatrophae JcFPS protein-active operationally is connected in expression regulation sequence, form Cortex jatrophae JcFPS protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 221-1246 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 JcFPS;
(3) be fit to express under the condition of Cortex jatrophae JcFPS protein polypeptide the reconstitution cell in the culturing step (2);
(4) isolate pure substantially polypeptide with Cortex jatrophae JcFPS protein-active.
10. one kind is utilized transgenic technology that the nucleotide sequence that coding has Cortex jatrophae JcFPS protein active polypeptide is transformed into plant to improve the method for terpene substances in the plant, it is characterized in that, as follows:
(1) nucleotide sequence of purifying that coding is had a polypeptide of Cortex jatrophae JcFPS protein-active operationally is connected in the expression of plants regulating and controlling sequence, formation contains the proteic plant expression vector of Cortex jatrophae JcFPS, shows at least 70% homology from the nucleotides sequence of Nucleotide 221-1246 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;
(3) by screening as antibiotic-screening, obtain transformant and final regeneration of transgenic plant and offspring thereof, comprise plant seed and plant tissue.
11., it is characterized in that the plant host of described step (2) is paddy rice, wheat, soybean, corn, tobacco, rape, Chinese sorghum, cotton, clover, Cortex jatrophae or Arabidopis thaliana by the described method of claim 10.
12. one kind and many skins of the described Cortex jatrophae JcFPS of claim 7 protein polypeptide specificity bonded antibody is characterized in that it comprises polyclonal antibody and monoclonal antibody.
13. a nucleic acid molecule is characterized in that, it comprises 8-100 continuous nucleotide in the described dna molecular of claim 1.
14. the method for Cortex jatrophae JcFPS nucleotide sequence is characterized in that it comprises the steps in the test sample; Hybridize with described probe of claim 10 and sample, whether detection probes combination takes place then; Described sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to Cortex jatrophae JcFPS 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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| CN102433349A (en) * | 2011-11-28 | 2012-05-02 | 山东省农业科学院作物研究所 | Triticum aestivum farnesyl phosphate synthase (TaFPS) gene as well as isolation colonizing and enzyme activity measuring method thereof |
| CN102776159A (en) * | 2012-07-24 | 2012-11-14 | 中国农业科学院作物科学研究所 | Protein associated with sesquiterpene synthesis and encoding gene and application thereof |
| CN102876689A (en) * | 2012-07-11 | 2013-01-16 | 浙江大学 | Tea tree FPS (famesyl diphosphate synthase) gene and application thereof |
| CN105642663A (en) * | 2014-10-21 | 2016-06-08 | 周杰 | Flora and fauna comprehensive utilization restoring method of heavy metal contaminated soil |
| CN106459174A (en) * | 2015-02-18 | 2017-02-22 | 麻省理工学院 | Water-soluble trans-membrane proteins and methods for the preparation and use thereof |
| CN110747196A (en) * | 2019-10-16 | 2020-02-04 | 中国科学院西双版纳热带植物园 | Tissue-specific promoter JcTM6 gene promoter expressed in plant flowers and application thereof |
| CN112980809A (en) * | 2021-03-17 | 2021-06-18 | 云南中烟工业有限责任公司 | Tobacco farnesyl pyrophosphate synthase gene and application thereof |
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- 2009-07-15 CN CN200910054817A patent/CN101798579A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102433349A (en) * | 2011-11-28 | 2012-05-02 | 山东省农业科学院作物研究所 | Triticum aestivum farnesyl phosphate synthase (TaFPS) gene as well as isolation colonizing and enzyme activity measuring method thereof |
| CN102876689A (en) * | 2012-07-11 | 2013-01-16 | 浙江大学 | Tea tree FPS (famesyl diphosphate synthase) gene and application thereof |
| CN102876689B (en) * | 2012-07-11 | 2014-06-18 | 浙江大学 | Tea tree FPS (famesyl diphosphate synthase) gene and application thereof |
| CN102776159A (en) * | 2012-07-24 | 2012-11-14 | 中国农业科学院作物科学研究所 | Protein associated with sesquiterpene synthesis and encoding gene and application thereof |
| CN105642663A (en) * | 2014-10-21 | 2016-06-08 | 周杰 | Flora and fauna comprehensive utilization restoring method of heavy metal contaminated soil |
| CN106459174A (en) * | 2015-02-18 | 2017-02-22 | 麻省理工学院 | Water-soluble trans-membrane proteins and methods for the preparation and use thereof |
| CN106459174B (en) * | 2015-02-18 | 2021-08-27 | 麻省理工学院 | Water-soluble transmembrane proteins and methods of making and using same |
| CN110747196A (en) * | 2019-10-16 | 2020-02-04 | 中国科学院西双版纳热带植物园 | Tissue-specific promoter JcTM6 gene promoter expressed in plant flowers and application thereof |
| CN112980809A (en) * | 2021-03-17 | 2021-06-18 | 云南中烟工业有限责任公司 | Tobacco farnesyl pyrophosphate synthase gene and application thereof |
| CN112980809B (en) * | 2021-03-17 | 2023-04-11 | 云南中烟工业有限责任公司 | Tobacco farnesyl pyrophosphate synthase gene and application thereof |
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