CN1563385A - Coded sequence of protein anti anaerobic enolase in Chinese cabbage - Google Patents
Coded sequence of protein anti anaerobic enolase in Chinese cabbage Download PDFInfo
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Abstract
A Chinese cabbage anti-anaerobic enol zymoprotein coding series is used in biological and gene engineering. The DNA molecular from it includes: coding an active polymorphic nucleotide series with cabbage enolase protein which has 70% homology with that from 20-1354 potentials of SEQ ID NO.3 or the said nucleotide series can be intercrossed with it, said coded series has the polymorphic of amino acid series shown in SEQ ID NO.3, or conservative variant or it active fragments or it derivative and said coding sequence has the nudeotide series from 20-1354 potentials of nucleotide from SEQ ID NO.3.
Description
Technical field
The present invention relates to be used for biology and gene engineering technology field.Particularly, the present invention relates to a kind of enolase protein encoding sequence and nucleotide sequence thereof of in Chinese cabbage, expressing.
Background technology
Glycolysis-is the metabolic integral part of cell ability, and it almost is present in all types of cells, no matter is prokaryotic cell prokaryocyte or eukaryotic cell.Under the anaerobism stress conditions, the carbohydrate metabolism approach of many plants transfers glycolytic pathway to from oxidation model, to satisfy the energy metabolism under the plant anoxia condition.Stress conditions has been induced several proteic synthetic participating in the glycolytic pathway, as enolase, aldehyde carboxylic acid, pyruvic carboxylase etc.Enolase (Enolase) is unique in a glycolytic cycle dehydrating step, main catalysis 2-phosphoglyceric acid forms phosphoenolpyruvic acid, under the katalysis of pyruvate kinase, form enol pyruvic acid subsequently, pyruvic acid enters plastosome and begins tricarboxylic acid cycle, and the energy requirement under the plant anoxia condition is provided.
Warp is to finding in the prior art literature search: magazine " Plant Science (plant science) " is 1999,146:41-51 has delivered article " Transcript levels of genes encoding variousglycolytic and fermentation enzymes change in response to abiotic stresses (transcriptional level of coding glycolytic ferment gene under the anaerobism stress conditions changes) ", though the document to the key enzyme enolase gene Enolase in the glycolytic pathway at salt tolerant, arid, expression under high temperature stress is handled is affirmed fully, but report up to now shows the salt tolerant of the transgenic tobacco plant of this gene, the effect that drought resisting and temperature are coerced the aspect does not obviously improve, and does not find to have with theme of the present invention the report of close ties document in addition so far as yet.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of Chinese cabbage anaerobic enolase protein encoding sequence is provided, it is coerced in the anti-anaerobism of vegetable cell have tangible effect, can obviously improve the resistance of vegetable cell under anoxia condition, the reduction anaerobism is coerced the infringement to plant.
The present invention is achieved by the following technical solutions, the isolated dna molecular of the present invention, this molecule comprises: coding has the nucleotide sequence of the active polypeptide of Chinese cabbage enolase protein matter, and shows at least 70% homology from the nucleotides sequence of Nucleotide 20-1354 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps described nucleotide sequence can with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 20-1354 position, described encoding sequence has the polypeptide of the aminoacid sequence shown in the SEQ ID NO.3, or its conservative property variation polypeptide or its active fragments, or its reactive derivative, described encoding sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 20-1354 position.
The anti-anaerobism of the isolated Chinese cabbage of the present invention is coerced class related protein polypeptide, it comprises: the polypeptide with SEQ ID NO.3 aminoacid sequence, this polypeptide is to have SEQ ID NO.3 polypeptide of sequence, this polypeptide can all can play a role under salt, low temperature, water logging and dormin are handled, and these environment stresses all can cause the conversion of plant materials oxidative metabolism approach.
Carrier of the present invention, it comprises described dna molecular, a kind of nucleic acid molecule, it comprises 8-100 continuous nucleotide in the described dna molecular.
Dna molecular transformed host cells of the present invention, it is an eukaryotic cell.
In the present invention, " isolating ", " purifying " DNA are meant, this DNA or fragment have been arranged in the sequence of its both sides under native state separates, and refers to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separates with follow its protein in cell.
In the present invention, refer to encode has the nucleotide sequence of the active polypeptide of Chinese cabbage enolase protein to term " the anti-anaerobism stress protein of Chinese cabbage (or polypeptide) encoding sequence ", as 20-1354 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 20-1354 position Nucleotide of SEQ ID NO.3 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.3 in 20-1354 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.3 of also encoding out.This term also comprises can be under the moderate stringent condition (70%-85% consistence), better under the height stringent condition among (85% above consistence) and the SEQ ID NO.3 from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 20-1354 position.This term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 20-1354 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 ID NO.3 with natural Chinese cabbage enolase 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 " anti-anaerobism stress protein of Chinese cabbage or polypeptide " refers to have the active SEQ ID of Chinese cabbage enolase protein NO.3 polypeptide of sequence.This term also comprises having and the variant form relevant identical function of natural Chinese cabbage enolase, SEQ ID NO.3 sequence.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 the active fragments and the reactive derivative of Chinese cabbage enolase protein.
The variant form of Chinese cabbage enolase polypeptide of the present invention comprises: the albumen that homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, DNA that can relevant DNA hybridization with the Chinese cabbage enolase under high or low rigorous condition are coded and the polypeptide or the albumen that utilize the antiserum(antisera) of Chinese cabbage enolase polypeptide to obtain.
In the present invention, " Chinese cabbage enolase conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO.3, has 10 at the most, and preferably at the most 8, more preferably 5 amino acid is replaced by similar performance or close amino acid 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 |
| 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
91％identity?in?1334nt?overlap
Query：20??atggccactatcaccgctgttaaagctaggcagatcttcgacagccgtggcaatcccacg?79
|||||?||||||||||?||||||?|||||?||||||||||||||?|||||?||||||||
Sbjct：81??atggctactatcaccgttgttaaggctagacagatcttcgacagtcgtggtaatcccacc?140
Query：80??gttgaggttgatgtacacacatcaagtggtgttaaggttacagcagcggttccgagtgga?139
||||||||||||?|?|||||?||||?||||?||||||||||||||||?|||||?||||||
Sbjct：141?gttgaggttgatatccacacgtcaaatggtattaaggttacagcagctgttccaagtgga?200
Query：140?gcttccaccggtatctacgaggctcttgagcttagggatggtggatcagactaccttgga?199
||||||||?||||||||?||||||||||||||?||||||||?|||||?||||||||||||
Sbjct：201?gcttccactggtatctatgaggctcttgagctgagggatggaggatctgactaccttgga?260
Query：200?aagggtgtctctaaggctgttggcaatgtgaactccatcatcggcccagcttcgattgga?259
||||||||?||||||||||||||||||||||||?||||||||||?|||||???|||||||
Sbjct：261?aagggtgtatctaaggctgttggcaatgtgaacaacatcatcgggccagcacttattgga?320
Query：260?aaagacccaactcagcagactgctattgacaacttcatggtccatgaacttgatggaacc?319
||?||||||||||||||||||||||||||||||||||||||||||||||||||?||||||
Sbjct：321?aaggacccaactcagcagactgctattgacaacttcatggtccatgaacttgacggaacc?380
Query：320?cagaacgaatggggatggtgcaagcaaaagcttggagccaatgctattcttgctgtatct?379
||?|||||?|||||?|||||||||||||||||||||||||||||?|||||||||||?|||
Sbjct：381?caaaacgagtgggggtggtgcaagcaaaagcttggagccaatgcgattcttgctgtgtct?440
Query：380??ctagctgtctgcaaagctggggctgttgtcagtggcattcctctctacaagcacattgcc?439
||?|||||||||||||||||||||||||||||?|||||||||||?|||||||||||||||
Sbjct：441??cttgctgtctgcaaagctggggctgttgtcagcggcattcctctatacaagcacattgcc?500
Query：440??aatcttgctggtaaccctaagattgtgctaccagttcctgcgttcaacgttatcaatggt?499
||?||||||||||||||?|||||||||||||||||||||||?||||||||?|||||||||
Sbjct：501??aaccttgctggtaaccccaagattgtgctaccagttcctgccttcaacgtcatcaatggt?560
Query：500??ggatcccatgccggaaacaagctcgccatgcaggagtttatgatcctccctgttggagct?559
|||||||||||||||||||||||?||?|||||||||||||||||||||||||||||||||
Sbjct：561??ggatcccatgccggaaacaagcttgctatgcaggagtttatgatcctccctgttggagct?620
Query：560??tcttctttcaaagaagccatgaaaatgggtgttgaagtttaccacaacttgaagtctgtg?619
||||||||||?||||||||||?|||||||||?||||||||||||?||||||||||||||
Sbjct：621??gcttctttcaaggaagccatgaagatgggtgtggaagtttaccaccacttgaagtctgtg?680
Query：620??attaagaagaagtatggacaggatgccaccaacgtcggtgatgaagggggctttgcacca?679
||||||||||||||?||?|||||||||||?||?||?|||||||||||?||?|||||||||
Sbjct：681??attaagaagaagtacggccaggatgccacaaatgttggtgatgaaggtgggtttgcacca?740
Query：680??aacattcaagaaaacaaggaaggtcttgaactgcttaagactgctatcgagaaggctgga?739
||||||||?||?||||||||?|||||||||?||||?||||||||||||||||||||||||
Sbjct：741??aacattcaggagaacaaggagggtcttgaattgctcaagactgctatcgagaaggctgga?800
Query：740??tacactggcaaggttgtcattgggatggatgttgccgcttctgagttctactcatctgac?799
||||||||?||||||||||||||?|||||||||||||||||?||||||||||||???|||
Sbjct：801??tacactggaaaggttgtcattggaatggatgttgccgcttcagagttctactcagaagac?860
Query：800??aagacttacgacttgaacttcaaagaagagaacaacaatggttctcagaagatttctgga?859
|||||?|||||||||||||||||||||||||||||||||||?|||||||||||||||||
Sbjct：861??aagacctacgacttgaacttcaaagaagagaacaacaatggctctcagaagatttctggt?920
Query：860??gatgctctcaaggacctgtacaagtccttcgttgctgagtacccaattgtgtccattgag?919
||||||||?||||||||||||||||||||?||?||||||||||||||?||||||||||||
Sbjct：921??gatgctctaaaggacctgtacaagtcctttgtcgctgagtacccaatcgtgtccattgag?980
Query：920??gacccatttgaccaagatgactgggagcactatgctaagatgaccgccgagtgtggagac?979
|||||||||||||||||||||||||||||||||||||||||||||?|?|||||||||??|
Sbjct：981??gacccatttgaccaagatgactgggagcactatgctaagatgaccactgagtgtggaacc?1040
Query：980??aatgttcagattgtcggtgatgatttgttggtcaccaaccccaagggagttgccaaggca?1039
|?||||||||||||||||||||||||||||||||?|||||||||?|||||||?||||||
Sbjct：1041?gaggttcagattgtcggtgatgatttgttggtcactaaccccaagagagttgctaaggca?1100
Query：1040?atcgcagaaaagtcttgcaatgctcttctcttgaaggttaatcaaatcgggtcagtaaca?1099
||||||||?||||||||||||||||||||?|||||||||||?||||||||?||?|||||
Sbjct：1101?atcgcagagaagtcttgcaatgctcttcttttgaaggttaaccaaatcggatctgtaacc?1160
Query：1100?gagagtattgaggcagtgaagatgtcaaagagagcaggatggggagtgatggctagccac?1159
||||||||?||||||||?||||||||?||||?||||||?||||||||||||?|?||||||
Sbjct：1161?gagagtatcgaggcagttaagatgtcgaagaaagcaggttggggagtgatgaccagccac?1220
Query：1160?cgaagtggtgaaaccgaggacaccttcatcgctgacttatccgttggcttgtctactgga?1219
||||||||||||||||||||||?|||||?|||||||||?|||||||||||||?||||||
Sbjct：1221?agaagtggtgaaaccgaggacacattcattgctgacttagccgttggcttgtccactgga?1280
Query：1220?caaatcaagactggagctccttgcagatccgagcgtcttgccaagtacaaccagcttttg?1279
||||||||?||?||?|||||||||||||||||||||||||||||||||||||||||||||
Sbjct：1281?caaatcaaaaccggtgctccttgcagatccgagcgtcttgccaagtacaaccagcttttg?1340
Query：1280?cgtattgaggaggagttgggatcagaggcagtttacgctggagccaacttccgcaagcct?1339
||||||||||||||||||||||||||||||?||||||||||||?||||||||||||?|||
Sbjct：1341?cgtattgaggaggagttgggatcagaggcaatttacgctggagtcaacttccgcaaacct?1400
Query：1340?gtggagccctacta?1353
|||||?||||||||
Sbjct：1401?gtggaaccctacta?1414
Query: the nucleotide sequence of Chinese cabbage enolase
Sbjct: the nucleotide sequence of Arabidopis thaliana enolase (NM_129209)
Table 2 is that the homology of the nucleotide sequence of Da Bai enolase protein of the present invention and Arabidopis thaliana enolase protein compares (GAP) table.
Table 3
95％identity?in?444aa?overlap，97％similarity?in?444aa?overlap
Query：20??MATITAVKARQIFDSRGNPTVEVDVHTSSGVKVTAAVPSGASTGIYEALELRDGGSDYLG?199
MATIT?VKARQIFDSRGNPTVEVD+HTS+G+KVTAAVPSGASTGIYEALELRDGGSDYLG
Sbjct：1???MATITVVKARQIFDSRGNPTVEVDIHTSNGIKVTAAVPSGASTGIYEALELRDGGSDYLG?60
Query：200?KGVSKAVGNVNSIIGPASIGKDPTQQTAIDNFMVHELDGTQNEWGWCKQKLGANAILAVS?379
KGVSKAVGNVN+IIGPA?IGKDPTQQTAIDNFMVHELDGTQNEWGWCKQKLGANAILAVS
Sbjct：61??KGVSKAVGNVNNIIGPALIGKDPTQQTAIDNFMVHELDGTQNEWGWCKQKLGANAILAVS?120
Query：380?LAVCKAGAVVSGIPLYKHIANLAGNPKIVLPVPAFNVINGGSHAGNKLAMQEFMILPVGA?559
LAVCKAGAVVSGIPLYKHIANLAGNPKIVLPVPAFNVINGGSHAGNKLAMQEFMILPVGA
Sbjct：121?LAVCKAGAVVSGIPLYKHIANLAGNPKIVLPVPAFNVINGGSHAGNKLAMQEFMILPVGA?180
Query：560?SSFKEAMKMGVEVYHNLKSVIKKKYGQDATNVGDEGGFAPNIQENKEGLELLKTAIEKAG?739
+SFKEAMKMGVEVYH+LKSVIKKKYGQDATNVGDEGGFAPNIQENKEGLELLKTAIEKAG
Sbjct：181?ASFKEAMKMGVEVYHHLKSVIKKKYGQDATNVGDEGGFAPNIQENKEGLELLKTAIEKAG?240
Query：740?YTGKVVIGMDVAASEFYSSDKTYDLNFKEENNNGSQKISGDALKDLYKSFVAEYPIVSIE?919
YTGKVVIGMDVAASEFYS?DKTYDLNFKEENNNGSQKISGDALKDLYKSFVAEYPIVSIE
Sbjct：241??YTGKVVIGMDVAASEFYSEDKTYDLNFKEENNNGSQKISGDALKDLYKSFVAEYPIVSIE?300
Query：920??DPFDQDDWEHYAKMTAECGDNVQIVGDDLLVTNPKGVAKAIAEKSCNALLLKVNQIGSVT?1099
DPFDQDDWEHYAKMT?ECG??VQIVGDDLLVTNPK?VAKAIAEKSCNALLLKVNQIGSVT
Sbjct：301??DPFDQDDWEHYAKMTTECGTEVQIVGDDLLVTNPKRVAKAIAEKSCNALLLKVNQIGSVT?360
Query：1100?ESIEAVKMSKRAGWGVMASHRSGETEDTFIADLSVGLSTGQIKTGAPCRSERLAKYNQLL?1279
ESIEAVKMSK+AGWGVM?SHRSGETEDTFIADL+VGLSTGQIKTGAPCRSERLAKYNQLL
Sbjct：361??ESIEAVKMSKKAGWGVMTSHRSGETEDTFIADLAVGLSTGQIKTGAPCRSERLAKYNQLL?420
Query：1280?RIEEELGSEAVYAGANFRKPVEPY?1351
RIEEELGSEA+YAG?NFRKPVEPY
Sbjct：421??RIEEELGSEAIYAGVNFRKPVEPY?444
Query: the aminoacid sequence of Chinese cabbage enolase
Sbjct: the aminoacid sequence of Arabidopis thaliana enolase (NP_181192)
Table 3 is that the homology of the aminoacid sequence of Chinese cabbage enolase protein of the present invention and Arabidopis thaliana enolase protein compares (FASTA) table.Wherein, identical amino acid marks with the amino acid monocase between two sequences.
The analogue of invention Chinese cabbage enolase protein or polypeptide.The difference of these analogues and natural Chinese cabbage enolase related 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.
(the not changing primary structure usually) form of modification 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 anti-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 Chinese cabbage enolase polypeptide of the present invention, Chinese cabbage enolase encoding sequence operationally can be connected in expression regulation sequence, thereby form Chinese cabbage enolase 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 Chinese cabbage enolase gene product, the existence of rna transcription thing in cell of promptly analyzing the Chinese cabbage enolase.
In addition, the nucleic acid molecule that can be used as probe provided by the invention, this molecule have 8-100 continuous nucleotide of Chinese cabbage enolase nucleotide coding sequence usually, preferably have 15-50 continuous nucleotide.This probe can be used for whether existing in the test sample the relevant nucleic acid molecule of coding Chinese cabbage enolase.
The method that whether has Chinese cabbage enolase related nucleotide sequences in the test sample of the present invention, 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 Chinese cabbage enolase associated nucleotide encoding 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 Chinese cabbage enolase nucleotide sequence of the present invention and aminoacid sequence, can be on the homology basis of nucleic acid homology or marking protein, relevant homologous gene of screening Chinese cabbage enolase or homologous protein.
In order to obtain the dot matrix of the Chinese cabbage cDNAs relevant with Chinese cabbage enolase genes involved, can screen Chinese cabbage cDNA library with dna probe, these probes are under low rigorous condition, use
32P relevant all or part of of Chinese cabbage enolase cooked the radioactivity mark and.The cDNA library that most is suitable for screening is the library from Chinese cabbage.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 of the gene family relevant with the Chinese cabbage enolase.
Chinese cabbage enolase associated nucleotide 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 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 Chinese cabbage enolase protein of the present invention,, can filter out the interactional material of relevant generation with the Chinese cabbage enolase, perhaps acceptor, inhibitor or antagonist etc. by various conventional screening methods.
The objective of the invention is to overcome deficiency of the prior art, a kind of Chinese cabbage enolase protein encoding sequence be provided, it is coerced in the anti-anaerobism of vegetable cell have tangible effect,
The present invention has substantive distinguishing features and marked improvement, and the present invention coerces in the anti-anaerobism of vegetable cell has tangible effect, can obviously improve the resistance under the vegetable cell anoxia condition, and the reduction anaerobism is coerced the infringement to plant.Therefore, the present invention has very big using value.
Embodiment
Below in conjunction with the concrete data of laboratory test, further set forth the present invention with specific embodiment:
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.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.
Embodiment 1
The clone of Chinese cabbage enolase gene
1. separate tissue (isolation)
Place 28 ℃ to germinate 24 hours in Chinese cabbage (kind is " spring king ") seed, be seeded in then in the greenhouse, when treating that the Chinese cabbage blade is the 3-5 sheet, prepare DNA extraction or RNA.
2.RNA separation (RNA isolation)
Get portion of tissue, grind, add the 1.5mL EP pipe that fills lysate, fully after the vibration, move in the glass homogenizer again with mortar.Move in the 1.5mL EP pipe extracted total RNA (CTAB method) after the homogenate.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 Arabidopis thaliana enolase gene, utilize homologous genes clone principle, adopt RACE method (GibcoBRL test kit) to carry out the cDNA full-length clone, divide three phases to carry out:
(1)RT-PCR
PCR[MA001 (SEQ ID NO.1)+MA002 (SEQ ID NO.2)] obtain the fragment of 628bp, reclaim, be connected on the pGEMT-Easy carrier, with SP6 or T7 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 knowing its nucleotide sequence and proteins encoded and known cress such as Arabidopis thaliana (Arabidopsis assulta) enolase gene is very high, so think that tentatively it is an enolase gene.
(2)3’-RACE
PCR[AP+MA301 (5 '-GACCCATTTGACCAAGATGA-3 ')] obtain MA3 ' (660bp), reclaim, be connected on the T-Easy carrier, with SP6 or T7 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 knowing its nucleotide sequence and proteins encoded and known cress such as Arabidopis thaliana (Arabidopsis assulta) enolase gene is very high, so think that tentatively it is a gene relevant with the enolase enolase.
(3)5’-RACE
First round PCR [AAP+MA501 (5 '-TGTGCTTGTAGAGAGGAATG-3 ')]
Second takes turns PCR[(AUAP+MA502 (5 '-GACAACAGCCCCAGCTTTGC-3 ')) obtain MA5 ' (about 409bp) (process is with (1))
With the overlap splicing of sequencing result, the fragment that discovery procedure (1) obtains is the complete coding region of this gene.
The gene that result's proof of BLAST newly obtains from Chinese cabbage really is a gene relevant with the Arabidopis thaliana enolase.
By being used in combination above-mentioned 3 kinds of methods, obtained the complete encoding sequence (SEQ ID NO.3) of candidate's Chinese cabbage enolase protein.
Embodiment 2
The sequence information of Chinese cabbage enolase gene and homology analysis
The length of the Chinese cabbage enolase full-length cDNA that the present invention is new is 1580bp, and detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 20-1354 position Nucleotide (1335 Nucleotide).Derive the aminoacid sequence of Chinese cabbage enolase according to full-length cDNA, totally 444 amino-acid residues, molecular weight is 47380.23 dalton, iso-electric point (pI) is 5.46.Detailed sequence is seen SEQ ID NO.3.
The full length cDNA sequence and the coded protein thereof of Chinese cabbage enolase 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 arabidopsis gene enolase (NM 129209) have 91% homogeny, (subordinate list 2) on nucleotide level; On amino acid levels, it and Arabidopis thaliana enolase gene (NP 181192) also have 95% homogeny (subordinate list 3).This shows that all there are higher homology in Chinese cabbage gene enolase and arabidopsis gene enolase on nucleic acid still is protein level.Arabidopsis gene enolase (NM 129209) has been proved to be under the anaerobism stress conditions obviously to strengthen and has expressed, and the effect wanted emphatically of performance, can think that Chinese cabbage gene enolase also has similar effect in anti-anaerobism aspect coercing.
Embodiment 3
Chinese cabbage gene enolase protein or polypeptide carry out the anti-anaerobism of eukaryotic cell expression and transfer-gen plant in tobacco identifies
The structure that contains the expression vector of goal gene (Chinese cabbage gene enolase)
Full length sequence (SEQ ID NO.3) according to Chinese cabbage gene enolase, design amplifies the primer that complete coding is read frame, and on the upstream and downstream primer, introduce restriction endonuclease sites (this is decided by the 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, cDNA is cloned into intermediate carrier (as pBluescript) with Chinese cabbage gene enolase, further be cloned into binary expression vector (as pBI121 and improved pCAMBIA2300), guaranteeing to identify good expression vector under the correct prerequisite of reading frame, again it is changed in the Agrobacterium, utilize leaf dish law technology transformation mode plant tobacco.
1. send out seedling: seed is with rinsed with sterile water 15-20 minute, uses 70% ethanol disinfection again 1 minute, sterilizes 10-12 minute with 0.1% mercuric chloride then.Use aseptic water washing 5 times at last again.Washed seed is blotted with thieving paper, put into the MS substratum.Illumination cultivation 5 days is treated that seedling is long just can cut seedling to 4-5 centimetre.
2. cut seedling: clip 0.5-1 centimetre hypocotyl small segment is put into pre-culture medium, cultivates 2 days.
Pre-culture medium: MS+6BA (0.2mg/l)+2.4D (1.2mg/l)
3. transform altogether and cultivate: the hypocotyl that will cultivate in advance 2 days is put into prior cultured bacterium liquid (OD value 0.4-0.6) and infected 3-5 minute.Then take out and put into the dark cultivation of pre-culture medium 2 days.
4. screening and culturing: cultivate altogether finish after, explant is put into screening culture medium.2 all subcultures once.The differentiation of callus formation and bud is arranged in 2-4 week.After treating green bud length to 2 centimetre, cutting-out is taken root.
Screening culture medium: MS+6BA (4.5mg/l)+NAA (0.1mg/l)+AgNO3 (6mg/l)+cb (250mg/l)+Kan (20mg/l)
Root media: MS+NAA (0.5mg/l)+cb (250mg/l)+Kan (5mg/l)
5. transformed plant is cultivated; After treating 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.
The anti-anaerobism that contains the transgenic tobacco plant of Chinese cabbage gene enolase is identified
In view of the coding enolase, be proved to be in anti-anaerobism as the enolase gene of Arabidopis thaliana and played a role aspect coercing, and the enolase of Chinese cabbage gene enolase transcriptional level and Arabidopis thaliana has higher homology, can further carry out anti-anaerobism to the transgenic tobacco plant that contains Chinese cabbage gene enolase and identify.With the seed of 100mM sodium-chlor and 4 ℃ of subzero treatment transfer-gen plants and transfer-gen plant (2m, 15m, 30m, 1 hour, 3 hours, 7 hours, 12 hours, 24 hours) back research enolase gene in transfer-gen plant expression and various processing to the growing state of plant.Northern blot analytical results proves, transfer-gen plant enolase transcriptional level is handled its expression amount of back at salt and descended, and its expression amount significantly strengthens after the subzero treatment, though and contrast non-transgenic plant expression amount also changes, be starkly lower than transfer-gen plant.The non-transgenic plant strain growth is slow in addition, and is withered and dead under salt and subzero treatment at last, and transgenic plant still can normal growth, and are just slow slightly than undressed plant strain growth.This proof Chinese cabbage gene enolase has more effective and the function in degeneration-resistant border widely than the enolase gene of Arabidopis thaliana, will can be used for utilizing in anti-anaerobic research of transgenic technology improvement plant and the industrialization production.
Embodiment 4
The copy number analysis of Chinese cabbage gene enolase in Chinese cabbage
Adopt ordinary method from the Chinese cabbage blade, to extract DNA (with reference to " molecular cloning ", Sambrook etc., 1989), cut DNA[20 μ g (microgram)/sample with BamH, EcoRI, XhoI and KpnI enzyme respectively] after, go to DNA on the Hybond membrane (nylon membrane) after.Use the Amersham Pharmacia Gene Images of company
TMContents CDP-Star
TMLabelling module (PRN3540), we are labeled as probe with the enolase gene coding region, hybridize (in 60 ℃ of hybridization 16 hours) then.Take out film, place film washing liquid I (1*SSC, 1%SDS) in, in 60 ℃ of rinsings 3 times, each 15 minutes.Change over to film washing liquid II (0.1*SSC, 1%SDS) in 60 ℃ of rinsings 3 times, each same 15 minutes.With X-ray sheet compressing tablet 60-90 minute, develop then, photographic fixing (method is with reference to Roche DIG labeled test kit specification sheets).Result (Southern blot) finds one to three different hybridization band to occur on Hybond membrane, illustrates that enolase gene is low copy gene in Chinese cabbage.
Embodiment 5
The expression pattern analysis that the different time of Chinese cabbage gene enolase under low temperature and condition of salt stress handled
1.RNA extraction: the Chinese cabbage seedling of the 4-5 sheet leaf of will having grown through 100mMNaCl and subzero treatment 2m, 15m, 30m, 1 hour, 3 hours, 7 hours, 12 hours and 24 hours, use TRIzol test kit (GIBCO BRL then, USA) extract also with reference to " molecular cloning " preparation chapters and sections (Sambrook etc., 1989) about RNA.
2.RNA quantitatively: with reference to " molecular cloning " (Sambrook etc., 1989), spectrophotometric instrumentation OD
260Rna content calculates: 1 OD
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) 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: 1) before the transfer, 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 the 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 the transfer, 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 1) hybridization solution in, 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, development, photographic fixing (method is with reference to Roche DIGlabeled test kit specification sheets) then.Northern hybridization shows: along with the low temperature and the prolongation in salt treatment time, the expression of enolase progressively strengthens respectively and weakens, and illustrates that enolase is adverse circumstance signal such as low temperature and salt abduction delivering, plays the part of important role in the anti-anaerobism of Chinese cabbage is coerced.
Sequence that the present invention relates to and mark apportion are as follows:
(1) information of SEQ ID NO.1
(i) sequence signature:
(A) length: 20bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: oligonucleotide
(iii). sequence description: SEQ ID NO.1
TGG?TGC?AAG?CAA?AAG?CTT?GG
(2) information of SEQ ID NO.2
(i) sequence signature:
(A) length: 20bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: oligonucleotide
(iii). sequence description: SEQ ID NO.2
GGT?CAT?CTT?AGC?ATA?GTG?CT
(3) information of SEQ ID NO.3
(i) sequence signature:
(A) length: 1554bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: Nucleotide
(iii) sequence description: SEQ ID NO.3
＜110〉Shanghai Communications University
＜120〉Chinese cabbage enolase protein encoding sequence
<160>2
<170>PatentIn?version?3.1
<210>1
<211>1580
<212>DNA
＜213〉Chinese cabbage (Brassica campestris)
<220>
<221>CDS
<222>(20)..(1354)
<223>
<400>1
tccagatctc?tactcaacc?atg?gcc?act?atc?acc?gct?gtt?aaa?gct?agg?cag????52
Met?Ala?Thr?Ile?Thr?Ala?Val?Lys?Ala?Arg?Gln
1???????????????5???????????????????10
atc?ttc?gac?agc?cgt?ggc?aat?ccc?acg?gtt?gag?gtt?gat?gta?cac?aca????100
Ile?Phe?Asp?Ser?Arg?Gly?Asn?Pro?Thr?Val?Glu?Val?Asp?Val?His?Thr
15??????????????????20??????????????????25
tca?agt?ggt?gtt?aag?gtt?aca?gca?gcg?gtt?ccg?agt?gga?gct?tcc?acc????148
Ser?Ser?Gly?Val?Lys?Val?Thr?Ala?Ala?Val?Pro?Ser?Gly?Ala?Ser?Thr
30??????????????????35??????????????????40
ggt?atc?tac?gag?gct?ctt?gag?ctt?agg?gat?ggt?gga?tca?gac?tac?ctt??????196
Gly?Ile?Tyr?Glu?Ala?Leu?Glu?Leu?Arg?Asp?Gly?Gly?Ser?Asp?Tyr?Leu
45??????????????????50??????????????????55
gga?aag?ggt?gtc?tct?aag?gct?gtt?ggc?aat?gtg?aac?tcc?atc?atc?ggc??????244
Gly?Lys?Gly?Val?Ser?Lys?Ala?Val?Gly?Asn?Val?Asn?Ser?Ile?Ile?Gly
60??????????????????65??????????????????70??????????????????75
cca?gct?tcg?att?gga?aaa?gac?cca?act?cag?cag?act?gct?att?gac?aac??????292
Pro?Ala?Ser?Ile?Gly?Lys?Asp?Pro?Thr?Gln?Gln?Thr?Ala?Ile?Asp?Asn
80??????????????????85??????????????????90
ttc?atg?gtc?cat?gaa?ctt?gat?gga?acc?cag?aac?gaa?tgg?gga?tgg?tgc??????340
Phe?Met?Val?His?Glu?Leu?Asp?Gly?Thr?Gln?Asn?Glu?Trp?Gly?Trp?Cys
95??????????????????100?????????????????105
aag?caa?aag?ctt?gga?gcc?aat?gct?att?ctt?gct?gta?tct?cta?gct?gtc??????388
Lys?Gln?Lys?Leu?Gly?Ala?Asn?Ala?Ile?Leu?Ala?Val?Ser?Leu?Ala?Val
110?????????????????115?????????????????120
tgc?aaa?gct?ggg?gct?gtt?gtc?agt?ggc?att?cct?ctc?tac?aag?cac?att??????436
Cys?Lys?Ala?Gly?Ala?Val?Val?Ser?Gly?Ile?Pro?Leu?Tyr?Lys?His?Ile
125?????????????????130?????????????????135
gcc?aat?ctt?gct?ggt?aac?cct?aag?att?gtg?cta?cca?gtt?cct?gcg?ttc??????484
Ala?Asn?Leu?Ala?Gly?Asn?Pro?Lys?Ile?Val?Leu?Pro?Val?Pro?Ala?Phe
140?????????????????145?????????????????150?????????????????155
aac?gtt?atc?aat?ggt?gga?tcc?cat?gcc?gga?aac?aag?ctc?gcc?atg?cag??????532
Asn?Val?Ile?Asn?Gly?Gly?Ser?His?Ala?Gly?Asn?Lys?Leu?Ala?Met?Gln
160?????????????????165?????????????????170
gag?ttt?atg?atc?ctc?cct?gtt?gga?gct?tct?tct?ttc?aaa?gaa?gcc?atg??????580
Glu?Phe?Met?Ile?Leu?Pro?Val?Gly?Ala?Ser?Ser?Phe?Lys?Glu?Ala?Met
175?????????????????180?????????????????185
aaa?atg?ggt?gtt?gaa?gtt?tac?cac?aac?ttg?aag?tct?gtg?att?aag?aag??????628
Lys?Met?Gly?Val?Glu?Val?Tyr?His?Asn?Leu?Lys?Ser?Val?Ile?Lys?Lys
190?????????????????195?????????????????200
aag?tat?gga?cag?gat?gcc?acc?aac?gtc?ggt?gat?gaa?ggg?ggc?ttt?gca??????676
Lys?Tyr?Gly?Gln?Asp?Ala?Thr?Asn?Val?Gly?Asp?Glu?Gly?Gly?Phe?Ala
205?????????????????210?????????????????215
cca?aac?att?caa?gaa?aac?aag?gaa?ggt?ctt?gaa?ctg?ctt?aag?act?gct??????724
Pro?Asn?Ile?Gln?Glu?Asn?Lys?Glu?Gly?Leu?Glu?Leu?Leu?Lys?Thr?Ala
220?????????????????225?????????????????230?????????????????235
atc?gag?aag?gct?gga?tac?act?ggc?aag?gtt?gtc?att?ggg?atg?gat?gtt??????772
Ile?Glu?Lys?Ala?Gly?Tyr?Thr?Gly?Lys?Val?Val?Ile?Gly?Met?Asp?Val
240?????????????????245?????????????????250
gcc?gct?tct?gag?ttc?tac?tca?tct?gac?aag?act?tac?gac?ttg?aac?ttc??????820
Ala?Ala?Ser?Glu?Phe?Tyr?Ser?Ser?Asp?Lys?Thr?Tyr?Asp?Leu?Asn?Phe
255?????????????????260?????????????????265
aaa?gaa?gag?aac?aac?aat?ggt?tct?cag?aag?att?tct?gga?gat?gct?ctc??????868
Lys?Glu?Glu?Asn?Asn?Asn?Gly?Ser?Gln?Lys?Ile?Ser?Gly?Asp?Ala?Leu
270?????????????????275?????????????????280
aag?gac?ctg?tac?aag?tcc?ttc?gtt?gct?gag?tac?cca?att?gtg?tcc?att?????916
Lys?Asp?Leu?Tyr?Lys?Ser?Phe?Val?Ala?Glu?Tyr?Pro?Ile?Val?Ser?Ile
285?????????????????290?????????????????295
gag?gac?cca?ttt?gac?caa?gat?gac?tgg?gag?cac?tat?gct?aag?atg?acc??????964
Glu?Asp?Pro?Phe?Asp?Gln?Asp?Asp?Trp?Glu?His?Tyr?Ala?Lys?Met?Thr
300?????????????????305?????????????????310?????????????????315
gcc?gag?tgt?gga?gac?aat?gtt?cag?att?gtc?ggt?gat?gat?ttg?ttg?gtc?????1012
Ala?Glu?Cys?Gly?Asp?Asn?Val?Gln?Ile?Val?Gly?Asp?Asp?Leu?Leu?Val
320?????????????????325?????????????????330
acc?aac?ccc?aag?gga?gtt?gcc?aag?gca?atc?gca?gaa?aag?tct?tgc?aat?????1060
Thr?Asn?Pro?Lys?Gly?Val?Ala?Lys?Ala?Ile?Ala?Glu?Lys?Ser?Cys?Asn
335?????????????????340?????????????????345
gct?ctt?ctc?ttg?aag?gtt?aat?caa?atc?ggg?tca?gta?aca?gag?agt?att?????1108
Ala?Leu?Leu?Leu?Lys?Val?Asn?Gln?Ile?Gly?Ser?Val?Thr?Glu?Ser?Ile
350?????????????????355?????????????????360
gag?gca?gtg?aag?atg?tca?aag?aga?gca?gga?tgg?gga?gtg?atg?gct?agc?????1156
Glu?Ala?Val?Lys?Met?Ser?Lys?Arg?Ala?Gly?Trp?Gly?Val?Met?Ala?Ser
365?????????????????370?????????????????375
cac?cga?agt?ggt?gaa?acc?gag?gac?acc?ttc?atc?gct?gac?tta?tcc?gtt?????1204
His?Arg?Ser?Gly?Glu?Thr?Glu?Asp?Thr?Phe?Ile?Ala?Asp?Leu?Ser?Val
380?????????????????385?????????????????390?????????????????395
ggc?ttg?tct?act?gga?caa?atc?aag?act?gga?gct?cct?tgc?aga?tcc?gag?????1252
Gly?Leu?Ser?Thr?Gly?Gln?Ile?Lys?Thr?Gly?Ala?Pro?Cys?Arg?Ser?Glu
400?????????????????405?????????????????410
cgt?ctt?gcc?aag?tac?aac?cag?ctt?ttg?cgt?att?gag?gag?gag?ttg?gga?????1300
Arg?Leu?Ala?Lys?Tyr?Asn?Gln?Leu?Leu?Arg?Ile?Glu?Glu?Glu?Leu?Gly
415?????????????????420?????????????????425
tca?gag?gca?gtt?tac?gct?gga?gcc?aac?ttc?cgc?aag?cct?gtg?gag?ccc????1348
Ser?Glu?Ala?Val?Tyr?Ala?Gly?Ala?Asn?Phe?Arg?Lys?Pro?Val?Glu?Pro
430?????????????????435?????????????????440
tac?tag?aagaagtaca?agcttttttg?aagcaaagtg?gtcttttgtg?acgcgaagag?????1404
Tyr
gatgagagtt?gttttggtca?ttttgcttaa?ataaaacact?acgcttctgt?tttttctgtg??1464
tttatgattt?gaacccttat?ttttgtaaga?gaaacctgag?atagctttgt?ctttatagac??1524
aacagcagcg?ttcttttata?atataatttc?ggctttactc?aaaaaaaaaa?aaaaaa??????1580
<210>2
<211>444
<212>PRT
＜213〉Chinese cabbage (Brassica campestris)
<400>2
Met?Ala?Thr?Ile?Thr?Ala?Val?Lys?Ala?Arg?Gln?Ile?Phe?Asp?Ser?Arg
l???????????????5???????????????????10??????????????????15
Gly?Asn?Pro?Thr?Val?Glu?Val?Asp?Val?His?Thr?Ser?Ser?Gly?Val?Lys
20??????????????????25??????????????????30
Val?Thr?Ala?Ala?Val?Pro?Ser?Gly?Ala?Ser?Thr?Gly?Ile?Tyr?Glu?Ala
35??????????????????40??????????????????45
Leu?Glu?Leu?Arg?Asp?Gly?Gly?Ser?Asp?Tyr?Leu?Gly?Lys?Gly?Val?Ser
50??????????????????55??????????????????60
Lys?Ala?Val?Gly?Asn?Val?Asn?Ser?Ile?Ile?Gly?Pro?Ala?Ser?Ile?Gly
65??????????????????70??????????????????75??????????????????80
Lys?Asp?Pro?Thr?Gln?Gln?Thr?Ala?Ile?Asp?Asn?Phe?Met?Val?His?Glu
85??????????????????90??????????????????95
Leu?Asp?Gly?Thr?Gln?Asn?Glu?Trp?Gly?Trp?Cys?Lys?Gln?Lys?Leu?Gly
100?????????????????105?????????????????110
Ala?Asn?Ala?Ile?Leu?Ala?Val?Ser?Leu?Ala?Val?Cys?Lys?Ala?Gly?Ala
115?????????????????120?????????????????125
Val?Val?Ser?Gly?Ile?Pro?Leu?Tyr?Lys?His?Ile?Ala?Asn?Leu?Ala?Gly
130?????????????????135?????????????????140
Asn?Pro?Lys?Ile?Val?Leu?Pro?Val?Pro?Ala?Phe?Asn?Val?Ile?Asn?Gly
145?????????????????150?????????????????155?????????????????160
Gly?Ser?His?Ala?Gly?Asn?Lys?Leu?Ala?Met?Gln?Glu?Phe?Met?Ile?Leu
165?????????????????170?????????????????175
Pro?Val?Gly?Ala?Ser?Ser?Phe?Lys?Glu?Ala?Met?Lys?Met?Gly?Val?Glu
180?????????????????185?????????????????190
Val?Tyr?His?Asn?Leu?Lys?Ser?Val?Ile?Lys?Lys?Lys?Tyr?Gly?Gln?Asp
195?????????????????200?????????????????205
Ala?Thr?Asn?Val?Gly?Asp?Glu?Gly?Gly?Phe?Ala?Pro?Asn?Ile?Gln?Glu
210?????????????????215?????????????????220
Asn?Lys?Glu?Gly?Leu?Glu?Leu?Leu?Lys?Thr?Ala?Ile?Glu?Lys?Ala?Gly
225?????????????????230?????????????????235?????????????????240
Tyr?Thr?Gly?Lys?Val?Val?Ile?Gly?Met?Asp?Val?Ala?Ala?Ser?Glu?Phe
245?????????????????250?????????????????255
Tyr?Ser?Ser?Asp?Lys?Thr?Tyr?Asp?Leu?Asn?Phe?Lys?Glu?Glu?Asn?Asn
260?????????????????265?????????????????270
Asn?Gly?Ser?Gln?Lys?Ile?Ser?Gly?Asp?Ala?Leu?Lys?Asp?Leu?Tyr?Lys
275?????????????????280?????????????????285
Ser?Phe?Val?Ala?Glu?Tyr?Pro?Ile?Val?Ser?Ile?Glu?Asp?Pro?Phe?Asp
290?????????????????295?????????????????300
Gln?Asp?Asp?Trp?Glu?His?Tyr?Ala?Lys?Met?Thr?Ala?Glu?Cys?Gly?Asp
305?????????????????310?????????????????315?????????????????320
Asn?Val?Gln?Ile?Val?Gly?Asp?Asp?Leu?Leu?Val?Thr?Asn?Pro?Lys?Gly
325?????????????????330?????????????????335
Val?Ala?Lys?Ala?Ile?Ala?Glu?Lys?Ser?Cys?Asn?Ala?Leu?Leu?Leu?Lys
340?????????????????345?????????????????350
Val?Asn?Gln?Ile?Gly?Ser?Val?Thr?Glu?Ser?Ile?Glu?Ala?Val?Lys?Met
355?????????????????360?????????????????365
Ser?Lys?Arg?Ala?Gly?Trp?Gly?Val?Met?Ala?Ser?His?Arg?Ser?Gly?Glu
370?????????????????375?????????????????380
Thr?Glu?Asp?Thr?Phe?Ile?Ala?Asp?Leu?Ser?Val?Gly?Leu?Ser?Thr?Gly
385?????????????????390?????????????????395?????????????????400
Gln?Ile?Lys?Thr?Gly?Ala?Pro?Cys?Arg?Ser?Glu?Arg?Leu?Ala?Lys?Tyr
405?????????????????410?????????????????415
Asn?Gln?Leu?Leu?Arg?Ile?Glu?Glu?Glu?Leu?Gly?Ser?Glu?Ala?Val?Tyr
420?????????????????425?????????????????430
Ala?Gly?Ala?Asn?Phe?Arg?Lys?Pro?Val?Glu?Pro?Tyr
435?????????????????440
Claims (4)
1, a kind of Chinese cabbage anaerobic enolase protein encoding sequence, it is characterized in that, isolated dna molecular, this molecule comprises: coding has the nucleotide sequence of the active polypeptide of Chinese cabbage enolase protein matter, and shows at least 70% homology from the nucleotides sequence of Nucleotide 20-1354 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps described nucleotide sequence can with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 20-1354 position, described encoding sequence has the polypeptide of the aminoacid sequence shown in the SEQ ID NO.3, or its conservative property variation polypeptide or its active fragments, or its reactive derivative, described encoding sequence has among the SEQID NO.3 nucleotide sequence from Nucleotide 20-1354 position.
2, Chinese cabbage anaerobic enolase protein encoding sequence according to claim 1, it is characterized in that the anti-anaerobism of the isolated Chinese cabbage of institute is coerced class related protein polypeptide, it comprises: the polypeptide with SEQ ID NO.3 aminoacid sequence, this polypeptide is to have SEQ ID NO.3 polypeptide of sequence
3, Chinese cabbage anaerobic enolase protein encoding sequence according to claim 1 is characterized in that, its carrier, and it comprises described dna molecular, a kind of nucleic acid molecule, it comprises 8-100 continuous nucleotide in the described dna molecular.
4, Chinese cabbage anaerobic enolase protein encoding sequence according to claim 1 is characterized in that, described dna molecular, and transformed host cells, it is an eukaryotic cell.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974537A (en) * | 2010-09-13 | 2011-02-16 | 华中农业大学 | Maize water-logging tolerance-related transcription factor gene zm-bRLZ, molecular marker and application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974537A (en) * | 2010-09-13 | 2011-02-16 | 华中农业大学 | Maize water-logging tolerance-related transcription factor gene zm-bRLZ, molecular marker and application |
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