CN1584032A - Protein coding sequence of protein kinase 3 activated by cell division protoplasm for brassia rape - Google Patents
Protein coding sequence of protein kinase 3 activated by cell division protoplasm for brassia rape Download PDFInfo
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- CN1584032A CN1584032A CN 200410024695 CN200410024695A CN1584032A CN 1584032 A CN1584032 A CN 1584032A CN 200410024695 CN200410024695 CN 200410024695 CN 200410024695 A CN200410024695 A CN 200410024695A CN 1584032 A CN1584032 A CN 1584032A
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Abstract
A cabbage rape antireversal signal transmitting kinase BnMPK3 protein coding sequence includes: coding polypeptide ribonucleotide sequence with cabbage rape BnMPK3 protein activity, having at least 70% homology for the ribonucleotide sequence and SEQ ID NO.3 of ribonucleotide sequence from No.154-1263 of ribonucleotide; the ribonucleotide sequence hybridizing with SEQ ID NO.3 of ribonucleotide sequence from No.154-1263 of ribonucleotide. It achieves drought-resistant and zoophobous function.
Description
Technical field
What the present invention relates to is a kind of albumen coded sequence that is used for technical field of biological genetic engineering, the former activated protein kinase 3 of particularly a kind of swede type rape cell fission (BnMPK3) albumen coded sequence.
Background technology
Farm crop can run into various natural disasteies and cause the underproduction in process of growth, as arid, stagnant waterlogging and disease and pest etc.When previous important breeding technique be various adversity genes are carried out farm crop genetic engineering modified, to obtain degeneration-resistant new crop varieties.Plant is coerced for the bad external environment of opposing, developed complicated signal transduction system and help oneself to spend current predicament in secular evolution naturally, wherein map kinase (Mitogen-actived protein kinase) signal transduction system has been played the part of important role in degeneration-resistant process.MAPKs swashs (MAPK) and map kinase kinases (mitogen-activated proteinkinase kinase by MAP, MAPKK) and map kinase kinase kinase (mitogen-activated pro2teinkinase kinase kinase, MAPKKK) 3 types of compositions.After vegetable cell is experienced environment-stress (as damage, arid, low temperature etc.) and the signal that grows (growth hormone, ethene, dormin etc.), can activate son by upstreams such as receptor protein kinases and activate MAPKKK, MAPKK, MAPK in turn.Wherein, MAPK is transcription factor and the functional gene that last kinases directly acts on the downstream in this transfer chain, makes plant show various anti-adversities, is a ring important in the plant signal transmittance process.
Through the prior art literature search is found, magazine " Proceeding Natural Academic Science, institute of United States of America U.S. natural science institute periodical " 2002,99 (24): 15812-1581717 has delivered article " Mitogen-activated protein kinase signaling inpostgermination arrest of development by abscisic acid (the plant hormone dormin is suppressing the plant germination later stage; the signal transmission of the former activated protein kinase of cell fission) ", abundant description has been done in expression under the plant hormone dormin is handled to gene M APK3, it is the positively related gene of dormin ABA abduction delivering, relevant with the flow of water of plant, but it be unclear that the salt tolerant of this gene, drought resisting and the pest-resistant effect that waits other environmental factors, not finding as yet so far in addition has the report of maintaining close ties with document with theme of the present invention.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, provide a kind of swede type rape cell fission former activated protein kinase 3 albumen coded sequences.The invention discloses and the closely-related protein gene of swede type rape BnMPK3 and swede type rape BnMPK3 protein sequence and nucleotide sequence thereof, and the application in utilizing transgenic technology improvement plant drought salt tolerant.
The present invention is achieved by the following technical solutions, at the isolated dna molecular of the present invention, this molecule comprises: coding has the nucleotide sequence of the polypeptide of swede type rape BnMPK3 protein active, and shows at least 70% homology from the nucleotides sequence of Nucleotide 154-1263 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 154-1263 position, described nucleotide coding 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 nucleotide coding sequence has among the SEQ IDNO.3 nucleotide sequence from Nucleotide 154-1263 position.
At the also isolated former activated protein kinase of the cell fission swede type rape MAPK class related protein polypeptide BnMPK3 that comprises of the present invention, 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, drought, cold, active oxygen and Whitfield's ointment are handled, and the ability of the various poor environments of opposing of plant is provided.
Described dna molecular is the dna molecular that carrier provided by the invention comprises, and is a kind of nucleic acid molecule, and it comprises 8-100 continuous nucleotide in the described dna molecular.
With described dna molecular transformed host cells, it is an eukaryotic cell in the present invention.
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, term " swede type rape degeneration-resistant signal transmit kinases " refer to the encode nucleotide sequence of polypeptide with swede type rape BnMPK3 protein-active is as 154-1263 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 154-1263 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 154-1263 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 moderate stringent condition (70-85% consistence), better height stringent condition (85% above consistence) down with SEQ ID NO.3 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 154-1263 position.This term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 154-1263 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.3 with natural swede type rape BnMPK3 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 " the encoding protein kinase sequence that the degeneration-resistant signal of swede type rape transmits " refers to have the SEQ ID NO.3 polypeptide of sequence of swede type rape BnMPK3 protein-active.This term also comprises having and the variant form relevant identical function of natural swede type rape BnMPK3, SEQ ID NO.4 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 proteic active fragments of swede type rape BnMPK3 and reactive derivative.
The variant form of swede type rape BnMPK3 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 swede type rape BnMPK3 under high or low rigorous condition are coded and the polypeptide or the albumen that utilize the antiserum(antisera) of swede type rape BnMPK3 polypeptide to obtain.
In the present invention, " swede type rape BnMPK3 conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO.3, have 10 at the most, preferably at the most 8, more preferably 5 amino acid is replaced by similar performance or close amino acid and is 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
90％identity?in?1113nt?overlap
Query：??150??gagagacatgaacaacgccggcggccaatatccagattttccggcggtgcagacgcacgg?209
||||||?|||||||?||?|||?||||||||??|?|||||||||||||||?|?||?|||||
Sbjct：??18???gagagaaatgaacaccggcggtggccaatacacggattttccggcggtggacactcacgg?77
Query：??210??aggacagttcataagctacgatatcttcggtagtttattcgaggtcacatccaagtaccg?269
|||||||||||||||?|||||||||||||||||||||||||||?|||||||?|||||?||
Sbjct：??78???aggacagttcataagttacgatatcttcggtagtttattcgagatcacatctaagtatcg?137
Query：270??tcctccgattgttccaatcggtcgaggcgcatacggcatcgtttgctctgtgttggattc?329
||||||||||?|?||||??|||||?||?||?||?||?|||||||||||||||||||||?|
Sbjct：138??tcctccgattataccaattggtcgtggagcttatggaatcgtttgctctgtgttggatac?197
Query：330??ggagacgaacgagctagtggcgatgaagaagatagccaacgccttcgataatcacatgga?389
||||||||||||||||||?|||||||||||||||||?||?||?||?||||||||?|||||
Sbjct：198??ggagacgaacgagctagtagcgatgaagaagatagctaatgcttttgataatcatatgga?257
Query：390??cgccaagcgtacacttcgcgagatcaagcttcttcgtcatctcgatcatgaaaacatcat?449
||??||||||||?|||||?|||||||||||||||||||||||?||||||||||||||?||
Sbjct：258??tgctaagcgtacgcttcgtgagatcaagcttcttcgtcatcttgatcatgaaaacattat?317
Query：450??agctataagagatgtggttcctccaccacttagaagacagttcagtgatgtttatatcgc?509
|||||||||||||||?|||||?||||||||?|||||||||||||||||||||||||||?|
Sbjct：318??agctataagagatgttgttccaccaccactaagaagacagttcagtgatgtttatatctc?377
Query：510??cactgagttaatggacactgatcttcatcaaatcatcagatccaaccagggcttgtctga?569
|||||?||||||||?||||||||||||||||||||||||||?||||||?|?||?||?||
Sbjct：378??tactgaattaatggatactgatcttcatcaaatcatcagatctaaccagagtttatcaga?437
Query：570??ggaacactgtcagtacttcctgtaccagcttcttcgagggctcaagtacatccactcggc?629
||||||||||||||||||?||||||||||?||||||||?||?|||||?||||||||?||
Sbjct：438??agaacactgtcagtacttcttgtaccagctacttcgaggactgaagtatatccactcagc?497
Query：630??taaagttatccacagggatctaaagcctagcaatcttctcctgaacgccaactgcgattt?689
|||??||||?||?||||||?|||||||?|||||||||||??|||||||?||?||||||||
Sbjct：498??taacattattcatagggatttaaagccgagcaatcttctgttgaacgcgaattgcgattt?557
Query：690??aaagatctgtgattttggtcttgctagacccacttcagagaacgagtttatgactgagta?749
||||||?||||||||?||||||||||||||?|||||||||||?||?||||||||||||||
Sbjct：558??aaagatttgtgatttcggtcttgctagacctacttcagagaatgattttatgactgagta?617
Query：750??tgttgttaccagatggtatagagcacctgagcttctgctgaactcatccgattacactgc?809
|||||||||?|||||||||||||||||||||||||||?|||||||?||?||||||||?||
Sbjct：618??tgttgttacgagatggtatagagcacctgagcttctgttgaactcttcagattacacagc?677
Query：810??tgctattgatgtttggtctgttggttgtatcttcatggagctcatgaacagaaagccttt?869
|||||||||||||||||||||||||||||||||?||||||||?|||||?|||||||||||
Sbjct：678??tgctattgatgtttggtctgttggttgtatctttatggagcttatgaatagaaagccttt?737
Query：870??gttccctggtaaagaccatgtccatcagatgcgcttattgacagagttgcttggcacacc?929
|||||||||||||||||||||?|||||?||||||||||||||||||||||||||||||||
Sbjct：738??gttccctggtaaagaccatgttcatcaaatgcgcttattgacagagttgcttggcacacc?797
Query：930??gacagaatcagatctcgggtttacacacaacgaggatgccaaaagatacatccggcagct?989
|||||||||?||||||||?|||||?|||||?||||||||?|||||||||||||||||?||
Sbjct：798??gacagaatctgatctcggttttactcacaatgaggatgcgaaaagatacatccggcaact?857
Query：990??tcccaacttccctcgccaaccgttagctaaactgttctctcatgttaactcattggccat?1049
||||||||||||?||?||?||??||||||||||?|||||||||||||||?||?|||||||
Sbjct：858??tcccaacttcccacgtcagcccttagctaaacttttctctcatgttaacccaatggccat?917
Query：1050?tgatctagttgacagaatgttgacgtttgaccccaacaaaagaatcactgttgaagaagc?1109
||||||?|||||||||||||||||||||||||||||||?||||||||||||||||?||||
Sbjct：918??tgatcttgttgacagaatgttgacgtttgaccccaacagaagaatcactgttgaacaagc?977
Query：1110?tctgaatcacccgtacctagccaagttgcacgacccgaatgatgagccaatctgtctaaa?1169
|||||||||||?||||||?||?||?|||||||||||||||||||||||||||||||?|||
Sbjct：978??tctgaatcaccagtaccttgctaaattgcacgacccgaatgatgagccaatctgtcaaaa?1037
Query：1170?gccattctctttcgactttgaacaacaacctctagacgaggaacagataaaagagatgat?1229
||||||||||||?||?||?||||||||?|||||?||?|||||||||||||||||||||||
Sbjct：1038?gccattctcttttgagttcgaacaacagcctctggatgaggaacagataaaagagatgat?1097
Query：1230?ctacagggaagccattgcactcaatccaacata?1262
||||???||||||||?|||||||||||||||||
Sbjct：1098?ctaccaagaagccatagcactcaatccaacata?1130
Query: the nucleotide sequence of swede type rape BnMPK3
Sbjct: the nucleotide sequence of Arabidopis thaliana AtMPK3 (AY090981)
Table 2 is that the homology of swede type rape BnMPK3 albumen of the present invention and the proteic nucleotide sequence of Arabidopis thaliana AtMPK3 compares (GAP) table.
Table 3
94％identity?in?370aa?overlap
Query：1????MNNAGGQYPDFPAVQTHGGQFISYDIFGSLFEVTSKYRPPIVPIGRGAYGIVCSVLDSET?60
MN??GGQY?DFPAV+THGGQFISYDIFGSLFE+TSKYRPPI+PIGRGAYGIVCSVLD+ET
Sbjct：1????MNTGGGQYTDFPAVETHGGQFISYDIFGSLFEITSKYRPPIIPIGRGAYGIVCSVLDTET?60
Query：61???NELVAMKKIANAFDNHMDAKRTLREIKLLRHLDHENIIAIRDVVPPPLRRQFSDVYIATE?120
NELVAMKKIANAFDNHMDAKRTLREIKLLRHLDHENIIAIRDVVPPPLRRQFSDVYI+TE
Sbjct：61???NELVAMKKIANAFDNHMDAKRTLREIKLLRHLDHENIIAIRDVVPPPLRRQFSDVYISTE?120
Query：121??LMDTDLHQIIRSNQGLSEEHCQYFLYQLLRGLKYIHSAKVIHRDLKPSNLLLNANCDLKI?180
LMDTDLHQIIRSNQ?LSEEHCQYFLYQLLRGLKYIHSA?+IHRDLKPSNLLLNANCDLKI
Sbjct：121??LMDTDLHQIIRSNQSLSEEHCQYFLYQLLRGLKYIHSANIIHRDLKPSNLLLNANCDLKI?180
Query：181??CDFGLARPTSENEFMTEYVVTRWYRAPELLLNSSDYTAAIDVWSVGCIFMELMNRKPLFP?240
CDFGLARPTSEN+FMTEYVVTRWYRAPELLLNSSDYTAAIDVWSVGCIFMELMNRKPLFP
Sbjct：181??CDFGLARPTSENDFMTEYVVTRWYRAPELLLNSSDYTAAIDVWSVGCIFMELMNRKPLFP?240
Query：241??GKDHVHQMRLLTELLGTPTESDLGFTHNEDAKRYIRQLPNFPRQPLAKLFSHVNSLAIDL?300
GKDHVHQMRLLTELLGTPTESDLGFTHNEDAKRYIRQLPNFPRQPLAKLFSHVN?+AIDL
Sbjct：241??GKDHVHQMRLLTELLGTPTESDLGFTHNEDAKRYIRQLPNFPRQPLAKLFSHVNPMAIDL?300
Query：301??VDRMLTFDPNKRITVEEALNHPYLAKLHDPNDEPICLKPFSFDFEQQPLDEEQIKEMIYR?360
VDRMLTFDPN+RITVE+ALNH?YLAKLHDPNDEPIC?KPFSF+FEQQPLDEEQIKEMIY+
Sbjct：301??VDRMLTFDPNRRITVEQALNHQYLAKLHDPNDEPICQKPFSFEFEQQPLDEEQIKEMIYQ?360
Query：361??EAIALNPTYA?370
EAIALNPTY
Sbjct：361??EAIALNPTYG?370
Query: the aminoacid sequence of swede type rape BnMPK3
Sbjct: the aminoacid sequence of Arabidopis thaliana AtMPK3 (CAB75493)
Table 3 is that the homology of the aminoacid sequence of swede type rape BnMPK3 albumen of the present invention and Arabidopis thaliana AtMPK3 compares (FASTA) table.Wherein, identical amino acid marks with the amino acid monocase between two sequences.
Invention also comprises the analogue of swede type rape BnMPK3 albumen or polypeptide.The difference of these analogues and natural swede type rape BnMPK3 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 swede type rape BnMPK3 polypeptide of the present invention, swede type rape BnMPK3 encoding sequence operationally can be connected in expression regulation sequence, thereby form swede type rape BnMPK3 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.
Also available Northern blotting and single stage method RT-PCR law technology are analyzed the expression of swede type rape BnMPK3 gene product, and whether and quantity the existence of rna transcription thing in cell of promptly analyzing swede type rape BnMPK3.
In addition, the present invention also provides a kind of nucleic acid molecule that can be used as probe, and this molecule has 8-100 continuous nucleotide of swede type rape BnMPK3 nucleotide coding sequence usually, preferably has 15-50 continuous nucleotide.This probe can be used for whether existing in the test sample the relevant nucleic acid molecule of coding swede type rape BnMPK3.
The present invention also provides the method that whether has swede type rape BnMPK3 related nucleotide sequences in the test sample, and 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 swede type rape BnMPK3 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 swede type rape BnMPK3 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 swede type rape BnMPK3 or homologous protein.
In order to obtain the dot matrix of the swede type rape cDNAs relevant with swede type rape BnMPK3 genes involved, can screen swede type rape cDNA library with dna probe, these probes are under low rigorous condition, use
32P relevant all or part of of swede type rape BnMPK3 cooked the radioactivity mark and.The cDNA library that most is suitable for screening is the library from swede type rape.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 swede type rape BnMPK3.
Swede type rape BnMPK3 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 swede type rape BnMPK3 albumen of the present invention,, can filter out the interactional material of relevant generation with swede type rape BnMPK3, perhaps acceptor, inhibitor or antagonist etc. by various conventional screening methods.
The present invention has substantive distinguishing features and marked improvement, and the present invention has tangible effect at anti-various environment-stress, can obviously be reduced in the loss of various farm crop on biological yield of planting on arid, water logging and the disease and pest soil.Arid, water logging and disease and pest can cause most of farm crop such as poor growth even death such as paddy rice, cotton, swede type rape and wheat, thereby make its obvious underproduction even No kernels or seeds are gathered, as in a year of scarcity.China exists large-area arid and semi-arid, saline and alkaline half saline and alkaline area, and also has arid and semi-arid, saline and alkaline half saline and alkaline area in the world wide, and the various agricultural chemicals of large-area in addition application are serious environment pollution also, and therefore, the present invention has very big using value.
Embodiment
Below in conjunction with test data in lab, 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 swede type rape BnMPK3 gene
1. separate tissue (isolation)
Swede type rape (kind is " a Shanghai oil 15 ") is buied from the market, places 28 ℃ to germinate 24 hours swede type rape, is seeded in then in the greenhouse, when treating that the swede type rape blade is the 3-5 sheet, prepares 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 to after the homogenate in the 1.5mL EP pipe, and extracted total RNA (TRIzol Reagents, GIBCO BRL, USA).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 AtMPK3 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[BP001 (SEQ ID NO.1)+BP002 (SEQ ID NO.2)] obtain PCR product (471bp) and reclaim, be connected on the pGEMT-Easy carrier, with SP6 or T7 as universal primer, adopt and stop thing fluorescent mark (Big-Dye, Perkin-Elmer, USA) method, (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 thaliana) AtMPK3 gene is very high, so think that tentatively it is a former activated protein kinase gene of cell fission.
(2)3’-RACE
PCR[AP+M301 (5 '-ATAGAGCACCTGAGCTTCTG-3 ')] obtain M3 ' (681bp), 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 thaliana) AtMPK3 gene is very high, so think that tentatively it is one and MAPK genes involved.
(3).5’-RACE
First round PCR[AAP+M501 (5 '-GCGTTCA (A/G) is AGAAGATTGCT-3 ' (C/G))]
Second takes turns PCR[(AUAP+M502 (5 '-TGGTGG (A/T) GGAAC (C/A) ACATCTC-3 ')] obtain M5 ' (about 473bp) (process with (1))
With the overlap splicing of sequencing result, find that the fragment that obtains is the complete coding region of this gene.
The gene that result's proof of BLAST newly obtains from swede type rape really is a gene relevant with Arabidopis thaliana AtMPK3.
By being used in combination above-mentioned 3 kinds of methods, obtained candidate's the proteic complete encoding sequence of swede type rape BnMPK3 (SEQ ID NO.3).
Embodiment 2
The sequence information and the homology analysis of swede type rape BnMPK3 gene
The length of the swede type rape BnMPK31 full-length cDNA that the present invention is new is 1464bp, and detailed sequence is seen SEQID NO.3, and wherein open reading frame is positioned at 154-1263 position Nucleotide (1114 Nucleotide).Derive the aminoacid sequence of swede type rape BnMPK3 according to full-length cDNA, totally 370 amino-acid residues, molecular weight is 42588.74 dalton, iso-electric point (pI) is 5.79.Detailed sequence is seen SEQ ID NO.3.
Full length cDNA sequence and the coded protein thereof of swede type rape BnMPK3 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 AtMPK3 have 90% homogeny on nucleotide level, (subordinate list 2); On amino acid levels, it and arabidopsis gene AtMPK3 (CAB75493) also have 94% homogeny (subordinate list 3).This shows that there are higher homology in swede type rape gene BnMPK3 and arabidopsis gene AtMPK3 on nucleic acid still is protein level.Arabidopsis gene AtMPK3 (CAB75493) has been proved to be obviously to strengthen under dehydration conditions and has expressed, and brings into play the effect of wanting emphatically, can think that swede type rape gene BnMPK3 also has similar effect aspect degeneration-resistant.
Embodiment 3
Swede type rape gene BnMPK3 albumen or polypeptide carry out the resistance of eukaryotic cell expression and transformed yeast in yeast identifies
The structure that contains the expression vector of goal gene (swede type rape gene BnMPK3 gene)
Full length sequence (SEQ ID NO.3) according to swede type rape gene BnMPK3, 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, with swede type rape gene BnMPK3 gene cDNA clone to expression vector (as pYES2.1), further transformed into escherichia coli TOP10 or DH5 α, guaranteeing to identify expression vector under the correct prerequisite of reading frame, utilize the LiAc method again with its transformed yeast INVSIC.
1. prepare: obtaining liq YPD substratum, 1 * TE, 1 * LiAc, 1 * LiAc/0.5 * TE, the salmon sperm dna of sex change comprises the pYES2.1 carrier of this gene, 1 * LiAc/40%/1 * TE does not contain the screening flat board of the substratum of uridylic and other reagent and vessel.
2. prepare the yeast competence: choose monoclonal yeast strain in the YPD substratum of 10ml, 30 ℃ are spent the night on shaking table.In the OD600 value is to be transferred in the 50ml YPD substratum in 0.4 o'clock to shake 2-4 hour.
Centrifugation cell under the 2500rpm suspends again with 1 * TE of 40ml.
Centrifugation cell under 2500rpm again with 1 * LiAc/0.5 * TE of 2ml suspension cell again, is placed 10min in room temperature.
3. transform: get the yeast suspension of 100 μ l, add the pYES2.1 carrier that 1 μ g built and the salmon sperm dna of 100 sex change, bathe 30min 30 ℃ of temperature then.
Add 88 μ l DMSO, mixing is then at 42 ℃ of heat shock 7min.
Centrifugal 10s precipitation yeast cell is removed supernatant liquor.Use 1ml 1 * TE to suspend again then.
The centrifugation yeast cell, resuspended with 50-100 μ l 1 * TE.
4. screening and culturing: above-mentioned resuspended liquid is tiled on the screening culture medium flat board.
5. the resistance of transformed yeast is identified: choose monoclonal yeast strain in the YPD substratum of 10ml, 30 ℃ are spent the night on shaking table.In the OD600 value is 0.4, and it is 1/3,1/9,1/27,1/81,1/243 that the yeast of getting 100 μ l dilutes respectively, gets 10 μ l then successively and selects on the YPGR culture medium flat plate that contains 600mM N.F,USP MANNITOL and 0.4mM t-BuOOH 30 ℃ of overnight incubation.
Contain the YPGR substratum of 600mM N.F,USP MANNITOL/liter: 1% yeast extract, 2% Tryptones, 2% agar, 2% semi-lactosi, 1% raffinose, 600mM N.F,USP MANNITOL
Contain the YPGR substratum of 0.4mM t-BuOOH/liter: 1% yeast extract, 2% Tryptones, 2% agar, 2% semi-lactosi, 1% raffinose, 0.4mM t-BuOOH
In view of coding BnMPK3, AtMPK3 gene as Arabidopis thaliana has been proved to be enhancing expression under dehydration conditions, and the AtMPK3 of swede type rape gene BnMPK3 transcriptional level and Arabidopis thaliana has higher homology, can further carry out resistance to the transformed yeast that contains swede type rape gene BnMPK3 and identify.Whether can improve the drought-resistant and oxidation resistant ability of zymic with research BnMPK3 gene behind 600mM N.F,USP MANNITOL and the 0.4mM t-BuOOH processing transformed yeast.The zymic growth result proves that transformed yeast BnMPK3 increment comparison on the substratum of 600mM N.F,USP MANNITOL is shone and handled many two orders of magnitude; The increment comparison is according to handling many orders of magnitude on the substratum of 0.4mM t-BuOOH.This proof swede type rape BnMPK3 gene can improve eukaryotic drought resisting and oxidation resistant ability really.And oxidation resistant ability is common and the ability of the waterlogging-resistant and disease and insect resistance of plant is closely related, so swede type rape BnMPK3 gene has more effective and the function in degeneration-resistant border widely, will can be used for utilizing during the research of the waterlogging and disease and insect resistance of transgenic technology improvement plant drought and industrialization produce.
Embodiment 4
The copy number analysis of swede type rape gene BnMPK3 in swede type rape
Adopt ordinary method from the swede type rape blade, to extract DNA (with reference to " molecular cloning ", Sambrook etc., 1989), cut DNA[13 μ g (microgram)/sample with HindIII Xba I BamH and EcoR I enzyme respectively] after, DNA is gone to after Hybond membrane (nylon membrane) goes up.Use the Amersham Pharmacia GeneImages of company
TMContents CDP-Star
TMLabelling module (PRN3540), we are labeled as probe with the BnMPK3 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 a spot of hybridization band to occur on Hybond membrane, illustrates that swede type rape gene BnMPK3 is low copy in swede type rape.
Embodiment 5
Swede type rape BnMPK3 gene is at NaCl, N.F,USP MANNITOL, H
2O
2The expression pattern analysis handled of different time
1.RNA extraction: the swede type rape seedling of the 3-5 sheet leaf of will having grown is through 200mM N.F,USP MANNITOL, 200mMNaCl and 200 μ M H
2O
2Handled 0 minute, 2 minutes, 5 minutes, 20 minutes, 1 hour, 2 hours, 6 hours, 12 hours and 24 hours, the RNA extraction agent box extracting RNA that provides according to Hua Shun company then, and carry out quantitatively.
2. adopt the single step reaction standard measure to carry out expression analysis, (TaKaRaBiotechnology Co., Ltd), concrete operations are carried out according to the test kit specification sheets to utilize One Step RNA PCR Kit.Each reaction comprises 2.5 μ l10 * One Step RNA PCR buffer, 5 μ l 25mM MgCl
25 μ l dNTP mixed solutions, 20U RNase inhibitor, 0.5U AMV Rtase XL, 0.5U AMV-Optimized Taq, special primer 1 (5 '-ATGAACAACGCCGGCGGCCA-3 ') and special primer 2 (5 '-CTAAGCATATGTTGGATTGAGTGC-3 ') each 10 μ Mol, the RNA 2 μ l that the various times handle replenish no RNase water then to 25 μ l.The PCR reaction conditions is as follows: 50 ℃ 30 minutes, 94 ℃ 2 minutes, carry out then 94 ℃ 30 seconds, totally 30 circulations in 1 minute of 60 ℃ of 30 seconds and 72 ℃, last 72 ℃ were extended 5 minutes.
3. after reaction finishes, get 15 μ l PCR reaction solutions and carry out electrophoresis in 1% sepharose, 5V/cm voltage electrophoresis was observed the DNA amount about 1 hour on gel imaging system.
The quantitative RT-PCR result shows that swede type rape BnMPK3 is at N.F,USP MANNITOL and H
2O
2Handle to begin after 5 minutes to start and express, after 6 hours, reach the climax in the treatment with mannitol, express subsequently and reduce; But H
2O
2Just reaching the climax in the processing after 2 hours reduces subsequently.Sodium-chlor can start the expression of BnMPK3 faster, promptly just starts at 2 minutes and expresses, and reached the climax reduce subsequently in 1 hour.These expression characteristic explanation swede type rapes BnMPK3 is typical signal transferrin, belongs to various adverse circumstances and handles the early expression gene, and start various and degeneration-resistant relevant expression of gene, improves the anti-adversity ability of plant.
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: 23bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: oligonucleotide
(iii). sequence description: SEQ ID NO.1
GCT(A/T/C/G)CTTCGTCA(C/T)(A/C/T)T(C/G/T)GATCATG
(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
GACCA(A/T)ACATC(A/G/T)AT(A/T/C/G)GC(A/T/C/G)GC
(3) information of SEQ ID NO.3
＜110〉Shanghai Communications University
＜120〉former activated protein kinase 3 albumen coded sequences of swede type rape cell fission
<130>2
<160>2
<170>PatentIn?version?3.2
<210>1
<211>1464
<212>DNA
<213>Brassica?napus
<220>
<221>CDS
<222>(154)..(1263)
<400>1
tcttctcatt?tcagtccctc?atctaactgt?aatataatta?tatatataaa?ggagtcacac????60
aataccttca?gattcactat?acctcaaagc?acaacaagtc?aagcttgaag?ttgacttagc???120
gcgtcatcga?ttgaaagaga?gagagagaga?gag?atg?aac?aac?gcc?ggc?ggc?caa????174
Met?Asn?Asn?Ala?Gly?Gly?Gln
1???????????????5
tat?cca?gat?ttt?ccg?gcg?gtg?cag?acg?cac?gga?gga?cag?ttc?ata?agc?????222
Tyr?Pro?Asp?Phe?Pro?Ala?Val?Gln?Thr?His?Gly?Gly?Gln?Phe?Ile?Ser
10?????????????????15??????????????????20
tac?gat?atc?ttc?ggt?agt?tta?ttc?gag?gtc?aca?tcc?aag?tac?cgt?cct?????270
Tyr?Asp?Ile?Phe?Gly?Ser?Leu?Phe?Glu?Val?Thr?Ser?Lys?Tyr?Arg?Pro
25?????????????????30?????????????????35
ccg?att?gtt?cca?atc?ggt?cgg?ggc?gca?tac?ggc?atc?gtt?tgc?tct?gtg?????318
Pro?Ile?Val?Pro?Ile?Gly?Arg?Gly?Ala?Tyr?Gly?Ile?Val?Cys?Ser?Val
40?????????????????45??????????????????50??????????????????55
ttg?gat?tcg?gag?acg?aac?gag?ctt?gtg?gcg?atg?aag?aag?ata?gcc?aac?????366
Leu?Asp?Ser?Glu?Thr?Asn?Glu?Leu?Val?Ala?Met?Lys?Lys?Ile?Ala?Asn
60?????????????????65??????????????????70
gcc?ttt?gat?aat?cac?atg?gac?gcc?aag?cgt?aca?ctt?cgc?gag?atc?aag?????414
Ala?Phe?Asp?Asn?His?Met?Asp?Ala?Lys?Arg?Thr?Leu?Arg?Glu?Ile?Lys
75?????????????????80??????????????????85
ctt?ctt?cgt?cat?ctc?gat?cat?gaa?aac?atc?ata?gct?ata?aga?gat?gtg????462
Leu?Leu?Arg?His?Leu?Asp?His?Glu?Asn?Ile?Ile?Ala?Ile?Arg?Asp?Val
90?????????????????95??????????????????100
gtt?cct?cca?cca?ctt?aga?aga?gag?ttc?agt?gat?gtt?tat?atc?gcc?act????510
Val?Pro?Pro?Pro?Leu?Arg?Arg?Glu?Phe?Ser?Asp?Val?Tyr?Ile?Ala?Thr
105?????????????????110????????????????115
gag?tta?atg?gac?act?gat?ctt?cat?caa?atc?atc?aga?tcc?aac?cag?ggc????558
Glu?Leu?Met?Asp?Thr?Asp?Leu?His?Gln?Ile?Ile?Arg?Ser?Asn?Gln?Gly
120?????????????????125????????????????130?????????????????135
ttg?tct?gag?gaa?cac?tgt?cag?tac?ttc?ctg?tac?cag?ctt?ctt?cga?ggg????606
Leu?Ser?Glu?Glu?His?Cys?Gln?Tyr?Phe?Leu?Tyr?Gln?Leu?Leu?Arg?Gly
140????????????????145?????????????????150
ctc?aag?tac?atc?cac?tcg?gct?aaa?gtt?atc?cac?agg?gat?cta?aag?cct????654
Leu?Lys?Tyr?Ile?His?Ser?Ala?Lys?Val?Ile?His?Arg?Asp?Leu?Lys?Pro
155????????????????160?????????????????165
agc?aat?ctt?ctc?ctg?aac?gcc?aac?tgc?gat?tta?aag?atc?tgt?gat?ttt????702
Ser?Asn?Leu?Leu?Leu?Asn?Ala?Asn?Cys?Asp?Leu?Lys?Ile?Cys?Asp?Phe
170?????????????????175????????????????180
ggt?ctt?gct?aga?ccc?act?tca?gag?aac?gag?ttt?atg?act?gag?tat?gtt????750
Gly?Leu?Ala?Arg?Pro?Thr?Ser?Glu?Asn?Glu?Phe?Met?Thr?Glu?Tyr?Val
185?????????????????190????????????????195
gtt?acc?aga?tgg?tat?aga?gca?cct?gag?ctt?ctg?ctc?aac?tca?tcg?gat????798
Val?Thr?Arg?Trp?Tyr?Arg?Ala?Pro?Glu?Leu?Leu?Leu?Asn?Ser?Ser?Asp
200?????????????????205????????????????210?????????????????215
tac?aca?gct?gct?ata?gat?gtt?tgg?tct?gtt?ggt?tgt?atc?ttt?atg?gag????846
Tyr?Thr?Ala?Ala?Ile?Asp?Val?Trp?Ser?Val?Gly?Cys?Ile?Phe?Met?Glu
220????????????????225?????????????????230
ctc?atg?aac?aga?aag?cct?ttg?ttc?cct?ggt?aaa?gac?cat?gtc?cat?cag????894
Leu?Met?Asn?Arg?Lys?Pro?Leu?Phe?Pro?Gly?Lys?Asp?His?Val?His?Gln
235????????????????240?????????????????245
atg?cgc?tta?ttg?aca?gag?ttg?ctt?ggc?acg?ccg?aca?gaa?tca?gat?ctc????942
Met?Arg?Leu?Leu?Thr?Glu?Leu?Leu?Gly?Thr?Pro?Thr?Glu?Ser?Asp?Leu
250????????????????255?????????????????260
ggg?ttt?aca?cac?aac?gag?gat?gcc?aaa?aga?tac?atc?cgg?cag?ctt?ccc????990
Gly?Phe?Thr?His?Asn?Glu?Asp?Ala?Lys?Arg?Tyr?Ile?Arg?Gln?Leu?Pro
265?????????????????270????????????????275
aac?ttc?cct?cgc?caa?ccg?tta?gct?aaa?ctg?ttc?tct?cat?gtt?aac?tca????1038
Asn?Phe?Pro?Arg?Gln?Pro?Leu?Ala?Lys?Leu?Phe?Ser?His?Val?Asn?Ser
280?????????????????285????????????????290?????????????????295
ttg?gcc?att?gat?cta?gtt?gac?aga?atg?ttg?acg?ttt?gac?ccc?aac?gaa????1086
Leu?Ala?Ile?Asp?Leu?Val?Asp?Arg?Met?Leu?Thr?Phe?Asp?Pro?Asn?Glu
300????????????????305?????????????????310
aga?atc?act?gtt?gaa?gaa?gct?ctg?aat?cac?ccg?tac?cta?gcc?aag?ttg????1134
Arg?Ile?Thr?Val?Glu?Glu?Ala?Leu?Asn?His?Pro?Tyr?Leu?Ala?Lys?Leu
315????????????????320?????????????????325
cac?gac?ccg?aat?gat?gag?cca?atc?tgt?cta?aag?cca?ttc?tct?ttc?gac????1182
His?Asp?Pro?Asn?Asp?Glu?Pro?Ile?Cys?Leu?Lys?Pro?Phe?Ser?Phe?Asp
330????????????????335?????????????????340
ttt?gaa?caa?caa?cct?cta?gac?gag?gaa?cag?ata?aaa?gag?atg?atc?tac????1230
Phe?Glu?Gln?Gln?Pro?Leu?Asp?Glu?Glu?Gln?Ile?Lys?Glu?Met?Ile?Tyr
345?????????????????350????????????????355
agg?gaa?gcc?att?gca?ctc?aat?cca?aca?tat?gct?tagaagagca?gcaaccgaat????1283
Arg?Glu?Ala?Ile?Ala?Leu?Asn?Pro?Thr?Tyr?Ala
360?????????????????365????????????????370
gaatggctaa?atatcctgcc?ctctagcatc?tttgcttgtt?cttttattca?tgtacctttt????1343
ttccatgtgt?aatcctattt?gtttgtttaa?cattgtatta?ctgctagtga?gtgagtgcat????1403
gtagctctca?tgtaaaagta?ctccagctac?gaaatattta?cttcacaaaa?aaaaaaaaaa????1463
a????????????????????????????????????????????????????????????????????1464
<210>2
<211>370
<212>PRT
<213>Brassica?napus
<400>2
Met?Asn?Asn?Ala?Gly?Gly?Gln?Tyr?Pro?Asp?Phe?Pro?Ala?Val?Gln?Thr
1???????????????5???????????????????10?????????????????15
His?Gly?Gly?Gln?Phe?Ile?Ser?Tyr?Asp?Ile?Phe?Gly?Ser?Leu?Phe?Glu
20??????????????????25?????????????????30
Val?Thr?Ser?Lys?Tyr?Arg?Pro?Pro?Ile?Val?Pro?Ile?Gly?Arg?Gly?Ala
35??????????????????40?????????????????45
Tyr?Gly?Ile?Val?Cys?Ser?Val?Leu?Asp?Ser?Glu?Thr?Asn?Glu?Leu?Val
50??????????????????55?????????????????60
Ala?Met?Lys?Lys?Ile?Ala?Asn?Ala?Phe?Asp?Asn?His?Met?Asp?Ala?Lys
65??????????????????70?????????????????75??????????????????80
Arg?Thr?Leu?Arg?Glu?Ile?Lys?Leu?Leu?Arg?His?Leu?Asp?His?Glu?Asn
85?????????????????90??????????????????95
Ile?Ile?Ala?Ile?Arg?Asp?Val?Val?Pro?Pro?Pro?Leu?Arg?Arg?Glu?Phe
100?????????????????105????????????????110
Ser?Asp?Val?Tyr?Ile?Ala?Thr?Glu?Leu?Met?Asp?Thr?Asp?Leu?His?Gln
115????????????????120?????????????????125
Ile?Ile?Arg?Ser?Asn?Gln?Gly?Leu?Ser?Glu?Glu?His?Cys?Gln?Tyr?Phe
130?????????????????135????????????????140
Leu?Tyr?Gln?Leu?Leu?Arg?Gly?Leu?Lys?Tyr?Ile?His?Ser?Ala?Lys?Val
145?????????????????150????????????????155?????????????????160
Ile?His?Arg?Asp?Leu?Lys?Pro?Ser?Asn?Leu?Leu?Leu?Asn?Ala?Asn?Cys
165????????????????170?????????????????175
Asp?Leu?Lys?Ile?Cys?Asp?Phe?Gly?Leu?Ala?Arg?Pro?Thr?Ser?Glu?Asn
180????????????????185?????????????????190
Glu?Phe?Met?Thr?Glu?Tyr?Val?Val?Thr?Arg?Trp?Tyr?Arg?Ala?Pro?Glu
195????????????????200?????????????????205
Leu?Leu?Leu?Asn?Ser?Ser?Asp?Tyr?Thr?Ala?Ala?Ile?Asp?Val?Trp?Ser
210????????????????215?????????????????220
Val?Gly?Cys?Ile?Phe?Met?Glu?Leu?Met?Asn?Arg?Lys?Pro?Leu?Phe?Pro
225?????????????????230????????????????235?????????????????240
Gly?Lys?Asp?His?Val?His?Gln?Met?Arg?Leu?Leu?Thr?Glu?Leu?Leu?Gly
245????????????????250?????????????????255
Thr?Pro?Thr?Glu?Ser?Asp?Leu?Gly?Phe?Thr?His?Asn?Glu?Asp?Ala?Lys
260????????????????265?????????????????270
Arg?Tyr?Ile?Arg?Gln?Leu?Pro?Asn?Phe?Pro?Arg?Gln?Pro?Leu?Ala?Lys
275????????????????280?????????????????285
Leu?Phe?Ser?His?Val?Asn?Ser?Leu?Ala?Ile?Asp?Leu?Val?Asp?Arg?Met
290????????????????295?????????????????300
Leu?Thr?Phe?Asp?Pro?Asn?Glu?Arg?Ile?Thr?Val?Glu?Glu?Ala?Leu?Asn
305?????????????????310????????????????315?????????????????320
His?Pro?Tyr?Leu?Ala?Lys?Leu?His?Asp?Pro?Asn?Asp?Glu?Pro?Ile?Cys
325????????????????330?????????????????335
Leu?Lys?Pro?Phe?Ser?Phe?Asp?Phe?Glu?Gln?Gln?Pro?Leu?Asp?Glu?Glu
340????????????????345?????????????????350
Gln?Ile?Lys?Glu?Met?Ile?Tyr?Arg?Glu?Ala?Ile?Ala?Leu?Asn?Pro?Thr
355????????????????360?????????????????365
Tyr?Ala
370
Claims (6)
1, former activated protein kinase 3 albumen coded sequences of a kind of swede type rape cell fission, it is characterized in that, isolated dna molecular, this molecule comprises: coding has the nucleotide sequence of the polypeptide of swede type rape BnMPK3 protein active, and shows at least 70% homology from the nucleotides sequence of Nucleotide 154-1263 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 154-1263 position,
2, former activated protein kinase 3 albumen coded sequences of swede type rape cell fission according to claim 1, it is characterized in that, described nucleotide coding 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
3, former activated protein kinase 3 albumen coded sequences of swede type rape cell fission according to claim 1 and 2 is characterized in that, described nucleotide coding sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 154-1263 position.
4, former activated protein kinase 3 albumen coded sequences of swede type rape cell fission according to claim 1, it is characterized in that, the also isolated former activated protein kinase of the cell fission swede type rape MAPK class related protein polypeptide BnMPK3 that comprises, it comprises: have the polypeptide of SEQ ID NO.3 aminoacid sequence, this polypeptide is to have SEQ ID NO.3 polypeptide of sequence.
5, former activated protein kinase 3 albumen coded sequences of swede type rape cell fission according to claim 1, it is characterized in that described dna molecular is the dna molecular that carrier comprises, be a kind of nucleic acid molecule, it comprises 8-100 continuous nucleotide in the described dna molecular.
6, former activated protein kinase 3 albumen coded sequences of swede type rape cell fission according to claim 4 is characterized in that, with described dna molecular transformed host cells, it is an eukaryotic cell.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105063085A (en) * | 2015-07-31 | 2015-11-18 | 江苏大学 | Cabbage type rape gene BnMPK3 and application thereof in resisting sclerotinia rot of colza |
| CN112280785A (en) * | 2020-10-30 | 2021-01-29 | 西南大学 | Application and method of Brassica napus BnMAPK2 gene |
| CN112795552A (en) * | 2021-03-10 | 2021-05-14 | 河南大学 | Application of Zm0001d024568 gene and encoding protein thereof in drought stress resistance of corn |
-
2004
- 2004-05-27 CN CN 200410024695 patent/CN1584032A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105063085A (en) * | 2015-07-31 | 2015-11-18 | 江苏大学 | Cabbage type rape gene BnMPK3 and application thereof in resisting sclerotinia rot of colza |
| CN105063085B (en) * | 2015-07-31 | 2018-08-10 | 江苏大学 | The application of cabbage type rape gene BnMPK3 and its anti-sclerotinia sclerotiorum |
| CN112280785A (en) * | 2020-10-30 | 2021-01-29 | 西南大学 | Application and method of Brassica napus BnMAPK2 gene |
| CN112280785B (en) * | 2020-10-30 | 2021-06-29 | 西南大学 | Application and method of Brassica napus BnMAPK2 gene |
| CN112795552A (en) * | 2021-03-10 | 2021-05-14 | 河南大学 | Application of Zm0001d024568 gene and encoding protein thereof in drought stress resistance of corn |
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