CN1600861A - Protein coded sequence of binding factor in ethane response element of cotton - Google Patents
Protein coded sequence of binding factor in ethane response element of cotton Download PDFInfo
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Abstract
This invention relates to cotton ethylene responsive element binding factor protein coding sequence. DNA molecule is obtd. by separation. Said molecules comprise: nucleotide sequence with its code having cotton ERF2 protein activity polypeptide, and having 70% homology with nucleotide sequence at positions of 139-1098 nucleotide of SEQ ID No.3, or, said nucleotide can be hybridized with nucleotide at positions of 139-1098 of SEQ ID No.3. This plants have advantages of: drought-resistance, salt-tolerance, high-temp. weather-resistance and anti-diseases.
Description
Technical field
What the present invention relates to is the encoding sequence in a kind of biological gene engineering field, particularly a kind of cotton ethylene response element binding factor protein encoding sequence.
Background technology
Plant hormone ethene is regulated multiple the coercing of plant and is reacted and the developmental character adaptation.Ethene participates in the various physiological processes of higher plant, as promote the tamato fruit maturation, wild-type Arabidopis thaliana tangible cell of performance after ethene is handled expands and is obstructed, the seed of sprouting is handled back performance triple effect at ethene: the top is crooked to increase, plumular axis phototropism expands, root nodule growth and cell fate decision etc.Injury, disease, water logging, high salt, arid, environment-stress such as low temperature can both be induced the higher plant synthesizing ethylene, by the Ethylene Signal Transduction process, induction phase is answered gene transcription, and environment-stress produces response to external world, has all found the Ethylene Signal Transduction process in higher plant.Binding factor in ethane response element (ERF2), it is the transcription factor in the Ethylene Signal Transduction process, it accepts the signal that Ethylene Signal approach middle and upper reaches are formed, and the initial relevant resistant gene that contains the GCC element at promoter region is transcribed, and plays an important role in the Ethylene Signal Transduction process.
Warp is to finding in the prior art literature search: magazine " The Plant Cell (vegetable cell) ", 2000,12:393-404 has delivered article " Arabidopsis ethylene-responsive element bindingfactors act as transcriptional activators or repressor of GCC box-mediated geneexpression (the Arabidopis thaliana binding factor in ethane response element is as the transcription activator or supression of GCC box regulate gene expression) ", though the document activates Ethylene Signal approach transcription factor or the Transcription of supression target gene is affirmed fully, but report up to now shows transgenic tobacco plant disease-resistant of this gene, ripe, the effect that salt tolerant 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 cotton ethylene response element binding factor protein encoding sequence is provided, make it on the disease-resistant salt tolerant of vegetable cell, have tangible effect, can obviously improve the resistance of vegetable cell under germ and osmotic stress condition, reduce the infringement of environment-stress 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 polypeptide of binding factor in ethane response element of cotton (ERF2) protein active, and shows at least 70% homology from the nucleotides sequence of Nucleotide 139-1098 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 139-1098 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 139-1098 position.
The isolated cotton disease resistance salt tolerant of the present invention class related protein polypeptide ERF2, it comprises: the polypeptide with SEQ IDNO.3 aminoacid sequence, preferably, 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 (ABA) are handled, but and the equal inducing plant body of these environment stresses produces ethene.And by the Ethylene Signal Transduction process, induction phase is answered gene transcription, resists external environment and coerces.
Carrier provided by the invention, it comprises above-mentioned dna molecular.A kind of nucleic acid molecule, it comprises 8-100 continuous nucleotide in the described dna molecular.
The present invention is with above-mentioned dna molecular transformed host cells, and 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, term " cotton ethylene response element binding factor protein (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with cotton ERF2 protein-active is as 139-1098 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 139-1098 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 139-1098 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 139-1098 position.This term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 139-1098 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 cotton EEF2 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 of Nucleotide, insertion-and/or replace, 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 " cotton ethylene response element binding factor protein or polypeptide " refers to have the SEQ ID NO.3 polypeptide of sequence of cotton ERF2 protein-active.This term also comprises having and the variant form relevant identical function of natural cotton ERF2, 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 proteic active fragments of cotton ERF2 and reactive derivative.
The variant form of cotton ERF2 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 cotton ERF2 under high or low rigorous condition are coded and the polypeptide or the albumen that utilize the antiserum(antisera) of cotton ERF2 polypeptide to obtain.
In the present invention, " cotton ERF2 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
82％identity?in?960nt?overlap
Query：130?agaaagaggaagaaccagtatcgtggaatccggcagcgcccatggggtaaatgggctgct?189
|||||||||||||??|||||?||?||?|||?|?|||||?||?|||||||??|||||?|||
Sbjct：375?agaaagaggaagaatcagtaccgggggatcagacagcgtccttggggtaagtgggcagct?434
Query：190?gagatccgtgacccaaggaaaggggttagggtctggttaggaactttcaatactgctgaa?249
||?||?|||||?|||||||||||??||?|?||||||?|?||?|||||||||?|?||?|||
Sbjct：435?gaaatacgtgatccaaggaaaggtattcgagtctggcttggtactttcaattcagccgaa?494
Query：250?gaagctgcgagagcttatgatgctgaggcacggagaattcgtggtaagaaagctaaggtg?309
||?||?||?||||||||||||||||||||?||?||?||??|?||?|||||||||||||||
Sbjct：495?gaggcagccagagcttatgatgctgaggcgcgaaggatcagaggcaagaaagctaaggtg?554
Query：310?aactt?314
|||||
Sbjct：555?aactt?559
Query: the nucleotide sequence of cotton Ethylene Response Element Binding Factor
Sbjct: the nucleotide sequence (AY044235) of tomato Ethylene Response Element Binding Factor
Table 2 is that the homology of cotton ERF2 albumen of the present invention and the proteic nucleotide sequence of tomato ERF compares (GAP) table.
Table 3
48％identity?in?319aa?overlap.65％similarity?in?319aa?overlap
Query：73??GSNSEKSMQFDGQAEKCAKRKRKNQYRGIRQRPWGKWAAEIRDPRKGVRVWLGTFNTXXX?252
G??S+?S???+?+A++?+KRKRKNQYRGIRQRPWGKWAAEIRDPRKG+RVWLGTFN+
Sbjct：78??GPKSVRSGDSNCEADRSSKRKRKNQYRGIRQRPWGKWAAEIRDPRKGIRVWLGTFNSAEE?137
Query：253?XXXXXXXXXXXIRGKKAKVNFPNETPRTSPKHAVKTNSQKPLSKSNLSPVQLNLDQNYNY?432
IRGKKAKVNFP+E?P?+??+?A+K?N?QK?L?+??L+?VQ?N+?????Y
Sbjct：138?AARAYDAEARRIRGKKAKVNFPDEAPVSVSRRAIKQNPQKALREETLNTVQPNM----TY?193
Query：433?LSQPEQEYFDTMGFVEEKPLVNQFAYVDPVPTSIDAG---SNQSDNAPLYFNSDQGSNSI?603
+S??+????D+??F?EEKP???Q+?+?+???T++D?G???+??S???+YF+SD+??SN+
Sbjct：194?ISNLDGGSDDSFSFFEEKPATKQYGFENVSFTAVDMGLGSVSPSAGTNVYFSSDEASNTF?253
Query：604?NCSDYGWGEQGARTPEISSILEASVVGEE--FLEDANPSKKLKPSSDNVMPAEDNSAKTL?777
+CSD+GW?E??ARTPEISS+L???+???E??F?+D?+P?KKLK?S???+??+??N+??TL
Sbjct：254?DCSDFGWAEPCARTPEISSVLSEVLETNETHFDDDSRPEKKLKSCSSTSLTVDGNTVNTL?313
Query：778?SDELLALDNQMKYFQMPPFIEGNWDATIDAFLNGDATQDGGNPMDLWNFDDFPTMAEGVF?957
S+EL?A?++QMK+?Q+?P++EGNWDA++DAFLN??A?QDGGN?MDLW+FDD?P++??G?+
Sbjct：314?SEELSAFESQMKFLQI-PYLEGNWDASVDAFLNTSAIQDGGNAMDLWSFDDVPSLMGGAY?372
Query: the aminoacid sequence of cotton Ethylene Response Element Binding Factor
Sbjct: the aminoacid sequence (AAK95687) of tomato Ethylene Response Element Binding Factor
Table 3 is that the homology of cotton ERF2 albumen of the present invention and the proteic aminoacid sequence of tomato ERF compares (FASTA) table.Wherein, identical amino acid marks with the amino acid monocase between two sequences.
The analogue of invention cotton ERF2 albumen or polypeptide.The difference of these analogues and natural cotton ERF2 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 cotton ERF2 polypeptide of the present invention, cotton ERF2 encoding sequence operationally can be connected in expression regulation sequence, thereby form cotton ERF2 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 cotton ERF2 gene product, the existence of rna transcription thing in cell of promptly analyzing cotton ERF2.
In addition, the nucleic acid molecule that can be used as probe provided by the invention, this molecule have 8-100 continuous nucleotide of cotton ERF2 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 cotton ERF2.
The method that whether has cotton ERF2 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 cotton ERF2 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 cotton ERF2 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 cotton ERF2 or homologous protein.
In order to obtain the dot matrix of the cotton cDNAs relevant with cotton ERF2 genes involved, can screen cotton cDNA library with dna probe, these probes are under low rigorous condition, use
32P relevant all or part of of cotton ERF2 cooked the radioactivity mark and.The cDNA library that most is suitable for screening is the library from cotton.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 cotton ERF2.
Cotton ERF2 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 cotton ERF2 albumen of the present invention,, can filter out the interactional material of relevant generation with cotton ERF2, 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 binding factor in ethane response element of cotton (ERF2) albumen coded sequence be provided, make it on the disease-resistant salt tolerant of vegetable cell, have tangible effect,
The present invention has substantive distinguishing features and marked improvement, and the present invention has tangible effect on the disease-resistant salt tolerant of vegetable cell, can obviously improve the resistance of vegetable cell under adverse environmental factor, reduces the infringement of environment-stress 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, according to normal condition, Sambrook equimolecular clone for example: the condition described in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), 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 cotton ERF2 gene
1. separate tissue (isolation)
Place 37 ℃ to germinate 24 hours cotton seeds, be seeded in then in the greenhouse, when treating that cotton leaf 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 tomato ERF 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[EA001 (SEQ ID NO.1)+EA002 (SEQ ID NO.2)] obtain the fragment of 185bp, 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 (GenBank+EMBL), the homology of knowing its nucleotide sequence and proteins encoded and known plant of Solanaceae such as tomato (Solanum tuberosum) ERF gene is very high, so think that tentatively it is an ERF gene.
(2)3’-RACE
PCR[AP+EA301 (5 '-AGAAAGAGGAAGAACCAGTA-3 ')] obtain EA3 ' (1138bp), 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 (GenBank+EMBL), the homology of knowing its nucleotide sequence and proteins encoded and known plant of Solanaceae such as tomato (Lycopersicon esculentum) ERF gene is very high, so think that tentatively it is a gene relevant with ERF.
(3)5’-RACE
First round PCR[AAP+EA501 (5 '-AAGTTCACCTTAGCTTTCTT-3 ')]
Second takes turns PCR[(AUAP+EA502 (5 '-TCATAAGCTCTCGCAGCTTC-3 ')) obtain EA5 '
(about 407bp) (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 cotton really is a gene relevant with tomato ERF.
By being used in combination above-mentioned 3 kinds of methods, obtained candidate's the proteic complete encoding sequence of cotton ERF2 (SEQ ID NO.3).
Embodiment 2
The sequence information and the homology analysis of cotton ERF2 gene
The length of the cotton ERF2 full-length cDNA that the present invention is new is 1405bp, and detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 139-1098 position Nucleotide (960 Nucleotide).Derive the aminoacid sequence of cotton ERF2 according to full-length cDNA, totally 319 amino-acid residues, molecular weight is 36187.35 dalton, iso-electric point (pI) is 4.87.Detailed sequence is seen SEQ ID NO.3.
Full length cDNA sequence and the coded protein thereof of cotton ERF2 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 tomato dna ERF (AY044235) have 82% homogeny, (subordinate list 2) on nucleotide level; On amino acid levels, it and tomato dna ERF (AAK95687) also have 48% homogeny (subordinate list 3).This shows that there are higher homology in cotton gene ERF2 and tomato dna ERF on nucleic acid still is protein level.Tomato dna ERF (AY044235) has been proved to be obviously to strengthen under pathogenic bacteria, high-salt stress condition and has expressed, and brings into play the effect of wanting emphatically, can think that cotton gene ERF2 also has similar effect aspect the disease-resistant salt tolerant.
Embodiment 3
Cotton gene ERF2 albumen or polypeptide carry out the disease-resistant salt tolerant of eukaryotic cell expression and transfer-gen plant in tobacco identifies
The structure that contains the expression vector of goal gene (cotton gene ERF2)
According to the full length sequence (SEQ ID NO.3) of cotton gene ERF2, design amplifies the primer that complete coding is read frame, and introduces restriction endonuclease sites (this is decided by the carrier of selecting for use) on the upstream and downstream primer respectively, so that construction of expression vector.Amplified production with acquisition among the embodiment 1 is a template, behind pcr amplification, cotton gene ERF2 cDNA is cloned into intermediate carrier (as pBluescript), 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 disease-resistant salt tolerant that contains the transgenic tobacco plant of cotton gene ERF2 is identified
In view of coding ERF, be proved to be as the ERF gene of tomato and played a role aspect the disease-resistant salt tolerant, and the ERF of cotton gene ERF2 transcriptional level and tomato has higher homology, can further carry out disease-resistant salt tolerant to the transgenic tobacco plant that contains cotton gene ERF2 and identify.Seed (3 hours, 7 hours, 12 hours, 24 hours, 48 hours, 72 hours) the back research ERF2 gene of handling transfer-gen plant and transfer-gen plant with pathogenic bacteria, water logging, ABA and high temperature (45 ℃) in transfer-gen plant expression and various processing to the growing state of plant.Northern blot analytical results proves that transfer-gen plant ERF2 transcriptional level its expression amount after pathogenic bacteria, water logging, ABA and pyroprocessing all has considerable change, 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 pathogenic bacteria, water logging, ABA and pyroprocessing at last, and transgenic plant still can normal growth, and are just slow slightly than undressed plant strain growth.This proof cotton gene ERF2 has more effective and the function in degeneration-resistant border widely than the ERF gene of tomato, will can be used for utilizing in the research and industrialization production of transgenic technology improvement plant disease-resistant salt tolerant.
Embodiment 4
The copy number analysis of cotton gene ERF2 in cotton
Adopt ordinary method from cotton leaf, to extract DNA (with reference to " molecular cloning ", Sambrook etc., 1989), use BamH, KpnI, SacI and XbaI enzyme cutting DNA[20 μ g (microgram)/sample respectively] after, DNA is gone to after Hybond membrane (nylon membrane) goes up.Use the Amersham Pharmacia Gene Images of company
TMContentsCDP-Star
TMLabelling module (PRN3540), we are labeled as probe with the ERF2 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) discovery is cut at different enzymes on the Hybond membrane and is respectively occurred two to five hybridization bands on the swimming lane, illustrates that the ERF2 gene is low copy gene in cotton.
Embodiment 5
The expression pattern analysis that the different time of cotton gene ERF2 under pathogenic bacteria, water logging, ABA and high temperature stress condition handled
1.RNA extraction: the cotton seedling of the 3-5 sheet leaf of will having grown is handled (3 hours, 7 hours, 12 hours, 24 hours, 48 hours, 72 hours) through pathogenic bacteria, water logging, ABA and high temperature (45 ℃), 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 pathogenic bacteria, water logging, ABA and the prolongation of pyroprocessing time, the expression of ERF2 all has considerable change, illustrate that ERF2 is adverse circumstance signal such as abduction deliverings such as pathogenic bacteria, water logging, ABA and high temperature, plays the part of important role in the cotton disease resistance salt tolerant.
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
AGA?AAG?AGG?AAG?AA(C/T)CAG?TAA?AT
(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
AAG?TTC?ACC?TTA?GCT?TTC?TT
(3) information of SEQ ID NO.3
(i). sequence signature:
(A) length: 1405bp
(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〉cotton ethylene response element binding factor protein encoding sequence
<160>2
<170>PatentIn?version?3.1
<210>1
<211>1405
<212>DNA
＜213〉cotton (Gossypium barbadense)
<220>
<221>CDS
<222>(139)..(1098)
<223>
<400>1
atttgagaat?cgatgcccgg?attaataatt?tctgggatgt?agtcactaaa?aaggcctttc?????60
tcatgatttt?cttgcagtta?aaggtcttaa?attatatttc?cttgtgacag?ttatgttgtg????120
atttgtagtt?tttcatgg?atg?agt?aat?gtt?tat?tta?tgg?ttc?atg?tat?ccg??????171
Met?Ser?Asn?Val?Tyr?Leu?Trp?Phe?Met?Tyr?Pro
1????????????????5??????????????????10
tac?gat?att?aaa?ttt?ttc?ttt?ttg?tgc?tct?atc?att?gaa?ggt?tcg?aac??????219
Tyr?Asp?Ile?Lys?Phe?Phe?Phe?Leu?Cys?Ser?Ile?Ile?Glu?Gly?Ser?Asn
15??????????????????20??????????????????25
tct?gaa?aag?tcc?atg?cag?ttc?gat?ggt?caa?gct?gag?aaa?tgt?gcg?aaa????267
Ser?Glu?Lys?Ser?Met?Gln?Phe?Asp?Gly?Gln?Ala?Glu?Lys?Cys?Ala?Lys
30??????????????????35??????????????????40
aga?aag?agg?aag?aac?cag?tat?cgt?gga?atc?cgg?cag?cgc?cca?tgg?ggt????315
Arg?Lys?Arg?Lys?Asn?Gln?Tyr?Arg?Gly?Ile?Arg?Gln?Arg?Pro?Trp?Gly
45??????????????????50??????????????????55
aaa?tgg?gct?gct?gag?atc?cgt?gac?cca?agg?aaa?ggg?gtt?agg?gtc?tgg????363
Lys?Trp?Ala?Ala?Glu?Ile?Arg?Asp?Pro?Arg?Lys?Gly?Val?Arg?Val?Trp
60??????????????????65??????????????????70??????????????????75
tta?gga?act?ttc?aat?act?gct?gaa?gaa?gct?gcg?aga?gct?tat?gat?gct????411
Leu?Gly?Thr?Phe?Asn?Thr?Ala?Glu?Glu?Ala?Ala?Arg?Ala?Tyr?Asp?Ala
80??????????????????85??????????????????90
gag?gca?cgg?aga?att?cgt?ggt?aag?aaa?gct?aag?gtg?aac?ttc?cct?aac????459
Glu?Ala?Arg?Arg?Ile?Arg?Gly?Lys?Lys?Ala?Lys?Val?Asn?Phe?Pro?Asn
95??????????????????100?????????????????105
gag?act?ccg?cgt?acc?tct?cca?aag?cat?gca?gtc?aag?aca?aat?tct?cag????507
Glu?Thr?Pro?Arg?Thr?Ser?Pro?Lys?His?Ala?Val?Lys?Thr?Asn?Ser?Gln
110?????????????????l15?????????????????120
aaa?cca?ctt?tcc?aag?tcg?aat?ttg?agc?cct?gtt?cag?cta?aat?ctc?gac????555
Lys?Pro?Leu?Ser?Lys?Ser?Asn?Leu?Ser?Pro?Val?Gln?Leu?Asn?Leu?Asp
125?????????????????130?????????????????135
cag?aat?tac?aat?tac?ttg?agc?cag?cct?gag?cag?gaa?tac?ttc?gat?acc????603
Gln?Asn?Tyr?Asn?Tyr?Leu?Ser?Gln?Pro?Glu?Gln?Glu?Tyr?Phe?Asp?Thr
140?????????????????145?????????????????150?????????????????155
atg?ggt?ttc?gta?gaa?gag?aag?cca?ctg?gtc?aat?cag?ttt?gca?tat?gtg????651
Met?Gly?Phe?Val?Glu?Glu?Lys?Pro?Leu?Val?Asn?Gln?Phe?Ala?Tyr?Val
160?????????????????165?????????????????170
gac?cct?gtt?cct?acg?tct?ata?gat?gct?gga?tct?aat?caa?tca?gat?aat????699
Asp?Pro?Val?Pro?Thr?Ser?Ile?Asp?Ala?Gly?Ser?Asn?Gln?Ser?Asp?Asn
175?????????????????180?????????????????185
gcc?ccc?ttg?tac?ttc?aat?tcg?gac?cag?gga?agt?aac?tcc?atc?aat?tgt????747
Ala?Pro?Leu?Tyr?Phe?Asn?Ser?Asp?Gln?Gly?Ser?Asn?Ser?Ile?Asn?Cys
190?????????????????195?????????????????200
tcc?gac?tat?ggc?tgg?gga?gaa?cag?ggt?gcc?aga?act?cct?gaa?ata?tca????795
Ser?Asp?Tyr?Gly?Trp?Gly?Glu?Gln?Gly?Ala?Arg?Thr?Pro?Glu?Ile?Ser
205?????????????????210?????????????????215
tcc?att?ctt?gaa?gct?tct?gta?gtg?ggt?gaa?gag?ttt?ctt?gag?gat?gct????843
Ser?Ile?Leu?Glu?Ala?Ser?Val?Val?Gly?Glu?Glu?Phe?Leu?Glu?Asp?Ala
220?????????????????225?????????????????230?????????????????235
aac?cct?agc?aag?aag?ctg?aaa?cca?agt?tct?gac?aat?gtt?atg?cct?gcc????891
Asn?Pro?Ser?Lys?Lys?Leu?Lys?Pro?Ser?Ser?Asp?Asn?Val?Met?Pro?Ala
240?????????????????245?????????????????250
gaa?gac?aac?tcc?gcg?aag?acc?ttg?tcg?gac?gag?ctg?ttg?gct?ttg?gac????939
Glu?Asp?Asn?Ser?Ala?Lys?Thr?Leu?Ser?Asp?Glu?Leu?Leu?Ala?Leu?Asp
255?????????????????260?????????????????265
aac?cag?atg?aaa?tac?ttc?caa?atg?ccg?cca?ttt?att?gaa?gga?aac?tgg????987
Asn?Gln?Met?Lys?Tyr?Phe?Gln?Met?Pro?Pro?Phe?Ile?Glu?Gly?Asn?Trp
270?????????????????275?????????????????280
gac?gcc?act?att?gat?gct?ttc?ctc?aat?gga?gat?gca?aca?cag?gat?ggt????1035
Asp?Ala?Thr?Ile?Asp?Ala?Phe?Leu?Asn?Gly?Asp?Ala?Thr?Gln?Asp?Gly
285?????????????????290?????????????????295
gga?aac?ccg?atg?gat?ctt?tgg?aac?ttt?gat?gat?ttc?cct?acc?atg?gcg????1083
Gly?Asn?Pro?Met?Asp?Leu?Trp?Asn?Phe?Asp?Asp?Phe?Pro?Thr?Met?Ala
300?????????????????305?????????????????310?????????????????315
gag?ggt?gtt?ttc?tga?gcgaactttc?cataataact?agtgtttgta?aataaagcaa????1138
Glu?Gly?Val?Phe
catgaatttg?gtcaaaatct?gttgtgaagt?tgaagtaaaa?accaagctat?atgcatgctt??1198
aagccttgcc?tgcactgctt?tcagaggttt?ttagtatgta?cccctttttt?atgtgttttt??1258
ttgtagactt?tggactaaat?tttaaatttg?agtgactgtataagtaattg?tgtc?tgaatt??1318
tgtttatgtt?tgaatactga?aaaacatatg?aatgttttaa?actctgctat?ttgtttctcc??1378
caaaaaaaaa???????????????????????aaaaaaaaaa?????????????????????aaaaaaa
1405
<210>2
<211>319
<212>PRT
＜213〉cotton (Gossypium barbadense)
<400>2
Met?Ser?Asn?Val?Tyr?Leu?Trp?Phe?Met?Tyr?Pro?Tyr?Asp?Ile?Lys?Phe
1????????????????5??????????????????10??????????????????15
Phe?Phe?Leu?Cys?Ser?Ile?Ile?Glu?Gly?Ser?Asn?Ser?Glu?Lys?Ser?Met
20??????????????????25??????????????????30
Gln?Phe?Asp?Gly?Gln?Ala?Glu?Lys?Cys?Ala?Lys?Arg?Lys?Arg?Lys?Asn
35??????????????????40??????????????????45
Gln?Tyr?Arg?Gly?Ile?Arg?Gln?Arg?Pro?Trp?Gly?Lys?Trp?Ala?Ala?Glu
50??????????????????55??????????????????60
Ile?Arg?Asp?Pro?Arg?Lys?Gly?Val?Arg?Val?Trp?Leu?Gly?Thr?Phe?Asn
65??????????????????70??????????????????75??????????????????80
Thr?Ala?Glu?Glu?Ala?Ala?Arg?Ala?Tyr?Asp?Ala?Glu?Ala?Arg?Arg?Ile
85??????????????????90??????????????????95
Arg?Gly?Lys?Lys?Ala?Lys?Val?Asn?Phe?Pro?Asn?Glu?Thr?Pro?Arg?Thr
100?????????????????105?????????????????110
Ser?Pro?Lys?His?Ala?Val?Lys?Thr?Asn?Ser?Gln?Lys?Pro?Leu?Ser?Lys
115?????????????????120?????????????????125
Ser?Asn?Leu?Ser?Pro?Val?Gln?Leu?Asn?Leu?Asp?Gln?Asn?Tyr?Asn?Tyr
130?????????????????135?????????????????140
Leu?Ser?Gln?Pro?Glu?Gln?Glu?Tyr?Phe?Asp?Thr?Met?Gly?Phe?Val?Glu
145?????????????????150?????????????????155?????????????????160
Glu?Lys?Pro?Leu?Val?Asn?Gln?Phe?Ala?Tyr?Val?Asp?Pro?Val?Pro?Thr
165?????????????????170?????????????????175
Ser?Ile?Asp?Ala?Gly?Ser?Asn?Gln?Ser?Asp?Asn?Ala?Pro?Leu?Tyr?Phe
180?????????????????185?????????????????190
Asn?Ser?Asp?Gln?Gly?Ser?Asn?Ser?Ile?Asn?Cys?Ser?Asp?Tyr?Gly?Trp
195?????????????????200?????????????????205
Gly?Glu?Gln?Gly?Ala?Arg?Thr?Pro?Glu?Ile?Ser?Ser?Ile?Leu?Glu?Ala
210?????????????????215?????????????????220
Ser?Val?Val?Gly?Glu?Glu?Phe?Leu?Glu?Asp?Ala?Asn?Pro?Ser?Lys?Lys
225?????????????????230?????????????????235?????????????????240
Leu?Lys?Pro?Ser?Ser?Asp?Asn?Val?Met?Pro?Ala?Glu?Asp?Asn?Ser?Ala
245?????????????????250?????????????????255
Lys?Thr?Leu?Ser?Asp?Glu?Leu?Leu?Ala?Leu?Asp?Asn?Gln?Met?Lys?Tyr
260?????????????????265?????????????????270
Phe?Gln?Met?Pro?Pro?Phe?Ile?Glu?Gly?Asn?Trp?Asp?Ala?Thr?Ile?Asp
275?????????????????280?????????????????285
Ala?Phe?Leu?Asn?Gly?Asp?Ala?Thr?Gln?Asp?Gly?Gly?Asn?Pro?Met?Asp
290?????????????????295?????????????????300
Leu?Trp?Asn?Phe?Asp?Asp?Phe?Pro?Thr?Met?Ala?Glu?Gly?Val?Phe
305?????????????????310?????????????????315
Claims (5)
1, a kind of cotton ethylene response element binding factor protein encoding sequence, it is characterized in that, isolated dna molecular, this molecule comprises: coding has the nucleotide sequence of the active polypeptide of cotton ethylene response element binding factor protein matter, and shows at least 70% homology from the nucleotides sequence of Nucleotide 139-1098 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 139-1098 position.
2, cotton ethylene response element binding factor protein encoding sequence according to claim 1, it is characterized in that, 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.
3, cotton ethylene response element binding factor protein encoding sequence according to claim 1 and 2 is characterized in that, described encoding sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 139-1098 position.
4, cotton ethylene response element binding factor protein encoding sequence according to claim 1 is characterized in that, comprises: 8-100 continuous nucleotide in the dna molecular.
5, according to claim 1 or 4 described cotton ethylene response element binding factor protein encoding sequences, it is characterized in that, described dna molecular transformed host cells, it is an eukaryotic cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410066430 CN1600861A (en) | 2004-09-16 | 2004-09-16 | Protein coded sequence of binding factor in ethane response element of cotton |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410066430 CN1600861A (en) | 2004-09-16 | 2004-09-16 | Protein coded sequence of binding factor in ethane response element of cotton |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100430415C (en) * | 2005-09-21 | 2008-11-05 | 中国农业科学院作物科学研究所 | Thinopyrum intermedium ERF-transcription factor and its coding gene and use |
| CN103305528A (en) * | 2013-05-07 | 2013-09-18 | 南京农业大学 | Ethylene response factor gene of cotton transcription factor |
| CN113373161A (en) * | 2021-08-03 | 2021-09-10 | 中国农业科学院棉花研究所 | Application of GhERF017 gene in regulating and controlling plant salt tolerance |
| WO2022082866A1 (en) * | 2020-10-20 | 2022-04-28 | 隆平生物技术(海南)有限公司 | Stress-resistant gene line acdwem and use thereof in improvement of salt tolerance, drought resistance and high temperature resistance of crops |
-
2004
- 2004-09-16 CN CN 200410066430 patent/CN1600861A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100430415C (en) * | 2005-09-21 | 2008-11-05 | 中国农业科学院作物科学研究所 | Thinopyrum intermedium ERF-transcription factor and its coding gene and use |
| CN103305528A (en) * | 2013-05-07 | 2013-09-18 | 南京农业大学 | Ethylene response factor gene of cotton transcription factor |
| CN103305528B (en) * | 2013-05-07 | 2014-10-22 | 南京农业大学 | Ethylene response factor gene of cotton transcription factor |
| WO2022082866A1 (en) * | 2020-10-20 | 2022-04-28 | 隆平生物技术(海南)有限公司 | Stress-resistant gene line acdwem and use thereof in improvement of salt tolerance, drought resistance and high temperature resistance of crops |
| CN113373161A (en) * | 2021-08-03 | 2021-09-10 | 中国农业科学院棉花研究所 | Application of GhERF017 gene in regulating and controlling plant salt tolerance |
| CN113373161B (en) * | 2021-08-03 | 2022-07-08 | 中国农业科学院棉花研究所 | Application of GhERF017 gene in regulating and controlling plant salt tolerance |
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