CN1475496A - Soybean ethylene response protein transcription factor and its coded gene and application - Google Patents
Soybean ethylene response protein transcription factor and its coded gene and application Download PDFInfo
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Abstract
A vinylresponder bindin transcription factor GmEREB of soybean and its coding gene are disclosed. Said GmEREB is the protein having the amino acid residue sequence of sequence 2 in sequence table. Said coding gene is one of such neucleotide sequences: the DNA sequence of sequence 1 in sequence table and the DNA sequence having more than 90% of homology to the DNA sequence limited by sequence 1 in sequence table and coding the same function protein. Said gene can be used to culture the stress-resistant plane.
Description
Technical field
The present invention relates to plant transcription factor and encoding gene thereof and application, particularly derive from transcription factor and encoding gene and the application of soybean.
Background technology
Biologies such as cause of disease, arid, saline and alkaline, low temperature and abiotic stress factor have material impact to growth and development of plant.Under environment stress, a series of Physiology and biochemistry can take place in the plant materials usually change.Plant experienced the variation of external environment before this by number of ways, and extracellular signal transferred to cell interior, chain reaction passes to transcription factor with signal through a series of phosphorylation level, transcription factor interacts by its specific function amino acid and goal gene again, startup produces the destination gene expression of replying to environment stress, thereby improves the resistance of plant.For example, some gene that contains the ERF class transcription factor in the conservative territory of ERF function can be accepted the environment-stress signal and start the adverse circumstance response gene, makes plant resistance to environment stress improve.Verified, ethene is one of regulatory factor of stress response, coercing the factor induces ethene intravital synthetic plant, some can be combined with the needed GCC zygote of a disease-resistant antigen gene expressed core sequence by the ethene inductive gene relevant with defence, and the expression of startup disease-resistant gene, reach disease-resistant and degeneration-resistant effect.
Soybean is improved its disease resistance and resistance and has important theory and realistic meaning as a kind of important oil plant and food crop.
Summary of the invention
The purpose of this invention is to provide ethylene response conjugated protein transcription factor that derives from soybean and encoding gene thereof with stronger degeneration-resistant effect.
The name of ethylene response conjugated protein transcription factor provided by the present invention is called GmEREB, be protein, or the amino acid residue sequence of sequence 2 is passed through replacement, disappearance or the interpolation of one or several amino-acid residue and has identical active by sequence 2 deutero-protein with the amino acid residue sequence of sequence 2 with sequence 2 amino acid residue sequences in the sequence table.
The protein that sequence 2 amino acid residue sequences are made up of 384 amino-acid residues in the sequence table.The ERF function that is transcription factor GmEREB from carbon teminal to 117-174 amino-acid residue of nitrogen end in the described sequence 2 is guarded the territory.
The encoding gene of ethylene response conjugated protein transcription factor GmEREB is one of following nucleotide sequences:
1) dna sequence dna of sequence 1 in the sequence table;
2) with sequence table in the dna sequence dna that limits of sequence 1 have 90% above homology, and the identical function protein DNA sequence of encoding.
The dna sequence dna of sequence 1 is by 1599 based compositions in the sequence table, and the reading frame of this gene is the 106th to the 1257th bit base, and its expression is induced by the instantaneous of ethene, arid, low temperature and high salt mainly.
Utilize any carrier that can guide foreign gene in plant, to express, with the gene transfered plant cell of encoding transcription factor GmEREB provided by the present invention, can obtain transgenic cell line and transfer-gen plant that arid, low temperature and high-salt stress tolerance are enhanced.Gene of the present invention can add any enhancing promotor or inducible promoter in being building up to plant expression vector the time before its transcription initiation Nucleotide.For the ease of transgenic plant cells or plant being identified and screening, can process employed carrier, as adding the alternative mark of plant.Carry GmEREB expression carrier of the present invention and can import vegetable cell by using conventional biotechnological meanss such as Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electroporation, by the plant transformed host both can be monocotyledons, also can be dicotyledons.Gene pairs of the present invention is cultivated the adversity resistant plant kind, particularly cultivates the plant variety of drought resisting, cold-resistant and anti-salt, and it is significant to improve crop yield.
Description of drawings
Fig. 1 is the vivoexpression of GmEREB gene in intestinal bacteria.
Fig. 2 is Southern hybridization analysis result.
Fig. 3 is the GmEREB allelic expression that Northern hybridization detects under the different stress conditions.
Embodiment
The clone of embodiment 1, soybean GmEREB transcription factor cDNA and sequential structure analysis
The soybean seedling of growing 10 days at room temperature placed on the filter paper of adequate thickness dehydration 5 hours after cleaning, collect plant and in liquid nitrogen, be ground to fine degree, place the 4mol/L guanidinium isothiocyanate, use acid phenol/chloroform extracting mixture again, get supernatant liquor, with the total RNA of isopropanol precipitating, repeat above-mentioned steps more once, RNA is dissolved in the DEPC water, be stored in-80 ℃ of (Davis etc., molecular biological basic skills: [Basic Methods inMolecular Biology], pp.777, APPLETON ﹠amp; LANCE, Norwalk, Connecticut, USA, 1994).
Get the total RNA of 1 μ g and carry out separately an independently step reverse transcription chain type amplified reaction (PCR) by 4 pairs of special degenerated primerses (four forward primer and a reverse primer combination).Article four, the 5 '-YRGIRQ-3 ' forward primer (Forward primers) corresponding to the conservative territory N-end of the AP2/EREBP of RAP2.2 and RAP2.12 is:
DF1：5’-TA(C/T)CGIGGIAT(A/C/T)CGICA(A/G)-3’
DF2：5’-TA(C/T)CGIGGIAT(A/C/T)AG(A/G)CA(A/G)-3’
DF3：5’-TA(C/T)AG(A/G)GGIAT(A/C/T)CGICA(A/G)-3’
DF4：5’-TA(C/T)AG(A/G)GGIAT(A/C/T)AG(A/G)CA(A/G)-3’
Article one, the reverse primer 5 '-AKVNFP-3 ' corresponding to the conservative territory C-end of AP2/EREBP (Reverseprimers) is: DR1:5 '-IGG (A/G) AA (A/G) TTIAC (C/T) TTIGC-3 '
In the above-mentioned primer, I (inosine) is a Trophicardyl.The PCR condition is: 4 ℃, and 3 minutes; 94 ℃, 30 seconds, 60 ℃, 30 seconds, 72 ℃, circulation in 1.5 minutes-30; 72 ℃, 10 minutes; 4 ℃ of preservations.The PCR product is connected to and is used for order-checking on the pMD18-T carrier.
Be template with the positive colony again, utilize a pair of Auele Specific Primer, DFR:5 '-CTGGCGGATTCCGCGATA-3 '; DRF:5 '-GCCAAGGTGAATTTCCCT-3 ' is by the 5 ' end and the 3 ' end of reverse transcription PCR reaction amplifying target genes.External then packing In vitro-GmEREB gene order, design a pair of Auele Specific Primer GmF:5 '-ATGTGTGGTGGTGCGATT-3 ' and GmR:5 '-GAAGACTCCT GCCATGGA-3 ' GmEREB-cDNA that increases again, the PCR product is connected in the pMD18-T carrier and checks order, one and the on all four cDNA clone of external packaging sequence have been obtained, called after GmEREB is the sequence in the sequence table 1.This gene inserts fragment 1599bp, contains the open reading frame (106 initial-1257 stop) of 1152bp, the polypeptide (sequence 2 in the sequence table) that 384 amino acid of encoding are formed, and its 5 ' end is 115bp, 3 ' end is 282bp.This gene contains the conservative territory of peculiar AP2/ER EBP structure in the typical plant transcription factor EREBP family, and this structural domain can interact with the cis-acting elements of element responsive to ethylene GCC zygote core and start the expression of purpose adversity gene.
Embodiment 2, the vivoexpression of soybean GmEREB gene in a big bacillus are identified
According to sequence 1 a pair of Auele Specific Primer the GmERFF:331 5 '-AAAAGAATTCTTTGGGTTCTCTCGCTCC-3 ' 348 of design and GmERFR:1044 5 '-AAAACTCGAGCACCACATCCTGCGAGTT 1027, obtain the cDNA fragment that comprises the conservative territory of AP2/EREBP structure of a 712bp by pcr amplification, and be inserted on the corresponding site of protein expression vector pGEX-4T-1 (GST), transform then in intestinal bacteria, induce in intestinal bacteria through IPTG, SDS-PAGE detects and shows that this gene can carry out external accurate translation in intestinal bacteria, the polyacrylate hydrogel electrophoretic band as shown in Figure 1, among the figure, M is Marker; 1, the 3rd, through the inductive bacterial strain; The 2nd, without the inductive bacterial strain.As can be seen from the figure, there is protein band to occur, do not have protein band to occur without the inductive bacterial strain through the inductive bacterial strain.
The number of copies analysis in soybean of embodiment 3, GmEREB gene
Utilization through the GmEREB cDNA coding region of isotropic substance phosphorus-32 mark for probe with through three kinds of different restriction enzyme (SalI, EcoRV and XhoI) digestion the soybean plants genomic dna carry out the Southern hybridization analysis, and (condition of high rigorous degree is: 0.5 * SSC to wash film through the rigorous degree of height, 0.1 * SDS, 65 ℃), the result as shown in Figure 2, the DNA of each digestion all has many hybrid belts to occur, and shows that the GmEREB gene is a multi-copy gene.
Embodiment 4, the expression characteristic of soybean GmEREB gene under the environment stress condition.
The soybean seedling in 2 week of growth places 4 ℃ of water, the 250mM NaCl aqueous solution and 2mM vinyl solution at room temperature to carry out illumination cultivation respectively, and it is that soybean seedling with 2 week of growth is placed on dehydration at room temperature on the filter paper that is enough to suck dry moisture only through washing that arid is handled.Above-mentioned all processing respectively 1 hour, 5 hours, 12 hours and sampling in 24 hours, are extracted total RNA and GmEREB cDNA probe hybridization.The result as shown in Figure 3, wherein A is the result that arid is handled, B is the result of 4 ℃ of subzero treatment, C is the result that 250mM NaCl handles, D is the result that 2mM ethene is handled, as can be seen from the figure, transcribing of gene GmEREB mainly is subjected to ethene and arid induced strong, and different degree is subjected to inducing of low temperature and salt.
＜160〉2＜210〉1＜211〉1599＜212〉DNA＜213〉 ( Glycine max ( L. ) Merr. )＜400〉1tgattgagcc aaagctttgt attttctgcg ataattttcc attgggtgga agaaagtctc 60aacctttatt cgaaagagca aggatctgag ttgagttgag tgatcatgtg tggtggtgcg 120attatctccg acttcatacc ggcaggtccc gccagcgggg cgcggcgcgt gaccgccgac 180atcctgtggc cgagtttgag gaagcgcttc tcgaagccgc tgctggacga tgatttcgag 240gctgggttca gagaattcaa ggatgattcg gaaatcgagg atgttgatga cgaggacgat 300gaagacgagg aggagttgaa gaagaagccc tttgggttct ctcgctccag caacaaggct 360gcttctaagc ctctctctcg tggagcaaca actgtgaaat ctgtggaatc aaaggggcaa 420gctgagaagt gtgccaagag aaagaggaag aaccagtatc gcggaatccg ccagcgtcca 480tggggaaagt gggctgctga gattcgcgac ccaagaaagg gggttcgtgt ttggcttgga 540actttcagca ctgctgaaga agctgcaaga gcttacgatg ctgaagcaag gaggatccgt 600ggcaagaaag ccaaggtgaa tttccctgat gagccttcag gcgctgcttc ctcaaaacgt 660ctcaaggcga atccagaggc tcagccaatg aagaaaaatc tgaactctgt gaagccgaaa 720ataaaccaga tgttcaattt tggtgacaat cttgagggct actacagccc tatagatcag 780gtggaacaga aaccactggt taaccagtat gttaaccgtg ccccgtttgc tggaaatgga 840gttcaagtct cacctgttac tccatctgct gatgttactg cttacttcag ctctgagcat 900tcgagcaact cgtttgatta ttctgacctt ggatggggtg aacaagtccc caagaccccc 960gagatctcat ccttgctttc tgctgctcct ttggagggtg ctgctgatca ggttcagaag 1020accaacaact cgcaggatgt ggtggctgca caagatgatt ctgcaaaaac cctttccgaa 1080gagcttgcag acattgaatc ccagctcaag ttctttgaga ccccttcttt tcttgatgaa 1140gcctgggctg atgctacatt ggcgtctttg ctcggcggag acgcaactca tgacgccgcc 1200ggaaacccta tgaacctttg gagcttcgac gacctgcctt ccatggcagg agtcttctga 1260acacccttta tctccccttt tatgtaaata aagctacaag aattgtgatc gtgatgttgg 1320tgatggagtc cacagccaag aaacctgctt aaagcttatg tggagtttat tttatcttgt 1380agctaatgca gtagtatagg actatatata ggtttttatt atagggtatc cttttgtgaa 1440ctcaaagacc tcgttttcag gggattttct gtttgatgtc cttaaggatt atcaattatg 1560ttatatatgg tcttggataa aaataaaaaa aaaaaaaaa 1599＜210〉2＜211〉384＜212〉PRT＜213〉 ( Glycine max ( L. ) Merr. )＜400〉2Met Cys Gly Gly Ala Ile Ile Ser Asp Phe Ile Pro Ala Gly Pro
5 10 15Ala?Ser?Gly?Ala?Arg?Arg?Val?Thr?Ala?Asp?Ile?Leu?Trp?Pro?Ser
20 25 30Leu?Arg?Lys?Arg?Phe?Ser?Lys?Pro?Leu?Leu?Asp?Asp?Asp?Phe?Glu
35 40 45Ala?Gly?Phe?Arg?Glu?Phe?Lys?Asp?Asp?Ser?Glu?Ile?Glu?Asp?Val
50 55 60Asp?Asp?Glu?Asp?Asp?Glu?Asp?Glu?Glu?Glu?Leu?Lys?Lys?Lys?Pro
65 70 75Phe?Gly?Phe?Ser?Arg?Ser?Ser?Asn?Lys?Ala?Ala?Ser?Lys?Pro?Leu
80 85 90Ser?Arg?Gly?Ala?Thr?Thr?Val?Lys?Ser?Val?Glu?Ser?Lys?Gly?Gln
95 100 105Ala?Glu?Lys?Cys?Ala?Lys?Arg?Lys?Arg?Lys?Ash?Gln?Tyr?Arg?Gly
110 115 120Ile?Arg?Gln?Arg?Pro?Trp?Gly?Lys?Trp?Ala?Ala?Glu?Ile?Arg?Asp
125 130 135Pro?Arg?Lys?Gly?Val?Arg?Val?Trp?Leu?Gly?Thr?Phe?Ser?Thr?Ala
140 145 150Glu?Glu?Ala?Ala?Arg?Ala?Tyr?Asp?Ala?Glu?Ala?Arg?Arg?Ile?Arg
155 160 165Gly?Lys?Lys?Ala?Lys?Val?Asn?Phe?Pro?Asp?Glu?Pro?Ser?Gly?Ala
170 175 180Ala?Ser?Ser?Lys?Arg?Leu?Lys?Ala?Ash?Pro?Glu?Ala?Gln?Pro?Met
185 190 195Lys?Lys?Asn?Leu?Asn?Ser?Val?Lys?Pro?Lys?Ile?Asn?Gln?Met?Phe
200 205 210Asn?Phe?Gly?Asp?Asn?Leu?Glu?Gly?Tyr?Tyr?Ser?Pro?Ile?Asp?Gln
215 220 225Val?Glu?Gln?Lys?Pro?Leu?Val?Asn?Gln?Tyr?Val?Asn?Arg?Ala?Pro
230 235 240Phe?Ala?Gly?Asn?Gly?Val?Gln?Val?Ser?Pro?Val?Thr?Pro?Ser?Ala
245 250 255Asp?Val?Thr?Ala?Tyr?Phe?Ser?Ser?Glu?His?Ser?Ser?Asn?Ser?Phe
260 265 270Asp?Tyr?Ser?Asp?Leu?Gly?Trp?Gly?Glu?Gln?Val?Pro?Lys?Thr?Pro
275 280 285Glu?Ile?Ser?Ser?Leu?Leu?Ser?Ala?Ala?Pro?Leu?Glu?Gly?Ala?Ala
290 295 300Asp?Gln?Val?Gln?Lys?Thr?Asn?Asn?Ser?Gln?Asp?Val?Val?Ala?Ala
305 310 315Gln?Asp?Asp?Ser?Ala?Lys?Thr?Leu?Ser?Glu?Glu?Leu?Ala?Asp?Ile
320 325 330Glu?Ser?Gln?Leu?Lys?Phe?Phe?Glu?Thr?Pro?Ser?Phe?Leu?Asp?Glu
335 340 345Ala?Trp?Ala?Asp?Ala?Thr?Leu?Ala?Ser?Leu?Leu?Gly?Gly?Asp?Ala
350 355 360Thr?His?Asp?Ala?Ala?Gly?Asn?Pro?Met?Asn?Leu?Trp?Ser?Phe?Asp
365 370 375Asp?Leu?Pro?Ser?Met?Ala?Gly?Val?Phe
380 384
Claims (10)
1, ethylene response conjugated protein transcription factor GmEREB, be protein, or the amino acid residue sequence of sequence 2 is passed through replacement, disappearance or the interpolation of one or several amino-acid residue and has identical active by sequence 2 deutero-protein with the amino acid residue sequence of sequence 2 with sequence 2 amino acid residue sequences in the sequence table.
2, transcription factor according to claim 1 is characterized in that: it is the protein with sequence 2 amino acid residue sequences in the sequence table.
3, transcription factor according to claim 2 is characterized in that: the ERF function that is transcription factor GmEREB from the nitrogen end to 117-174 amino-acid residue conserved domain of carbon teminal in the described sequence 2 is guarded the territory.
4, the encoding gene of ethylene response conjugated protein transcription factor GmEREB is one of following nucleotide sequences:
1) dna sequence dna of sequence 1 in the sequence table;
2) with sequence table in the dna sequence dna that limits of sequence 1 have 90% above homology, and the identical function protein DNA sequence of encoding.
5, gene according to claim 4 is characterized in that: the encoding gene of described ethylene response conjugated protein transcription factor GmEREB is the dna sequence dna of sequence 1 in the sequence table.
6, gene according to claim 5 is characterized in that: the reading frame of this gene is for holding the 106th to the 1257th bit base from 3 '.
7, contain the described expression carrier of claim 4.
8, the clone that contains the described gene of claim 4.
9, the application of the described gene of claim 4 in cultivating degeneration-resistant border plant variety.
10, application according to claim 9 is characterized in that: described adverse circumstance is drought injury, cold damage and salt damage.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100349916C (en) * | 2005-11-22 | 2007-11-21 | 中国科学院遗传与发育生物学研究所 | PHD transcription factor of soybean and its coding gene and usage |
| CN100395337C (en) * | 2004-06-11 | 2008-06-18 | 中国农业科学院作物育种栽培研究所 | Plant ethylene response component binding protein and its coding gene |
| CN100395338C (en) * | 2004-06-11 | 2008-06-18 | 中国农业科学院作物育种栽培研究所 | Ethylene response component binding protein and its coding gene |
| 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 |
| CN102336826A (en) * | 2011-10-10 | 2012-02-01 | 吉林大学 | Transcription factor ERF related to soybean stress, coding gene thereof and application of coding gene |
| CN102516377A (en) * | 2012-01-12 | 2012-06-27 | 吉林大学 | Soybean ethylene responsive factor (ERF) transcription factor, and coding gene and salt tolerance application thereof |
| CN109021084A (en) * | 2018-08-06 | 2018-12-18 | 华中农业大学 | Trifoliate orange Cold resistant genes PtrERF109 and its application in plant cold resistance genetic improvement |
| CN109929019A (en) * | 2019-04-12 | 2019-06-25 | 东北农业大学 | A kind of and plant salt tolerance alkali GAP-associated protein GAP GsERF7 and its encoding gene and application |
| CN114107317A (en) * | 2021-10-22 | 2022-03-01 | 宁波大学 | Peach fruit ethylene response factor PpRAP2.12 gene and cloning method and application thereof |
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2002
- 2002-08-14 CN CN 02128818 patent/CN1216904C/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100395337C (en) * | 2004-06-11 | 2008-06-18 | 中国农业科学院作物育种栽培研究所 | Plant ethylene response component binding protein and its coding gene |
| CN100395338C (en) * | 2004-06-11 | 2008-06-18 | 中国农业科学院作物育种栽培研究所 | Ethylene response component binding protein and its coding gene |
| 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 |
| CN100349916C (en) * | 2005-11-22 | 2007-11-21 | 中国科学院遗传与发育生物学研究所 | PHD transcription factor of soybean and its coding gene and usage |
| CN102336826A (en) * | 2011-10-10 | 2012-02-01 | 吉林大学 | Transcription factor ERF related to soybean stress, coding gene thereof and application of coding gene |
| CN102516377A (en) * | 2012-01-12 | 2012-06-27 | 吉林大学 | Soybean ethylene responsive factor (ERF) transcription factor, and coding gene and salt tolerance application thereof |
| CN109021084A (en) * | 2018-08-06 | 2018-12-18 | 华中农业大学 | Trifoliate orange Cold resistant genes PtrERF109 and its application in plant cold resistance genetic improvement |
| CN109929019A (en) * | 2019-04-12 | 2019-06-25 | 东北农业大学 | A kind of and plant salt tolerance alkali GAP-associated protein GAP GsERF7 and its encoding gene and application |
| CN109929019B (en) * | 2019-04-12 | 2021-06-04 | 东北农业大学 | Plant saline-alkali tolerance associated protein GsERF7, and coding gene and application thereof |
| CN114107317A (en) * | 2021-10-22 | 2022-03-01 | 宁波大学 | Peach fruit ethylene response factor PpRAP2.12 gene and cloning method and application thereof |
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