CN1216905C - Transcription factor for regulating and controlling dry land cotton anti contrary property and its coded gene and application - Google Patents
Transcription factor for regulating and controlling dry land cotton anti contrary property and its coded gene and application Download PDFInfo
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Abstract
The present invention discloses a transcription factor for regulating and controlling the stress resistance of upland cotton, a coded gene thereof and an application thereof. The stress resistant transcription factor of the present invention, which is called GhDREB, is protein with an amino acid residue sequence of sequence 2 in a sequence list, or a protein which is used for substituting, deleting or adding one or a plurality of amino acid residues of the amino acid residue sequence of sequence 2, has the same activity with the amino acid residue sequence of sequence 2, and is derived from sequence 2. The coded gene of the stress resistant transcription factor GhDREB is one of the following nucleotide sequences: 1) a DNA sequence of sequence 1 in the sequence list, and 2) a DNA sequence which has more than of 90 % of homology with the DNA sequence limited by sequence 1 in the sequence list and codes the same functional protein. The gene of the present invention has important meanings for breeding stress resistant plant varieties, especially drought resistant plant varieties, cold resistant plant varieties and salt resistant plant varieties, and increasing crop yield.
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 upland cotton.
Background technology
Biologies such as cause of disease, arid, saline and alkaline, low temperature, freeze injury, waterlogging and the abiotic environment factor of coercing have material impact to growth and development of plant.Improve the resistance of reverse of crop, except utilizing traditional breeding method, one of field that the molecular genetic breeding has become present scientific worker to be paid close attention to.Under the environment stress environment, 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.Verified, some transcripton of DREB class can be accepted the environment-stress signal and start the adverse circumstance response gene, improves the resistance of reverse of plant.In plant, the conservative territory of AP2/EREBP function is the specificity structure of DREB class transcription factor.
Cotton is improved its resistance and has important economic value as a kind of important cash crop, is the emphasis of cotton breeding research.
Summary of the invention
The purpose of this invention is to provide transcription factor that derives from upland cotton and encoding gene thereof with strong adverse-resistant characteristic.
Anti-reverse transcription factor names provided by the present invention is GhDREB, be protein with sequence 2 amino acid residue sequences in the sequence table, or with the amino acid residue sequence of sequence 2 through replacement, disappearance or the interpolation of 1 or several amino acid residue and have identical active by sequence 2 deutero-protein with the amino acid residue sequence of sequence 2.
The protein that sequence 2 amino acid residue sequences are made up of 153 amino-acid residues in the sequence table.The ERF function that is transcription factor GhDREB from the nitrogen end to 29-96 amino-acid residue conserved domain of carbon teminal in the described sequence 2 is guarded the territory.
The encoding gene of anti-reverse transcription factor GhDREB is the dna sequence dna with sequence 1 nucleotide sequence in the sequence table.
The dna sequence dna of sequence 1 is by 866 based compositions in the sequence table, and the reading frame of this gene is that it is expressed and mainly is subjected to inducing of arid, low temperature, high salt and external dormin, ethene from 5 ' end the 106th to the 565th bit base.
Utilize any carrier that can guide foreign gene in plant, to express, derive and next pBI101 as pBIN19 and by it, pBI121 and pBI221 serial carrier (Bevan, 1984 nucleic acids research, 12:8711-8721), with the gene transfered plant cell of encoding transcription factor GhDREB 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.Expression vector pBI121-GhDREB is the expression vector that has GhDREB cDNA of the present invention that utilizes conventional molecular biology method to make up, and its gene mapping as shown in Figure 1.Gene of the present invention can add any enhancing promotor or inducible promoter, as cauliflower mosaic virus (CaMV 35S) and Ubiquitin promotor etc. 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 the antibiotic marker thing (gentamicin, kantlex etc.) that adds the alternative mark (gus gene, luciferase genes etc.) of plant or have resistance.Carry that GhDREB expression carrier of the present invention can conventional biotechnological means imports vegetable cell by using that Ti (the Tumor-induced-cancer is induced) plasmid, Ri (Root-induced-root induction) plasmid, plant viral vector, directly DNA conversion, microinjection, electricity are led etc., 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 structure of GhDREB expression vector.
Fig. 2 is the vivoexpression of GhDREB gene in intestinal bacteria.
Fig. 3 is Southern hybridization analysis result.
Fig. 4 is the GhDREB allelic expression that Northern hybridization detects under the different stress conditions.
Embodiment
The clone and the sequential analysis of embodiment 1, cotton GhDREB transcription factor cDNA
The cotton seedling of growing 10 days is at room temperature handled after washing only.It is that seedling was placed on the filter paper of adequate thickness dehydration 5 hours that arid is handled, collecting plant is ground to fine degree, places the 4mol/L guanidinium isothiocyanate in the mortar that liquid nitrogen is housed, use acid phenol/chloroform extracting mixture again, get supernatant liquor, with the total RNA of isopropanol precipitating, repeat above-mentioned steps more once, after after 75% the washing with alcohol drying RNA is dissolved in DEPC water, be stored in-80 ℃ of ((Davis etc., molecular biological basic skills: [Basic Methods in MolecularBiology], pp.777, APPLETON ﹠amp; LANCE, Norwalk, Connecticut, USA, 1994).
Get the 12 couple special degenerated primers of the total RNA of 1 μ g by following described three forward primers and the combination of four reverse primers and carry out separately an independently step reverse transcription chain type amplified reaction (PCR), the conservative territory of amplification AP2/EREBP.
Article 3, guard 5 '-GIRMRK-3 ' forward primer (Forwardprimers) of territory N-end corresponding to AP2/EREBP:
GDF1：5’-GGIAT(A/C/T)CGIATGCGIAA(A/G)-3’；
GDF2：5’-GGIAT(A/C/T)CGIATGAG(A/G)AA(A/G)-3’；
GDF3：5’-GGIAT(A/C/T)AG(A/G)ATGCGIAA(A/G)-3’；
Article 4, guard 5 '-ARLNFP-3 ' reverse primer (Reverseprimers) of territory C-end corresponding to AP2/EREBP:
GDR1：5’-IGG(A/G)AA(A/G)TTIAGICGIGC-3’；
GDR2：5’-IGG(A/G)AA(A/G)TTIAG(C/T)CTIGC-3’；
GDR3：5’-IGG(A/G)AA(A/G)TT(T/C)AA(T/C)CTIGC-3’；
GDR4：5’-IGG(A/G)AA(A/G)TT(T/C)AAICGIGC-3’。
In above-mentioned primer, I is hypoxanthylic acid (inosine), and the PCR condition is: 94 ℃, and 3 minutes; 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 1.5 minutes-30 the circulation; 72 ℃ 10 minutes; 4 ℃ of preservations.The PCR product is connected to and is used for order-checking on the pMD18-T carrier, is that template is utilized a pair of Auele Specific Primer again with the positive colony:
GhDF1R：5’-CTTCCTCATCCTTATCCC-3’；
GhDR1F:5 '-GCCAGGCTCAACTT CCCT-3 ', (the PCR program is seen kit D6122 to 5 ' end by reverse transcription PCR reaction amplifying target genes, Takara Biotech. (Dalian) Co.Ltd) and 3 ' end (the PCR program is seen kit D6121, Takara Biotech. (Dalian) Co.Ltd).External then packing Invitro-GmDREB gene order designs a pair of Auele Specific Primer again
GhDREBFd：5’-AAAA
GAATTCATGGAGCTAGGTGATTGTTG-3’(107-126)；
GhDREBRe：5’-AAAA
CTCGAGATCTTCATCAGAACTGTCAG-3’(562-546)；
94 ℃, 3 minutes; 94 ℃ 1 minute, 58 ℃ 1 minute, 72 ℃ 1.5 minutes-30 the circulation; 72 ℃ 10 minutes; 4 ℃ of preservations, amplification GhDREB cDNA, the PCR product is connected to the pMD18-T carrier and checks order, and has obtained one and the on all four cDNA clone of external packaging sequence, called after GhDREB.It is 866bp that this gene inserts fragment, the open reading frame that contains 459bp, the polypeptide that 153 amino acid of encoding are formed, contain a nuclear signal location conservative region and an activation domain conservative region that is rich in acidic amino acid that is rich in basic aminoacids respectively at aminoterminal and carboxyl terminal, its 5 ' end is 106bp, and 3 ' end is 179bp.
The structure of embodiment 2, the fusion expression vector that is used to transform.
The structure of GhDREB cDNA expression vector molecular biology method routinely carries out.Utilize GhDREB cDNA to carry out pcr amplification and be inserted into CaMV35S promotor back in the pBI121 binary expression vector GhDREB coding region by the BamHI of forward primer end and the SacI site of reverse primer end as template, obtain a fusion expression vector pBI121-GhDREB (collection of illustrative plates as shown in Figure 1), through enzyme cut identify to insert fragment errorless after, transform in Agrobacterium, extract plasmid again and cut affirmation through enzyme and successfully transform in Agrobacterium, this fusion expression vector can directly be used for the particularly gene transformation of cotton of plant by transgenic technology.Wherein, the LUC luciferase genes is as the marker of the positive transfer-gen plant of screening.
Embodiment 3, the vivoexpression of cotton GhDREB gene in a big bacillus are identified
GhDREB cDNA coding region is connected on the expression vector pGEX-4T-1, induces in intestinal bacteria through IPTG, SDS-PAGE detects and shows that this gene can carry out external accurate translation in intestinal bacteria.And there do not have protein expression to take out of without the inductive bacterial strain to be existing.The polyacrylate hydrogel electrophoretic band as shown in Figure 2, 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.
Embodiment 4, the analysis of GhDREB gene in the cotton gene group.
Utilize GhDREB cDNA as probe, with through different restriction enzyme (EcoRI, EcoRV, P-PstI and XhoI) postdigestive cotton genomic dna carries out the Southern hybridization analysis under 65 ℃ of conditions, and (condition of high rigorous degree is: 0.5 * SSC to wash film under the rigorous degree of height, 0.1 * SDS, 65 ℃), the result as shown in Figure 3, except that the genomic dna with EcoRV and XhoI (because the two locates a site respectively on 395 and 510 position) digestion presents faint two hybrid belts, other a hybrid belt all only occurs, shows that the GhDREB gene is a single copy gene in the cotton gene group, perhaps exists the GhDREB autoploid of low copy.
Embodiment 5, the expression characteristic of cotton GhDREB gene under the environment stress condition
The cotton seedling in 2 week of growth is placed 4 ℃ of water respectively, the NaCl aqueous solution of 250mM, and 100 carry out illumination cultivation in μ M dormin (ABA) solution, it is that cotton 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, and respectively 1 hour, 3 hours, 5 hours, 7 hours, 12 hours and sampling in 24 hours, extract total RNA, carry out the Northern hybridization analysis with GhDREB cDNA probe.The result as shown in Figure 4, 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 acid-treated result that comes off, and as can be seen from the figure, transcribing of gene GhDREB mainly is subjected to low temperature, salt and drought-induced expression, and external dormin and second is rare also can induce this expression of gene.
Sequence table
<160>2
<210>1
<211>866
<212>DNA
＜213〉Gossypium upland cotton (Gossypium hirsutum L.)
<400>1
tttttctcct?tcttatggct?tacaagttat?agtttccaga?aacaagggga?aataaaaaaa?60
aatccatttt?gttttaggat?tttggtcttc?ttttatcttt?tgggtcatgg?agctaggtga?120
ttgttgttta?acatcaagtc?cagcaagcgg?agagaagcga?aagctgcata?ggacacagca?180
aaaggagaaa?ccattcagag?ggataaggat?gaggaagtgg?ggaaagtggg?tcgctgaaat?240
cagagaaccc?aacaagcggt?ccaggatttg?gcttggttct?tacaccaccc?ctgtagccgc?300
cgctcgtgct?tatgacacag?ccgttttcta?cttgcggggt?ccttccgcca?ggctcaactt?360
ccctgacctc?atattccaag?aagacgagct?tagggatatc?tcagccgctt?ccatacgcaa?420
gaaagcaacc?gaggtggggg?ctaaagtcga?cgctttacag?acctcactcc?atcatgcttc?480
tgcttcatca?tcggaatcat?cgaaccctac?tcgagttttc?cgtaaacctg?atttgaacaa?540
gtaccctgac?agttctgatg?aagattgaaa?acaatataat?agtttaccat?aacccaaaaa?600
cctataactg?ttcatactcg?ccactttttt?tttttcccca?cttatgtctt?tttgattctc?660
tcatggaata?gcaagtgccc?aaagaataga?atcaataaaa?aaatggattg?attctctgtt?720
tgataaggtt?atgaatgttt?tactttggga?atacaagttt?agtttatggc?atgtaacttt?780
taacatgaat?ggatattgat?gaaacaggga?tatatatcgc?tgagttagta?aatactccta?840
attccttgac?caaaaaaaaa?aaaaaa 866
<210>1
<211>153
<212>PRT
＜213〉Gossypium upland cotton (Gossypium hirsutum L.)
<400>2
Met?Glu?Leu?Gly?Asp?Cys?Cys?Leu?Thr?Ser?Ser?Pro?Ala?Ser?Gly
5 10 15
Glu?Lys?Arg?Lys?Leu?His?Arg?Thr?Gln?Gln?Lys?Glu?Lys?Pro?Phe
20 25 30
Arg?Gly?Ile?Arg?Met?Arg?Lys?Trp?Gly?Lys?Trp?Val?Ala?Glu?Ile
35 40 45
Arg?Glu?Pro?Asn?Lys?Arg?Ser?Arg?Ile?Trp?Leu?Gly?Ser?Tyr?Thr
50 55 60
Thr?Pro?Val?Ala?Ala?Ala?Arg?Ala?Tyr?Asp?Thr?Ala?Val?Phe?Tyr
65 70 75
Leu?Arg?Gly?Pro?Ser?Ala?Arg?Leu?Asn?Phe?Pro?Asp?Leu?Ile?Phe
80 85 90
Gln?Glu?Asp?Glu?Leu?Arg?Asp?Ile?Ser?Ala?Ala?Ser?Ile?Arg?Lys
95 100 105
Lys?Ala?Thr?Glu?Val?Gly?Ala?Lys?Val?Asp?Ala?Leu?Gln?Thr?Ser
110 115 120
Leu?His?His?Ala?Ser?Ala?Ser?Ser?Ser?Glu?Ser?Ser?Asn?Pro?Thr
125 130 135
Arg?Val?Phe?Arg?Lys?Pro?Asp?Leu?Asn?Lys?Tyr?Pro?Asp?Ser?Ser
140 145 150
Asp?Glu?Asp
153
Claims (7)
1, anti-reverse transcription factor GhDREB, be protein with sequence 2 amino acid residue sequences in the sequence table, or with the amino acid residue sequence of sequence 2 through replacement, disappearance or the interpolation of 1 or several amino acid residue and have identical active by sequence 2 deutero-protein with the amino acid residue sequence of sequence 2.
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, the encoding gene of anti-reverse transcription factor GhDREB, it has the nucleotide sequence of sequence 1 in the sequence table.
4, contain the described expression carrier of claim 3.
5, expression vector according to claim 4 is characterized in that: described expression vector is pBI121-GhDREB.
6, the transgenic cell line that contains the described gene of claim 3.
7, the application of the described gene of claim 3 in cultivating drought resisting, cold-resistant, salt-resistant plant kind.
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| CN021288712A CN1216905C (en) | 2002-08-16 | 2002-08-16 | Transcription factor for regulating and controlling dry land cotton anti contrary property and its coded gene and application |
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| CN021288712A CN1216905C (en) | 2002-08-16 | 2002-08-16 | Transcription factor for regulating and controlling dry land cotton anti contrary property and its coded gene and application |
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| CN1475497A CN1475497A (en) | 2004-02-18 |
| CN1216905C true CN1216905C (en) | 2005-08-31 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101456908B (en) * | 2008-12-31 | 2011-10-05 | 中国科学院遗传与发育生物学研究所 | Transcription factor protein and coding gene thereof and application |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7598429B2 (en) | 2001-04-18 | 2009-10-06 | Mendel Biotechnology, Inc. | Transcription factor sequences for conferring advantageous properties to plants |
| CN103159840B (en) * | 2013-03-28 | 2014-06-11 | 浙江省萧山棉麻研究所 | Anti-reverse transcription factor PsDREB1 from peony, as well as encoding gene and application thereof |
| JP7091237B2 (en) * | 2016-03-24 | 2022-06-27 | 株式会社日清製粉グループ本社 | Detection method of genetically modified crops |
| CN112111508A (en) * | 2019-06-20 | 2020-12-22 | 新疆农业科学院核技术生物技术研究所（新疆维吾尔自治区生物技术研究中心） | Cotton stress-tolerant gene GhCBF and coding protein and application thereof |
| CN113151307B (en) * | 2021-06-11 | 2022-09-30 | 云南中烟工业有限责任公司 | Gene related to tobacco ethylene response transcription factor and application thereof |
-
2002
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101456908B (en) * | 2008-12-31 | 2011-10-05 | 中国科学院遗传与发育生物学研究所 | Transcription factor protein and coding gene thereof and application |
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| CN1475497A (en) | 2004-02-18 |
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