CN1184231C - Wheat TaDREB, its code gene and method for culturing adverse-resistant plant - Google Patents
Wheat TaDREB, its code gene and method for culturing adverse-resistant plant Download PDFInfo
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- CN1184231C CN1184231C CNB011365692A CN01136569A CN1184231C CN 1184231 C CN1184231 C CN 1184231C CN B011365692 A CNB011365692 A CN B011365692A CN 01136569 A CN01136569 A CN 01136569A CN 1184231 C CN1184231 C CN 1184231C
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Abstract
The present invention provides new wheat TaDREB genes which comprise DREB transcription factors of an AP2/EREBP structural domain. The present invention also provides a method for cultivating adverse resisting plant by using the genes.
Description
Technical field
The present invention relates to a kind of AP
2/ EREBP class transcription factor DREB specifically describes, and the present invention relates to a kind of AP that derives from wheat (Triticum aestivum)
2/ EREBP class transcription factor DREB, called after TaDREB.The invention still further relates to the gene of this class transcription factor of coding, contain the carrier of this genoid and utilize this genoid to cultivate the method for plant with adverse resistance.
Technical background
The variation of physical chemical factor in the environment, for example: arid, saline and alkaline and damage to plants caused by sudden drop in temperature and make the extensive underproduction of farm crop.Therefore, cultivating the resistance of reverse crop is one of major objective of plant husbandry.Higher plant cell can have number of ways to experience the variation of physico-chemical parameter in the external environment, thereby the signal that born of the same parents are outer becomes intracellular signal, phosphorylation cascade reaction by series passes to transcription factor with signal, transcription factor remakes and is used for functional gene, thereby the expression of gene that the startup adverse circumstance is replied improves the resistance of reverse of plant.In plant, AP
2Class in the/EREBP class DREB transcription factor family can be accepted the environment-stress signal and start the adverse circumstance response gene.Wheat (Triticum aestivum) is an important crops.Cultivate and resist cold, salt tolerant, drought-enduring crop new variety are one of vital tasks in the Wheat Production.The contriver has cloned DREB transcription factor, called after TaDREB from wheat.This expression of gene is subjected to arid, salt, cold inducing.Make up the expression vector that is applicable to single, double cotyledon plant respectively, transformed unifacial leaf model plant paddy rice.The tangible resistance of reverse of transfer-gen plant tool.
Summary of the invention
An object of the present invention is to provide a kind of TaDREB transcription factor with stronger anti-reverse effect, this transcription factor derives from wheat (Triticum aestivum), and its aminoacid sequence is shown in Fig. 1 and SEQ ID NO:2.Sequence shown in Fig. 1 has been represented DREB transcription factor family in the wheat.Should be pointed out that transcription factor of the present invention is carried out one or more amino acid whose replacements, insertion and/or lack resulting functional analogue also reaching purpose of the present invention.Thereby the present invention also comprises with the aminoacid sequence shown in the SEQ ID NO:2 having at least 90% homology, preferably has at least 95% homology, but has the functional analogue of wheat DREB transcription factor activity simultaneously.
Another object of the present invention provides the gene of a kind of encoding transcription factor TaDREB, has represented a kind of DREB transcription factor family in the wheat at the gene shown in Fig. 1 and the SEQ ID NO:1, and they all contain conservative AP
2/ EREBP structural domain sequence.But be not limited to listed sequence.
A further object of the present invention provides a kind of method of cultivating anti-contrary plant, and this method comprises the gene constructed plant expression vector with transcription factor TaDREB of the present invention; Transform plant tissue with the expression vector that makes up; The plant transformed tissue cultivating is become plant.Described conversion can be undertaken by agriculture bacillus mediated or particle bombardment.
Another purpose of the present invention provides a kind of expression vector (Fig. 2) that contains mentioned TaDREB class transcription factor.Can use any expression vector that can guide foreign gene in plant, to express.These plant expression vectors include but not limited to, the double base agrobacterium vector, for example Bin19 (Bevan, M., 1984, nucleic acids research, 12:8711-8721), the carrier that contains the promotor of DRE element and be used for the monocotyledons micropellet bombardment.Carrier also can comprise 3 ' untranslated zone.3 ' untranslated zone is meant a kind of part of gene, but it contain a kind of comprise the polyadenylic acid signal and any other effect mRNA processing or the dna fragmentation of genetic expression.Polyadenylic acid signal guidance polyadenylic acid joins 3 ' end of mRNA precursor.The example in 3 ' zone comprises that containing the Agrobacterium crown-gall nodule induces (Ti) plasmid gene (for example rouge alkali synthetase (Nos gene)), and the 3 ' non-translational region of transcribing of plant gene (for example big storage protein gene).3 ' untranslated zone of DREB transcription factor of the present invention can be used to express in plant.
Carrier of the present invention also can contain suitable promotor.Can use any strong promoter in the present invention.These promotors include but not limited to cauliflower mosaic virus (CAMV 35S) and Ubiqutin promotor.It can use separately or be used in combination with other plant promoter.
Expression vector of the present invention also can comprise enhanser when needed, no matter be translational enhancer or transcriptional enhancer.These enhanser zones include but not limited to ATG initiator codon and neighboring region.Initiator codon must with the reading frame homophase of encoding sequence, to guarantee the translation of whole sequence.The translation control signal can be multiple different source with initiator codon, can be natural or synthetic.Translation initiation region can be from the transcription initiation zone, or from structure gene.
For the ease of transgenic plant cells or plant being identified and screening, can process employed carrier, comprise adding the alternative mark of plant.Spendable selected marker comprises the enzyme to antibiotics resistance, and microbiotic comprises gentamicin, Totomycin, kantlex etc.Equally, can use the enzyme that produces by the compound of colour-change (for example GUS) or the identification of luminous (for example luciferase) fork-like farm tool used in ancient China, or anti-chemical reagent (for example weedkiller).In addition, also can any selection markers.
Expression vector of the present invention can be by using Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, importing such as electroporation vegetable cell.The summary of these technology is consulted Weissbach and Weissbach, molecular biology of plants method (Method for PlantMolecular Biology), Academy Press, New York VIII, pp.421-463 (1988); With Geiserson and Corey, molecular biology of plants (Plant Molecular Biology), second edition (1988).
Can use method plant transformed host of the present invention to comprise monocotyledons and dicotyledons, for example: paddy rice, wheat, corn, cucumber, tomato, willow, turfgrass, lucerne place etc.
Brief Description Of Drawings
Fig. 1 represents the Nucleotide and the deduced amino acid of TaDREB gene;
Fig. 2 represents the structure of TaDREB plant expression vector;
Fig. 3 represents that difference coerces down the TaDREB expression of gene feature that detects through RT-PCR;
Fig. 4 represents the compositional analysis of TaDREB gene in the wheat cdna group.
Embodiment
One, wheat cDNA library construction and transcription factor TaDREB cDNA clone, sequential analysis
Little wheat seeds after 14 days, take out in basin, soak 3-5 hour in 2.0%NaCl solution.The cDNA library construction is undertaken by Sambrook (1987) method.Collect fresh complete stool blade 1g and grind in liquid nitrogen, be suspended from the 4mol/L sulphur hydracid guanidine, mixture adds dehydrated alcohol and precipitates total RNA with acid phenol, chloroform extracting in the supernatant, afterwards, water-soluble.Obtain mRNA through the processing of mRNA purification kit.Get 2ug mRNA and carry out reverse transcription reaction, and then carry out two chains and synthesize.Double-stranded cDNA is connected the back phosphorylation with the EcoR1 joint, (Sephacryl-400) removes unnecessary joint through the preparative centrifugation post, the dephosphorization pExcell that cuts with enzyme then, and carrier (Pharmacia) connects, and gets 5ul connection product adding packaging protein, builds up the cDNA library.
With Arabidopis thaliana DREB cDNA is that probe has screened 4.5 * 10
5The spot that do not increase obtains 3 positive spots, and it is 1.29kb that its skewer is gone into fragment.This fragment comprises the open reading frame of 834bp, the polypeptide that coding is made up of 278 amino acid, and its 5 ' end is 251bp, 3 ' end is 173bp.Wheat TaDREB cDNA complete sequence and aminoacid sequence are seen Fig. 1.
The structure of two .TaDREB cDNA plant expression plasmids and agrobacterium mediation converted Arabidopis thaliana
The structure of TaDREB cDNA plant expression vector carries out according to a conventional method.Binary expression vector pB1121 contains the fragment of 35S promoter and translational enhancer TMV.The plant expression vector that will contain 35S promoter directly transforms Agrobacterium, and environmental Co1-0 (the Arabidopis thaliana biology resource center) plant of Arabidopis thaliana is used for Agrobacterium-mediated Transformation, and agrobacterium strains is EH105 (Inst. of Genetics and Development Biology, CAS).
1. vegetable material is prepared
(1) with the vernalization 72 hours under 4 ℃ of conditions of Arabidopis thaliana Co1-0 seed.With 1/3 * B
5Nutritive medium soaks the plantation basin that vermiculite (vermiculite) is housed, after planting in 23 ℃ of cultivations.
(2) treat plant blossom after, cut off its major branch top, promote the side shoot development.In 4-6 after beta pruning days, set about preparing Agrobacterium-mediated Transformation.
2. Agrobacterium is cultivated
The expression of exogenous gene carrier electric shock of having that (3) will build transforms Agrobacterium, and PCR and enzyme are cut and checked and shake bacterium after errorless.
(4) Agrobacterium is inoculated in the 5ml LB+50 μ g/ml rifampin+50 μ g/ml kantlex substratum, shakes bacterium and spend the night.Rolling bottle to 1 rose in the LB substratum in second day, and 28 ℃ are cultured to OD
600About=0.8.
(5) centrifugal collection thalline transforms damping fluid suspension Agrobacterium with 1 liter.About 300-400ml can transform 2 basin plant.
3. vacuumize conversion
(6) 4-6 after the beta pruning days plant is inverted in contains in the vial that transforms damping fluid, vacuumize, kept 0.05Mpa pressure 5-10 minute.
(7) take out the plantation basin, be sidelong 24 hours.It is upright to plant basin then, cultivates plant to solid by usual method, the results mature seed.
Three, the resistance of reverse of gene plant is identified
Transfer-gen plant and adjoining tree be transplanted to respectively contain 150mM, continued growth in the MS substratum of 200mM and 250mMNaCl, under a plurality of salt concn, transfer-gen plant grows good and adjoining tree wiltings of turning white gradually, all death of back all around.
Four analyses of .TaDREB gene in the wheat cdna group
With TaDREB is probe, carries out Southern Blot and analyze under the rigorous degree of difference, when washing film under 65 ℃, presents many hybrid belts, shows a plurality of copies that exist the TaDREB gene in the wheat cdna group.Banding pattern when washing film under 45 ℃ of low rigorous conditions is consistent with 65 ℃, shows that the TaDREB gene does not have other homology analogue (Fig. 4) in the wheat cdna group.
Five, the expression characteristic of wheat TaDREB in adverse circumstance
With 2.0%NaCl, 20%PEG and 30mM ABA solution and 4 ℃ of subzero treatment wheats 1 day respectively 0,2, were collected blade in 5,24 hours, extracted that RNA makes RT-PCR and Northern analyzes.The result shows that transcribing of TaDREB is subjected to salt in leaf, and arid and ABA coerce and the inducing in various degree of low temperature adverse circumstance (Fig. 3).
Sequence table
＜110 〉
1Institute of Genetics, Academia Sinica
Tsing-Hua University
＜120〉method of wheat TaDREB, its encoding gene and cultivation plant with adverse resistance
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caaaaccaag?gcggcggcag?cggggcggga?gagcggggag?caccgaccga?caccggccga 60
cagggtgggc?tgcatgcgga?gctgaggcga?ggcgaggcga?ggcggggaga?gatccggcgc 120
gggtgccacc?gccggccggc?cgcgggagat?ctggttggtg?gcgccgcccg?gataagggag 180
aggcggcgag?gggagagcag?ccgggggaga?ccgaggcgag?aggagatctc?tctcgtccct 240
cttctcgctc?c?atg?gag?acc?ggg?ggt?agc?aag?cgg?gaa?gga?gac?tgc?ccc 290
Met?Glu?Thr?Gly?Gly?Ser?Lys?Arg?Glu?Gly?Asp?Cys?Pro
1 5 10
ggg?cag?gaa?agg?aag?aag?aaa?gtg?cgc?agg?aga?agc?act?ggt?cct?gat 338
Gly?Gln?Glu?Arg?Lys?Lys?Lys?Val?Arg?Arg?Arg?Ser?Thr?Gly?Pro?Asp
15 20 25
tcg?gtt?gct?gaa?acc?atc?aag?aag?tgg?aag?gag?gaa?aac?cag?aag?ctc 386
Ser?Val?Ala?Glu?Thr?Ile?Lys?Lys?Trp?Lys?Glu?Glu?Asn?Gln?Lys?Leu
30 35 40 45
cag?caa?gag?aat?gga?tcc?cgg?aaa?gca?ccg?gcc?aag?ggt?tcc?aag?aaa 434
Gln?Gln?Glu?Asn?Gly?Ser?Arg?Lys?Ala?Pro?Ala?Lys?Gly?Ser?Lys?Lys
50 55 60
ggg?tgc?atg?gca?ggg?aaa?gga?ggt?cca?gag?aat?tca?aac?tgc?gct?tac 482
Gly?Cys?Met?Ala?Gly?Lys?Gly?Gly?Pro?Glu?Asn?Ser?Asn?Cys?Ala?Tyr
65 70 75
cgc?ggt?gtg?agg?cag?agg?acg?tgg?ggg?aaa?tgg?gtt?gct?gag?atc?cgt 530
Arg?Gly?Val?Arg?Gln?Arg?Thr?Trp?Gly?Lys?Trp?Val?Ala?Glu?Ile?Arg
80 85 90
gag?ccc?aac?cgt?ggc?aat?cgg?ctg?tgg?ctt?ggt?tca?ttc?cct?acc?gca 578
Glu?Pro?Asn?Arg?Gly?Asn?Arg?Leu?Trp?Leu?Gly?Ser?Phe?Pro?Thr?Ala
95 100 105
gtc?gaa?gct?gca?cgt?gca?tat?gat?gat?gcg?gca?agg?gca?atg?tat?ggc 626
Val?Glu?Ala?Ala?Arg?Ala?Tyr?Asp?Asp?Ala?Ala?Arg?Ala?Met?Tyr?Gly
110 115 120 125
gcc?aaa?gca?cgt?gtc?aac?ttc?tca?gag?cag?tcc?ccg?gat?gcc?aac?tct 674
Ala?Lys?Ala?Arg?Val?Asn?Phe?Ser?Glu?Gln?Ser?Pro?Asp?Ala?Asn?Ser
130 135 140
ggt?tgc?acg?ctg?gca?cct?cca?ttg?ccg?atg?tct?aat?ggg?gca?acc?gct 722
Gly?Cys?Thr?Leu?Ala?Pro?Pro?Leu?Pro?Met?Ser?Asn?Gly?Ala?Thr?Ala
145 150 155
gcg?tca?cat?cct?tct?gat?ggg?aag?gat?gaa?tcg?gag?tct?cct?cct?tct 770
Ala?Ser?His?Pro?Ser?Asp?Gly?Lys?Asp?Glu?Ser?Glu?Ser?Pro?Pro?Ser
160 165 170
ctt?atc?tca?aat?gcg?ccg?aca?gct?gcg?ctg?cat?cgg?tct?gat?gct?aag 818
Leu?Ile?Ser?Asn?Ala?Pro?Thr?Ala?Ala?Leu?His?Arg?Ser?Asp?Ala?Lys
175 180 185
gat?gag?tct?gag?tct?gca?ggg?acc?gtg?gca?cgt?aag?gtg?aaa?aaa?gaa 866
Asp?Glu?Ser?Glu?Ser?Ala?Gly?Thr?Val?Ala?Arg?Lys?Val?Lys?Lys?Glu
190 195 200 205
gtg?agc?aat?gat?ttg?aga?agt?acc?cat?gag?gag?cac?aag?acc?ctg?gaa 914
Val?Ser?Asn?Asp?Leu?Arg?Ser?Thr?His?Glu?Glu?His?Lys?Thr?Leu?Glu
210 215 220
gta?tcc?caa?cca?aaa?ggg?aag?gct?tta?cat?aaa?gca?gcg?aac?gta?agt 962
Val?Ser?Gln?Pro?Lys?Gly?Lys?Ala?Leu?His?Lys?Ala?Ala?Asn?Val?Ser
225 230 235
tat?gat?tac?ttc?aac?gtc?gag?gaa?gtt?ctt?gac?atg?ata?att?gtg?gaa 1010
Tyr?Asp?Tyr?Phe?Asn?Val?Glu?Glu?Val?Leu?Asp?Met?Ile?Ile?Val?Glu
240 245 250
ttg?agt?gct?gat?gta?aaa?atg?gaa?gca?cat?gaa?gag?tac?caa?gat?ggt 1058
Leu?Ser?Ala?Asp?Val?Lys?Met?Glu?Ala?His?Glu?Glu?Tyr?Gln?Asp?Gly
255 260 265
gat?gat?ggg?ttt?agt?ctt?ttc?tca?tat?tagggtttta?gctatgaggg 1105
Asp?Asp?Gly?Phe?Ser?Leu?Phe?Ser?Tyr
270 275
ttgcagtcat?gcggagcaat?agggataact?ttcattctag?ctgctaggaa?atacttcaaa 1165
tctgcaaccc?gaagctttgt?agtcacttat?ggttttcatc?ttactggaga?gaatagcttt 1225
ataccataag?tcaacgggta?caagaagttg?tcctgtgcgt?tgagttcatg?tactatggta 1285
aaagttg 1292
<210>2
<211>278
<212>PRT
<213>Triticum?aestivum
<400>2
Met?Glu?Thr?Gly?Gly?Ser?Lys?Arg?Glu?Gly?Asp?Cys?Pro?Gly?Gln?Glu
1 5 10 15
Arg?Lys?Lys?Lys?Val?Arg?Arg?Arg?Ser?Thr?Gly?Pro?Asp?Ser?Val?Ala
20 25 30
Glu?Thr?Ile?Lys?Lys?Trp?Lys?Glu?Glu?Asn?Gln?Lys?Leu?Gln?Gln?Glu
35 40 45
Asn?Gly?Ser?Arg?Lys?Ala?Pro?Ala?Lys?Gly?Ser?Lys?Lys?Gly?Cys?Met
50 55 60
Ala?Gly?Lys?Gly?Gly?Pro?Glu?Asn?Ser?Asn?Cys?Ala?Tyr?Arg?Gly?Val
65 70 75 80
Arg?Gln?Arg?Thr?Trp?Gly?Lys?Trp?Val?Ala?Glu?Ile?Arg?Glu?Pro?Asn
85 90 95
Arg?Gly?Asn?Arg?Leu?Trp?Leu?Gly?Ser?Phe?Pro?Thr?Ala?Val?Glu?Ala
100 105 110
Ala?Arg?Ala?Tyr?Asp?Asp?Ala?Ala?Arg?Ala?Met?Tyr?Gly?Ala?Lys?Ala
115 120 125
Arg?Val?Asn?Phe?Ser?Glu?Gln?Ser?Pro?Asp?Ala?Asn?Ser?Gly?Cys?Thr
130 135 140
Leu?Ala?Pro?Pro?Leu?Pro?Met?Ser?Asn?Gly?Ala?Thr?Ala?Ala?Ser?His
145 150 155 160
Pro?Ser?Asp?Gly?Lys?Asp?Glu?Ser?Glu?Ser?Pro?Pro?Ser?Leu?Ile?Ser
165 170 175
Asn?Ala?Pro?Thr?Ala?Ala?Leu?His?Arg?Ser?Asp?Ala?Lys?Asp?Glu?Ser
180 185 190
Glu?Ser?Ala?Gly?Thr?Val?Ala?Arg?Lys?Val?Lys?Lys?Glu?Val?Ser?Asn
195 200 205
Asp?Leu?Arg?Ser?Thr?His?Glu?Glu?His?Lys?Thr?Leu?Glu?Val?Ser?Gln
210 215 220
Pro?Lys?Gly?Lys?Ala?Leu?His?Lys?Ala?Ala?Asn?Val?Ser?Tyr?Asp?Tyr
225 230 235 240
Phe?Asn?Val?Glu?Glu?Val?Leu?Asp?Met?Ile?Ile?Val?Glu?Leu?Ser?Ala
245 250 255
Asp?Val?Lys?Met?Glu?Ala?His?Glu?Glu?Tyr?Gln?Asp?Gly?Asp?Asp?Gly
260 265 270
Phe?Ser?Leu?Phe?Ser?Tyr
275
Claims (8)
1. AP who derives from wheat
2/ EREBP class transcription factor TaDREB, it has the aminoacid sequence shown in the SEQ IDNO:2.
2. AP of the described wheat of claim 1 that encodes
2The gene of/EREBP class transcription factor TaDREB.
3. according to the described gene of claim 2, it has the nucleotide sequence shown in the SEQ ID NO:1.
4. plant expression vector that contains the gene of claim 2 or 3 described TaDREB transcription factors.
5. according to the described plant expression vector of claim 4, wherein, described plant expression vector is the double base agrobacterium vector.
6. a method of cultivating the resistance of reverse plant comprises the gene constructed plant expression vector with claim 2 or 3 described TaDREB transcription factors; With the expression vector transformed plant cells that makes up; With the plant transformed cell culture is become plant.
7. in accordance with the method for claim 6, wherein, described conversion is undertaken by agriculture bacillus mediated or particle bombardment.
8. in accordance with the method for claim 6, wherein said plant is monocotyledons or dicotyledons.
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100344759C (en) * | 2005-11-08 | 2007-10-24 | 天津师范大学 | cDNA sequence of dehydrolysis responding transcription factor DREB gene of wheal |
| CN102234320B (en) * | 2010-04-27 | 2013-07-31 | 中国农业科学院作物科学研究所 | Plant stress-tolerant associated protein TaDREB4B and encoding gene and application thereof |
| CN103755793B (en) * | 2014-01-28 | 2016-08-24 | 中国科学院新疆生态与地理研究所 | The most drought-enduring 4 DREB transcription factor of the red moss of tooth rib and encoding gene thereof |
| CN104910263B (en) * | 2014-03-12 | 2018-03-13 | 中国农业科学院作物科学研究所 | Plant stress tolerance correlative protein TaPPR and its encoding gene and application |
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