CN1223606C - Rice DREB transcription factor and its encoding gene and use - Google Patents
Rice DREB transcription factor and its encoding gene and use Download PDFInfo
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Abstract
The present invention discloses a paddy rice DREB type transcription factor, a coding gene and the application thereof, more specifically a paddy rice DREB type clod and drought resisting transcription factor, a coding gene and the application thereof. The aim of the present invention is to provide the paddy rice DREB type transcription factor and the coding gene thererof, and the transcription factor has the adjusting and controlling effects on a cold and drought resisting reaction of plants. The name of the DREB type transcription factor is OsDREB, and the DREB type transcription factor comprises an amino acid residue sequence of a sequence 2 in a sequence table or protein, wherein the protein replaces, deletes or adds an amino acid residue sequence of the sequence 2 by one or a plurality of amino acid residues; furthermore, the protein has the same activity as the amino acid residue sequence of the sequence 2 and is derived from the sequence 2. A coding gene of a reverse transcription factor resisting factor OsDREB is one of the following nucleotide sequences: 1) a DNA sequence of a sequence 1 in the sequence table; 2) a DNA sequence has more than 90% of homology with a DNA sequence limited by the sequence 1 in the sequence table and codes protein with the same functions. The OsDREB gene has the significance in cold and drought resisting plant variety cultivation.
Description
Technical field
The present invention relates to rice DREB transcription factor and encoding gene thereof and application, particularly relate to rice DREB cold-resistant and drought-enduring transcription factor and encoding gene and application.
Background technology
Plant can be subjected to many Effect of Environmental in process of growth, damage to plants caused by sudden drop in temperature with arid to cause the extensive underproduction of farm crop.The crop varieties of cultivating resistance of reverse is one of major objective of agricultural cience and farming techniques research always.The variation of biological physical efficiency recipient cell external environment condition also is delivered to it in cell by number of ways, thereby corresponding reaction is made in variation to external world.Transcription factor when organism experiences environment stress, can be regulated and control the expression of a series of downstream genes as a kind of regulatory gene, thereby strengthens the tolerance of organism to adverse circumstance, reaches the effect that the opposing unsuitable environmental condition is coerced.In plant, the DREB class transcription factor in the AP2/EREBP family can be accepted the environment-stress signal and start the expression of adverse circumstance response gene.
Paddy rice (Oryza Sativa) is one of most important food crop, cultivates and resists cold, and drought-enduring kind is significant to improving rice yield.
Summary of the invention
The purpose of this invention is to provide DREB class transcription factor and encoding gene thereof with strong adverse-resistant characteristic, this transcription factor has regulating and controlling effect in plant cold resistance and drought resisting reaction.
The anti-reverse transcription factor provided by the present invention derives from paddy rice, and name is called OsDREB, is the protein with sequence 2 amino acid residue sequences in the sequence table.
The protein that sequence 2 amino acid residue sequences are made up of 281 amino-acid residues in the sequence table, its conservative AP2/EREBP structural domain sequence is to carbon teminal 82-138 amino acids residue from the nitrogen end.
The encoding gene of anti-reverse transcription factor OsDREB is the dna sequence dna of sequence 1 in the sequence table.
The dna sequence dna of sequence 1 is by 1169 based compositions in the sequence table, and the reading frame of this gene is from 3 ' end the 91st to the 936th bit base, and it is expressed and mainly is subjected to inducing of low temperature and arid.
Class transcription factor OsDREB provided by the present invention has and DRE cis-acting elements bonded ability, and can activate the expression of downstream gene.Its encoding gene all contains conservative AP2/EREBP structural domain sequence.
Gene with encoding transcription factor OsDREB provided by the present invention, use any expression vector that can guide foreign gene in plant, to express (these plant expression vectors comprise double base agrobacterium vector and the carrier that is used for the monocotyledons micropellet bombardment) transformed plant, can obtain the transfer-gen plant that low temperature and drought stress tolerance are enhanced.Carrier also can comprise 3 ' untranslated zone, but 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 has the Agrobacterium crown-gall nodule to induce (Ti) plasmid gene (as kermes synthetic enzyme Nos gene), plant gene (as the soybean storage protein gene) 3 ' non-translational region of transcribing etc.3 ' untranslated zone of OsDREB gene of the present invention can be used to express in plant.Gene of the present invention can add any strong promoter or inducible promoter in being building up to plant expression vector the time before its transcription initiation Nucleotide.These promotors are a lot, and as cauliflower mosaic virus (CAMV 35S) and Ubiqutin promotor etc., they can use separately or be used in combination with other plant promoter.Gene of the present invention also can use enhanser in being building up to plant expression vector the time, comprises translational enhancer or transcriptional enhancer.These enhanser zones can be ATG initiator codon and neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the translation of whole sequence.Translation control signal and initiator codon can be multiple different sourcess such as natural or synthetic.Translation initiation region can be from transcription initiation zone or 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.The selected marker of using can be the gene of coding to the antibiotics resistance enzyme, and microbiotic comprises gentamicin, Totomycin, kantlex etc.Also can be to produce the enzyme of colour-change or the gene of luminophor, as GUS, luciferase, can also be the gene of anti-chemical reagent (for example weedkiller).Certainly, also can any selection markers.
Carry OsDREB expression carrier of the present invention and can import vegetable cell (Weissbach by using conventional biotechnological meanss such as Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electroporation, 1998, Method for Plant Molecular Biology VIII, Academy Press, Now York, pp.411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2
NdEdition).
Can use the plant expression vector that comprises OsDREB gene of the present invention to transform plant host (both can be monocotyledons, it also can be dicotyledons, as: paddy rice, wheat, corn, cucumber, tomato, willow, turfgrass, clover etc.), cultivate cold-resistant and plant variety drought resisting.
The present invention will be further described below in conjunction with specific embodiment.
Description of drawings
After Fig. 1 is the two reporter cells of OsDREB gene transformation yeast, the growing state on 0mM 3-AT SD/-His-Ura-Trp flat board and 30mM 3-AT SD/-His-Ura-Trp flat board.
After Fig. 2 was the two reporter cells of OsDREB gene transformation yeast, the mensuration of beta-galactose-glycoside enzyme activity detected the situation of the expression intensity of LacZ reporter gene.
The result that Fig. 3 forms in rice genome for Southern hybridization analysis OsDREB gene.
Fig. 4 is the RT-PCR detected result of OsDREB gene expression status under low temperature, drought-induced condition.
Embodiment
The structure of embodiment 1, rice cDNA library
Paddy rice (Oryza Sativa) kind upland rice 502 plant cultures are on 1/2 MS solid medium, place (28 ℃ of illumination boxs, illumination in 12 hours, 12 hours dark) cultivated 10-14 days, get complete stool quick-frozen in liquid nitrogen, grinding, be suspended among the TRIzole (Gibco), mixture is with acid phenol, chloroform extracting, add the total RNA of isopropanol precipitating in the supernatant, soluble in water.Obtain mRNA through the processing of mRNA purification kit.Get 1ug mRNA and carry out reverse transcription reaction, and then synthetic second chain.Double-stranded cDNA is connected the back phosphorylation with the EcoRI-NotI-BamHI joint, remove unnecessary joint through preparative centrifugation post (Sephacryl-400), be connected (concrete steps see Stratagene HybriZAP-1.1 XR Library Construction Kit for details) then with the pAD-GAL4 carrier.
Embodiment 2, contain the acquisition of the two reporter cells of yeast of DRE cis-acting elements
To by the HindIII restriction enzyme site it be connected into 4 repetitions from the isolating DRE cis-acting elements of forming by 75bp of rd29A promoter region, obtain to contain the dna fragmentation of 4 series connection multiple DRE elements.The upstream that resulting dna fragmentation is inserted the PminHIS3 promotor of pHISi-1 carrier, and obtain can be on the SD substratum that lacks Histidine the yeast conversion body of normal growth.Simultaneously this dna fragmentation is building up to the upstream of the PCYC1 promotor of pLacZi carrier, carrier is transformed in the yeast conversion body that contains the pHISi-1 carrier then.Like this, lack at the same time on the substratum of Histidine and uridylic, select to obtain to contain the yeast conversion body of pHISi-1 and pLacZi carrier.Obtain to contain the two report of the yeast daughter cell of mutant DRE cis-acting elements with same method.HIS and LacZ gene are as 2 reporter genes, and after the DRE of its upstream element was by the identification of DREB transcription factor, its expression intensity can significantly strengthen.Detect HIS expression of gene intensity by 3-AT (Histidine synthetic enzyme competitive inhibitor), the mensuration by the beta-galactose-glycoside enzyme activity detects LacZ expression of gene intensity simultaneously.Fig. 1 has shown behind the two report of the coding DREB class transcription factor OsDREB transformed yeast of the present invention daughter cell at the growing state that contains on the SD/-His-Ura-Trp flat board of 3-AT.Wherein A represents that the yepGAP empty carrier transforms the two reporters of wDRE yeast; B represents that the yepGAP empty carrier transforms the two reporters of mDRE yeast; C represents the two reporters of OsDREB gene transformation wDRE yeast; D represents the two reporters of OsDREB gene transformation mDRE yeast.Fig. 2 is the result that the galactoside enzyme activity detects.As can be seen from the figure, when the upstream was the wDRE element, yeast conversion body galactoside enzyme activity obviously strengthened; If its upstream is the mDRE cis-acting elements, then its expression can't be enhanced.
The clone of the screening of embodiment 3, rice cDNA library and encoding transcription factor OsDREB gene
The two report of the yeast daughter cell that utilization contains the DRE cis-acting elements screens rice cDNA library, (the concrete operations step is seen Clontech Mathchmakerone-hybrid system) on the flat board of the SD/-His-Ura-Leu that contains 30mM 3-AT.Resulting positive colony is cultivated in the YPD liquid nutrient medium, extracts plasmid transformation escherichia coli.The cDNA fragment of being inserted is downcut from carrier, be building up in the YepGAP Yeast expression carrier.Because no GAL4 transcriptional activation zone on this carrier, so only can activate downstream reporter gene expressed proteins again and could on the flat board of the SD/-His-Ura-Leu of 30mM 3-AT, grow with the DRE combination of elements.For such positive colony, its cDNA fragment is building up on the SK+ carrier again, obtained the nucleotide sequence of encoding transcription factor OsDREB gene through order-checking.The OsDREB gene comprises the open reading frame of 846bp, the polypeptide that coding is made up of 281 amino acid, and its 5 ' end is 90bp, 3 ' end is the sequence in the sequence table 1 for 233bp.
Embodiment 4, the analysis of OsDREB gene in rice genome
Extract the genome DNA of rice varieties upland rice 502, digest with EcoRI, NotI, 4 kinds of restriction enzymes of XbaI, XhoI respectively.Postdigestive DNA is forwarded on the N+Hybond film, make probe, carry out the Southern hybridization analysis with the OsDREB of part total length.Under the rigorous degree condition of height, wash (65 ℃ of films, 0.1 * SSC 0.1%SDS) obtains 1-2 hybridization band, as shown in Figure 3, on behalf of EcoRI, N, E represent NotI, Xb to represent XbaI, Xh to represent XhoI among the figure, and the result shows that the OsDREB gene exists with low copy form in rice genome.
Embodiment 5, the expression characteristic of rice Os dreb gene in adverse circumstance
To 10-14 days rice plant of growth on 1/2 MS substratum, 4 ℃ of following subzero treatment 6 hours, or under drought condition, handled 6 hours, through liquid nitrogen flash freezer, extract RNA and make RT-PCR and analyze.Be contrast with paddy rice rRNA in the RT-PCR reaction, with the unanimity of template cDNA concentration in the control PCR reaction.The result as shown in Figure 4, M be C represent sample without adverse circumstance handle, L represents that sample represents that through subzero treatment 6 hours, D sample handled 6 hours through arid, shows that transcribing of OsDREB is subjected to inducing of low temperature and arid.
Sequence table
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Ile?Lys?Trp?Trp?Lys?Glu?Gln?Asn?Gln?Lys?Leu?Gln?Glu?Glu?Asn?Ser
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125 130 135
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140 145 150
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170 175 180
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235 240 245
gtt?gtt?gag?atg?ata?att?gtt?gaa?tta?agt?gct?gat?cag?aaa?acg?gaa 882
Val?Val?Glu?Met?Ile?Ile?Val?Glu?Leu?Ser?Ala?Asp?Gln?Lys?Thr?Glu
250 255 260
gta?cat?gaa?gag?tac?caa?gag?gga?gat?gat?ggg?ttt?agc?ctt?ttc?tcc 930
Val?His?Glu?Glu?Tyr?Gln?Glu?Gly?Asp?Asp?Gly?Phe?Ser?Leu?Phe?Ser
265 270 275 280
tat?tag?agtagtagtc?atgctgcggg?tcaataggaa?tatttcattc?tagctgctag 986
Tyr
gggatacttc?aaatatctgc?aacctgaagc?tttgtagtca?tttacggttt?tcgtcttact 1046
gggtaatagc?tttatatata?ctataagcca?actggtacaa?gaagttgtac?tgtgtgttga 1106
gtgcactgtg?gtaaaaatga?atctatattt?aatgagctta?ctctgtcaaa?aaaaaaaaaa 1166
aaa 1169
<210>2
<211>281
<212>PRT
<213〉paddy rice belongs to paddy rice (Oryza sativa L.)
<400>2
Met?Leu?Phe?Arg?Phe?Val?Ser?Cys?Asn?Val?Gln?Leu?Cys?Gly?Ile?Ile
1 5 10 15
Glu?Leu?Pro?His?Trp?Val?Arg?Lys?Lys?Arg?Thr?Arg?Arg?Lys?Ser?Asp
20 25 30
Gly?Pro?Asp?Ser?Ile?Ala?Glu?Thr?Ile?Lys?Trp?Trp?Lys?Glu?Gln?Asn
35 40 45
Gln?Lys?Leu?Gln?Glu?Glu?Asn?Ser?Ser?Arg?Lys?Ala?Pro?Ala?Lys?Gly
50 55 60
Ser?Lys?Lys?Gly?Cys?Met?Ala?Gly?Lys?Gly?Gly?Pro?Glu?Asn?Ser?Asn
65 70 75 80
Cys?Ala?Tyr?Arg?Gly?Val?Arg?Gln?Arg?Thr?Trp?Gly?Lys?Trp?Val?Ala
85 90 95
Glu?Ile?Arg?Glu?Pro?Asn?Arg?Gly?Arg?Arg?Leu?Trp?Leu?Gly?Ser?Phe
100 105 110
Pro?Thr?Ala?Leu?Glu?Ala?Ala?His?Ala?Tyr?Asp?Glu?Ala?Ala?Arg?Ala
115 120 125
Met?Tyr?Gly?Pro?Thr?Ala?Arg?Val?Asn?Phe?Ala?Asp?Asn?Ser?Thr?Asp
130 135 140
Ala?Asn?Ser?Gly?Cys?Thr?Ser?Ala?Pro?Ser?Leu?Met?Met?Ser?Asn?Gly
145 150 155 160
Pro?Ala?Thr?Ile?Pro?Ser?Asp?Glu?Lys?Asp?Glu?Leu?Glu?Ser?Pro?Pro
165 170 175
Phe?Ile?Val?Ala?Asn?Gly?Pro?Ala?Val?Leu?Tyr?Gln?Pro?Asp?Lys?Lys
180 185 190
Asp?Val?Leu?Glu?Arg?Val?Val?Pro?Glu?Val?Gln?Asp?Val?Lys?Thr?Glu
195 200 205
Gly?Ser?Asn?Gly?Leu?Lys?Arg?Val?Cys?Gln?Glu?Arg?Lys?Asn?Met?Glu
210 215 220
Val?Cys?Glu?Ser?Glu?Gly?Ile?Val?Leu?His?Lys?Glu?Val?Asn?Ile?Ser
225 230 235 240
Tyr?Asp?Tyr?Phe?Asn?Val?His?Glu?Val?Val?Glu?Met?Ile?Ile?Val?Glu
245 250 255
Leu?Ser?Ala?Asp?Gln?Lys?Thr?Glu?Val?His?Glu?Glu?Tyr?Gln?Glu?Gly
260 265 270
Asp?Asp?Gly?Phe?Ser?Leu?Phe?Ser?Tyr
275 280
Claims (5)
1, the DREB class transcription factor OsDREB of paddy rice, it has the amino acid residue sequence of sequence 2 in the sequence table.
2, the encoding gene of rice DREB transcription factor OsDREB, it is the dna sequence dna of sequence 1 in the sequence table.
3, gene according to claim 2 is characterized in that: the reading frame of this gene is for holding the 91st to the 936th bit base from 5 '.
4, contain the described expression carrier of claim 2.
5, the clone that contains the described expression vector of claim 4.
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CN1223606C true CN1223606C (en) | 2005-10-19 |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN101062943B (en) * | 2007-04-29 | 2010-09-08 | 北京未名凯拓农业生物技术有限公司 | Rice stress tolerance related DREB transcription factor and its coding gene and application |
Families Citing this family (10)
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CN1332980C (en) * | 2004-12-10 | 2007-08-22 | 中国科学院上海生命科学研究院 | Paddy rice anti contravariance related gene-anchor series repetitive protein gene and its application |
CN100362104C (en) * | 2004-12-21 | 2008-01-16 | 华中农业大学 | Using gene of transcriptional factor OSNACX of paddy to increase drought resistance and salt tolerant abilities of plants |
CN100366634C (en) * | 2004-12-30 | 2008-02-06 | 中国农业科学院作物育种栽培研究所 | Binding protein of response component of plant dehydration, and coding genes and application |
CN1328288C (en) * | 2005-10-10 | 2007-07-25 | 中国科学院植物研究所 | Rice DREB transcription factor and its coding gene and application |
CN1313486C (en) * | 2005-11-08 | 2007-05-02 | 北京北方杰士生物科技有限责任公司 | Plant DREB transcription factor and its coding gene and use |
CN100357319C (en) * | 2006-01-10 | 2007-12-26 | 中国科学院植物研究所 | Barbadosnut cold-induced transcription factor, its encoding gene and uses |
WO2009127441A2 (en) * | 2008-04-16 | 2009-10-22 | Universität Potsdam | Transcription factors involved in drought stress in plants |
CN101486757B (en) * | 2009-03-06 | 2012-03-14 | 中国农业科学院作物科学研究所 | Plant chloroplast development associated protein, and coding gene and use thereof |
CN102373217A (en) * | 2010-08-12 | 2012-03-14 | 上海市农业生物基因中心 | Paddy DREBs (dehydration-responsive element binding) transcription factor and application thereof |
CN111500595B (en) * | 2020-04-30 | 2020-12-25 | 中国科学院华南植物园 | Ephedra sinica gene CeDREB1 and application thereof |
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CN101062943B (en) * | 2007-04-29 | 2010-09-08 | 北京未名凯拓农业生物技术有限公司 | Rice stress tolerance related DREB transcription factor and its coding gene and application |
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