CN1491960A - 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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- CN1491960A CN1491960A CNA021295182A CN02129518A CN1491960A CN 1491960 A CN1491960 A CN 1491960A CN A021295182 A CNA021295182 A CN A021295182A CN 02129518 A CN02129518 A CN 02129518A CN 1491960 A CN1491960 A CN 1491960A
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Abstract
The present invention discloses rice DREB transcription factor and its encoding gene and application, and aims at providing transcription factor with regulation effect in plant cold and drought reaction. The DREB transcription factor, named OsDREB, has the amino acid residue sequence of the Sequence 2 in the sequence list or protein derived from the Sequence 2 with the amino acid residue sequence possessing one or several amino acid residues substituted, defaulted or added and thus the same activity as the amino acid residue sequence of Sequence 2. The encoding gene of OsDREB is one of the nucleotide sequences: the DNA sequence of Sequence 1 in the sequence list, and DNA sequence with homology over 90 % with the DNA sequence of Sequence 1 in the sequence list and encoding protein of the same function. The OsDREB gene of the present invention has important significance in breeding cold and drought resisting plant variety.
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, name is called OsDREB, 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 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 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 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 the 1/2MS 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.
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.
To 10-14 days rice plant of growth on the 1/2MS 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
<160> 2
<210> 1
<211> 1169
<212> DNA
＜213〉paddy rice belongs to paddy rice (Oryza sativa L.)
<220>
<221> CDS
<222> (91)..(936)
<223>
<400> 1
cttaccgttg?attgctgata?gcctccttga?tttttggaca?aatgaaaagg?attccgcttt 60
tggtttgttg?tttttcataa?tttcaattgc?atg?ctg?ttt?cga?ttt?gtg?tct?tgc 114
Met?Leu?Phe?Arg?Phe?Val?Ser?Cys
1 5
aat?gtt?cag?ctt?tgt?gga?att?att?gag?tta?cct?cat?tgg?gtc?agg?aag 162
Asn?Val?Gln?Leu?Cys?Gly?Ile?Ile?Glu?Leu?Pro?His?Trp?Val?Arg?Lys
10 15 20
aag?aga?acg?cga?agg?aaa?agc?gat?ggc?cct?gat?tca?atc?gct?gaa?acc 210
Lys?Arg?Thr?Arg?Arg?Lys?Ser?Asp?Gly?Pro?Asp?Ser?Ile?Ala?Glu?Thr
25 30 35 40
atc?aag?tgg?tgg?aag?gag?caa?aac?cag?aag?ctc?cag?gag?gag?aat?agc 258
Ile?Lys?Trp?Trp?Lys?Glu?Gln?Asn?Gln?Lys?Leu?Gln?Glu?Glu?Asn?Ser
45 50 55
tcc?agg?aaa?gcg?cca?gcc?aag?ggg?tcc?aag?aaa?ggg?tgc?atg?gct?ggg 306
Ser?Arg?Lys?Ala?Pro?Ala?Lys?Gly?Ser?Lys?Lys?Gly?Cys?Met?Ala?Gly
60 65 70
aaa?gga?ggt?ccg?gaa?aat?tca?aat?tgt?gct?tac?cgc?ggt?gtc?agg?caa 354
Lys?Gly?Gly?Pro?Glu?Asn?Ser?Asn?Cys?Ala?Tyr?Arg?Gly?Val?Arg?Gln
75 80 85
cgg?aca?tgg?ggt?aag?tgg?gtg?gct?gag?atc?cgt?gaa?cca?aac?cgt?gga 402
Arg?Thr?Trp?Gly?Lys?Trp?Val?Ala?Glu?Ile?Arg?Glu?Pro?Asn?Arg?Gly
90 95 100
agg?cgc?cta?tgg?cta?gga?tca?ttt?cct?act?gcg?ctg?gag?gct?gcg?cat 450
Arg?Arg?Leu?Trp?Leu?Gly?Ser?Phe?Pro?Thr?Ala?Leu?Glu?Ala?Ala?His
105 110 115 120
gca?tac?gat?gag?gcg?gca?agg?gca?atg?tat?ggt?ccc?aca?gca?cgt?gtc 498
Ala?Tyr?Asp?Glu?Ala?Ala?Arg?Ala?Met?Tyr?Gly?Pro?Thr?Ala?Arg?Val
125 130 135
aat?ttt?gca?gat?aat?tcc?aca?gat?gcc?aac?tct?ggc?tgc?aca?tca?gca 546
Asn?Phe?Ala?Asp?Asn?Ser?Thr?Asp?Ala?Asn?Ser?Gly?Cys?Thr?Ser?Ala
140 145 150
cct?tca?ttg?atg?atg?tct?aat?ggg?ccg?gcc?act?ata?cct?tct?gat?gag 594
Pro?Ser?Leu?Met?Met?Ser?Asn?Gly?Pro?Ala?Thr?Ile?Pro?Ser?Asp?Glu
155 160 165
aag?gat?gag?ctg?gaa?tct?cct?cct?ttc?atc?gtg?gct?aat?ggg?cca?gct 642
Lys?Asp?Glu?Leu?Glu?Ser?Pro?Pro?Phe?Ile?Val?Ala?Asn?Gly?Pro?Ala
170 175 180
gtg?ttg?tat?cag?cct?gat?aag?aag?gat?gtg?ttg?gaa?cgt?gta?gtc?cct 690
Val?Leu?Tyr?Gln?Pro?Asp?Lys?Lys?Asp?Val?Leu?Glu?Arg?Val?Val?Pro
185 190 195 200
gag?gtg?cag?gat?gtt?aaa?aca?gaa?ggg?agc?aat?ggc?ttg?aaa?cgt?gtt 738
Glu?Val?Gln?Asp?Val?Lys?Thr?Glu?Gly?Ser?Asn?Gly?Leu?Lys?Arg?Val
205 210 215
tgt?cag?gag?cgg?aag?aat?atg?gag?gta?tgt?gaa?tca?gaa?ggg?atc?gtt 786
Cys?Gln?Glu?Arg?Lys?Asn?Met?Glu?Val?Cys?Glu?Ser?Glu?Gly?Ile?Val
220 225 230
tta?cac?aaa?gaa?gtg?aac?ata?agt?tat?gat?tat?ttc?aat?gtc?cat?gaa 834
Leu?His?Lys?Glu?Val?Asn?Ile?Ser?Tyr?Asp?Tyr?Phe?Asn?Val?His?Glu
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 (8)
1, the DREB class transcription factor OsDREB of paddy rice, it have the amino acid residue sequence of sequence 2 in the sequence table or with the amino acid residue sequence of sequence 2 through replacement, disappearance or the interpolation of one 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 has the amino acid residue sequence of sequence 2 in the sequence table.
3, the encoding gene of rice DREB transcription factor OsDREB, it 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.
4, gene according to claim 3 is characterized in that: the encoding gene of described rice DREB transcription factor OsDREB is the dna sequence dna of sequence 1 in the sequence table.
5, gene according to claim 4 is characterized in that: the reading frame of this gene is for holding the 91st to the 936th bit base from 5 '.
6, contain the described expression carrier of claim 3.
7, the clone that contains the described gene of claim 3.
8, the application of the described gene of claim 3 in cultivating cold-resistant and drought-resistant plant.
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| 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 |
| 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 |
| 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 |
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| 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 |
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| WO2009127441A3 (en) * | 2008-04-16 | 2010-03-11 | Universität Potsdam | Transcription factors involved in drought stress in plants |
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| 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 |
| CN111500595A (en) * | 2020-04-30 | 2020-08-07 | 中国科学院华南植物园 | Ephedra sinica gene CeDREB1 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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