CN104762305A

CN104762305A - Gene TaUreG related to wheat drought resisting and application thereof

Info

Publication number: CN104762305A
Application number: CN201510207339.3A
Authority: CN
Inventors: 张玮; 孙丽静; 韩洁; 纪军; 李辉; 李俊明
Original assignee: Institute of Genetics and Developmental Biology of CAS; Institute of Grain and Oil Crops of Hebei Academy of Agriculture and Forestry Sciences
Current assignee: Institute of Genetics and Developmental Biology of CAS; Institute of Grain and Oil Crops of Hebei Academy of Agriculture and Forestry Sciences
Priority date: 2015-04-28
Filing date: 2015-04-28
Publication date: 2015-07-08
Anticipated expiration: 2035-04-28
Also published as: CN104762305B

Abstract

The invention discloses a gene TaUreG related to wheat drought resisting. The gene TaUreG has a cDNA nucleotide sequence shown by SEQ ID NO:1 or a gDNA nucleotide sequence shown by SEQ ID NO:2. The plant genetic engineering technology is applied, the gene TaUreG related to wheat drought resisting is cloned in wheat for the first time, and through real-time fluorescence quantification PCR detection, the result shows that expression of the gene TaUreG is adjusted by drought stress and can play an important role in the early stage of adapting to drought stress by the wheat; expression of the gene TaUreG is adjusted by ABA and H2O2, and it is proved that TaUreG takes part in response to drought stress of the wheat through a signal transduction pathway depended by ABA and takes part in a signal transduction pathway with the ROS level lifted due to drought stress. Therefore, when wheat varieties are cultivated, wheat plants containing the gene have a high drought resistant ability, which is of great significance in screening the drought resistant wheat variety, cultivating high-drought-resistance transgenic wheat and increasing the planting yield of the wheat in a drought environment.

Description

The gene TaUreG relevant to Drought-resistance in Wheat and application thereof

Technical field

The present invention relates to technical field of biological genetic engineering, specifically a kind of and Drought-resistance in Wheat genes involved taUreGand application.

Background technology

At present, global climate warms and causes soil drought degree more and more serious, and this forms directly threat to grain-production.Wheat is one of topmost food crop in the world, the second largest food crop of Ye Shi China.Plant can produce a series of protection mechanism when tackling drought stress, the molecular mechanism of research Drought-resistance in Wheat, and cultivating Drought resistant Wheat new variety is improve a main path of wheat yield, for guarantee national food security and Water resources security significant.

Plant produces a series of basic reaction under drought stress, comprises the suppression of metabolism and growth level, the change of gene expression dose, the change of hormone in vivo level, the generation of induction and suppression signal transduction pathway, the accumulation of solubility permeation protective agent, reactive oxygen species (ROS) level raise the plasma membrane oxidation etc. caused.The signal transduction pathway of the ind DREB transcription factor mediation of Drought signal relies on mainly through dormin (ABA) and ABA regulates the expression (Yoshida et al. ABA-dependent and ABA-independent signaling in response to osmotic stress in plants. 2014) of downstream gene.Research shows, plant can produce a series of protection mechanism when tackling drought stress; Drought stress can induce the expression of many plant genes, and the expression product of some gene directly can strengthen stress tolerance, and some can be responded to and the genetic expression of transduction stress signals-modulating.In recent years, along with genomics and molecular biological development, the relevant gene of a collection of drought resisting has been cloned from wheat by Chinese and overseas scholars, as the Expression of a wheat MYB gene in transgenic tobacco enhances resistance to Ralstonia solanacearum of Liu et al. report in 2011, and to drought and salt stresses; The Wheat WRKY genes of Niu et al. report in 2012 taWRKY2and taWRKY19regulate abiotic stress tolerance in transgenic Arabidopsis plants; The Over-expression of of Qin et al. report in 2012 taMYB33encoding a novel wheat MYB transcription factor increases salt and drought tolerance in Arabidopsis; Molecular characterization of novel disclosed in Tang et al. in 2012 taNACgenes in wheat and overexpression of taNAC2aconfers drought tolerance in tobacco; These are and screen Drought resistant Wheat kind, improve wheat planting output under drought environment has established solid basis.

Summary of the invention

The object of this invention is to provide a kind of and Drought-resistance in Wheat genes involved taUreGand application.

A kind of gene relevant to Drought-resistance in Wheat taUreG, it has the cDNA nucleotide sequence shown in SEQ ID NO: 1 or the gDNA nucleotide sequence shown in SEQ ID NO: 2.

GDNA of the present invention comprises 7 exons and 6 introns.

A kind of gene relevant to Drought-resistance in Wheat taUreG, the proteins encoded of this gene is as shown in SEQ ID NO: 3.

The present invention clones described gene taUreGupstream primer taUreG-F1nucleotide sequence as shown in SEQ ID NO: 4, downstream primer taUreG-R1nucleotide sequence as shown in SEQ ID NO: 5.

Gene of the present invention is positioned on wheat 1A karyomit(e).

Gene source provided by the invention in wheat section agriculture 9204, according to sequence structure feature, called after taUreG, its cDNA sequence total length 945 bp, comprise one long be the open reading frame of 855 bp, 284 amino acid of encoding; GDAN sequence 2546 bp, is made up of 7 exons and 6 introns.From 5' end, the length of exon is followed successively by 77 bp, 204 bp, 61 bp, 202 bp, 122 bp, 105 bp, 84 bp, the length of intron is followed successively by 86 bp, 600 bp, 336 bp, 345 bp, 129 bp, 105 bp, and gene structure is shown in Fig. 1.

The gene that drought resisting disclosed by the invention is relevant taUreGwhen being analyzed by real-time PCR detection, its upstream primer taUreG-F2nucleotide sequence as shown in SEQ ID NO: 8, downstream primer taUreG-R2nucleotide sequence as shown in SEQ ID NO: 9; Internal reference upstream primer taActin-F2nucleotide sequence as shown in SEQ ID NO: 10, internal reference downstream primer taActin-R2nucleotide sequence as shown in SEQ ID NO: 11, its amplification system is: SYBR ^?premix Ex Taq II(2 ×) 10 μ l, Forward Primer(10 μM) 0.8 μ l, Reverse Primer(10 μM) 0.8 μ l, ROX Reference Dye II(50 ×) 0.4 μ l, cDNA template 2 μ l, ddH ₂o 6 μ l; Amount to 20 μ l; Its PCR response procedures: 95 DEG C of denaturation 30 s; 95 DEG C of sex change 5 s, anneal and extend 34 s for 60 DEG C, 40 circulations.

The present invention section used agriculture 9204 is Approved variety, passes through Hebei province crop varietal approval committee in 2002; Within 2003, by national variety certification, variety certification is numbered state and examines wheat 2003037.

Due to gene disclosed by the invention taUreGthere is drought resistance, thus can by described nucleotide sequence as SEQ ID NO: 1 or its coded protein the gene as shown in SEQ ID NO.3 taUreGproceed to Wheat Tissue, thus cultivate into drought tolerance wheat.

Invention applies plant gene engineering technology, from wheat, clone the gene relevant to Drought-resistance in Wheat first taUreG, detected by real-time fluorescence quantitative PCR, its result shows, described gene taUreGexpression be subject to the adjustment of drought stress, the early stage of drought stress can be adapted at wheat and just playing an important role; And described gene taUreGexpression equally by ABA and H ₂o ₂adjustment, explanation taUreGbe that the signal transduction pathway relied on by ABA take part in the response of wheat to drought stress, and take part in the signal transduction pathway of the ROS level rising that drought stress causes; taUreGthe excavation participating in Drought-resistance in Wheat mechanism is that the utilization of this gene in Drought-resistance in Wheat breeding provides more reliable evidence.Show thus, contain taUreGthe wheat plant of gene has stronger drought-resistant ability, the gene relevant to Drought-resistance in Wheat taUreGcan be applied in seed selection Drought resistant Wheat kind; Especially can by the gene of described nucleotide sequence as shown in SEQ ID NO:1 taUreGproceed to Wheat Tissue and cultivate the transgenic wheat with high drought resistance.

Accompanying drawing explanation

Fig. 1 is taUreGgene structure figure.Square frame represents exon, and lines represent intron.ATG and TAG is respectively initiator codon and terminator codon.

Fig. 2 is for utilizing China spring nulli-tetrasomes system pair taUreGcarry out the result of chromosomal localization.CS represents China spring; M represents marker; With taActin(can increase A, B, D group) is the integrity of internal reference proof DNA.

Fig. 3 is real-time fluorescence quantitative PCR pair taUreGthe analytical results of expression characteristic under ABA process different time.With taActinfor internal reference.

Fig. 4 is real-time fluorescence quantitative PCR pair taUreGat H ₂o ₂the analytical results of expression characteristic under process different time.With taActinfor internal reference.

Fig. 5 is real-time fluorescence quantitative PCR pair taUreGthe analytical results of expression characteristic under PEG-6000 manual simulation Osmotic treatment different time.With taActinfor internal reference.

Embodiment

Following embodiment is convenient to understand the present invention better, but does not limit the present invention.

Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.

Embodiment 1 Drought-resistance in Wheat genes involved taUreGclone

1, wheat taUreGthe electronic cloning of cDNA sequence

(1) with the paddy rice that NCBI submits to osUreG(HM369059) gene order is probe, search wheat est database, by the sequence DNAMAN software splicing obtained, predicts, to obtain complete wheat to open reading frame (ORF) taUreGcDNA sequence;

(2) a pair special primer is filtered out according to the full length cDNA sequence Primer5 software design that obtains taUreG-F1with taUreG-R1, be respectively used to wheat taUreGcDNA and gDNA gene clone.Its upstream and downstream primer is respectively:

Upstream primer taUreG-F1: 5 '-CCTTCCACTTCCAAGTCTGG-3 ' (SEQ ID NO:4)

Downstream primer taUreG-R1: 5 '-CATACGTGAACACATGCCCG-3 ' (SEQ ID NO:5).

2, Trizol(Invitrogen is utilized) extract total serum IgE, concrete steps are as follows:

(1) liquid nitrogen grinding 50-100 mg wheat leaf blade, transfers to after grinding in the EP pipe containing 1 ml Trizol reagent, fully mixes, and room temperature leaves standstill 5 min; (2) add 0.2 ml chloroform, fully mix, room temperature leaves standstill 2-3 min; At 4 DEG C, centrifugal 15 min of 12000 × g; (3) aqueous phase colourless for upper strata is transferred in clean 1.5 ml EP pipes, add 0.5 ml Virahol, after mixing, room temperature places 10 min; At 4 DEG C, centrifugal 10 min of 12000 × g; (4) remove supernatant, by precipitation 1 ml 75% ethanol purge, at 4 DEG C, centrifugal 5 min of 7500 × g, will precipitate air-dry afterwards; (5) precipitation is dissolved in the deionized water of appropriate RNase-free, 60 DEG C of dissolution 10 min; Obtain required total serum IgE; Can micro-spectrophotometer NanoDrop ND-2000 Spectrotometer detection by quantitative, the integrity of electrophoretic analysis RNA.

3, utilize plant genome DNA to extract test kit (sky root, DP305) and extract DNA, concrete steps are as follows:

(1) get wheat leaf agreement that contracts a film or TV play to an actor or actress 100 mg, add liquid nitrogen and fully mill; (2) ground powder is transferred to rapidly in the centrifuge tube that 700 μ l, 65 DEG C of preheating damping fluid GP1 are housed in advance and (before experiment, in the GP1 of preheating, add mercaptoethanol, its final concentration is made to be 0.1%), after putting upside down mixing rapidly, centrifuge tube is placed on 65 DEG C of water-bath 20 min, puts upside down centrifuge tube in water-bath process with biased sample for several times; (3) add 700 μ l chloroforms, fully mix, centrifugal 5 min of 12000 rpm; (4) carefully previous step gained upper strata aqueous phase is proceeded in a new centrifuge tube, add 700 μ l damping fluid GP2, fully mix; (5) proceed in adsorption column CB3 by the liquid of mixing, centrifugal 30 s of 12000 rpm, discard waste liquid; (6) in adsorption column CB3, add 500 μ l damping fluid GD, centrifugal 30 s of 12000 rpm, outwell waste liquid, adsorption column CB3 are put into collection tube; (7) in adsorption column CB3, add 600 μ l rinsing liquid PW, centrifugal 30 s of 12000 rpm, outwell waste liquid, adsorption column CB3 are put into collection tube; (8) repetitive operation step (7); (9) put back in collection tube by adsorption column CB3, centrifugal 2 min of 12000 rpm, outwell waste liquid, adsorption column CB3 are placed in room temperature and place several minutes, thoroughly to dry rinsing liquid remaining in sorbing material; (10) adsorption column CB3 is proceeded in a clean centrifuge tube, the unsettled dropping in middle part to adsorption film 50-200 μ l elution buffer TE, room temperature places 2-5 min, centrifugal 2 min of 12000 rpm, by solution collection in centrifuge tube, be required DNA.

4, utilize GoldScript cDNA synthetic agent box (Invitrogen, c81401190) to carry out the first chain cDNA to synthesize, concrete steps are as follows:

(1) add successively in 0.2 ml centrifuge tube step 2 extract total serum IgE 5 μ l(200 ng/ μ l), 10 mM dNTP mix 1 μ l, Oligo dT Primer 1 μ l, DEPC water 3 μ l; (2) above-mentioned RNA/ primer mixture is placed on 65 DEG C and hatches 5 min, be then placed on 1-2 min on ice; (3) in another pipe, preparation 2 × reaction mixture, is sequentially added into following component: 10 × RT damping fluid 2 μ l, 25 mM MgCl ₂4 μ l, 0.1 M DTT 2 μ l, restructuring RNase inhibitor (40U/ μ l) 1 μ l; (4) in each RNA/ primer premixed liquid of above-mentioned (2) step, add 9 μ l 2 × reaction premixed liquids, softly mix, centrifugal 30 s of 7500 rpm; Hatch 2 min for (5) 42 DEG C; 1 μ l GoldScript RT is added in each pipe; Hatch 50 min for 42 DEG C; Hatch 15 min, termination reaction, cooled on ice for (6) 70 DEG C; Centrifugal 30 s of (8) 7500 rpm, add 1 μ l RNaseH, hatch 20 min for 37 DEG C ,-20 DEG C of preservations in each pipe.

5, sequence amplification is carried out in polymerase chain reaction (PCR) reaction

With taUreG-F1with taUreG-R1primer carries out pcr amplification to wheat cDNA and gDNA respectively;

Its PCR amplification system is 50 μ l, comprising: 5 × PrimeSTAR GXL Buffer 10 μ l, dNTP Mixture 4 μ l, upstream primer taUreG-F1(10 μMs) 2.5 μ l, downstream primer taUreG-R1(10 μMs) 2.5 μ l, template cDNA or gDNA 2 μ l, PrimeSTAR GXL DNA Polymerase(TaKaRa, R050Q) 0.5 μ l, ddH ₂o 28.5 μ l.

Its pcr amplification reaction program: 98 DEG C of denaturation 2 min; 98 DEG C of sex change 10 s, 58 DEG C of annealing 15 s, 68 DEG C extend 3 min, 30 circulations; 68 DEG C extend 10 min; 4 DEG C of preservations.

6, reclaimed by pcr amplification product purifying, use sepharose to reclaim test kit (sky root, DP209), concrete steps are as follows:

(1) column equilibration step: add 500 μ l balance liquid BL to (adsorption column puts into collection tube) in adsorption column CA2, centrifugal 1 min of 12000 rpm, outwells the waste liquid in collection tube, placed back in by adsorption column in collection tube; (2) single target DNA band is cut from sepharose put into clean centrifuge tube, take weight; (3) in blob of viscose, add equimultiple bulk solution PN, 50 DEG C of water-baths are placed, and constantly leniently spin upside down centrifuge tube therebetween, to guarantee that blob of viscose fully dissolves; (4) added in an adsorption column CA2 (adsorption column puts into collection tube) by previous step gained solution, room temperature places 2 min, and the centrifugal 30-60 s of 12000 rpm, outwells the waste liquid in collection tube, adsorption column CA2 is put into collection tube; (5) in adsorption column CA2, add 600 μ l rinsing liquid PW, the centrifugal 30-60 s of 12000 rpm, outwells the waste liquid in collection tube, adsorption column CA2 is put into collection tube; This step of repetitive operation; (6) put back in collection tube by adsorption column CA2, centrifugal 2 min of 12000 rpm, eliminate rinsing liquid as far as possible; Adsorption column CA2 is placed in room temperature and places several minutes, dry up hill and dale; (7) be put into by adsorption column CA2 in a clean centrifuge tube, to the appropriate elution buffer EB of the unsettled dropping in adsorption film mid-way, room temperature places 2 min; 12000 rpm are centrifugal, and 2 min collect DNA solution.

7, gene clone

Get the product after 4 μ l PCR purifying recovery, add 1 μ l pEASY-Blunt Cloning carrier (Quan Shijin, CB101-01), mix gently, at temperature is 20 DEG C-37 DEG C, reacts 5 min, completes connection; Product conversion e.colistraindh5α will be connected, scribble 8 μ l IPTG(500 mM on surface), 40 μ l X-gal(20 mg/ml) the dull and stereotyped 37 DEG C of grow overnight of kantlex (50 μ g/ml) LB; Select white colony, select positive colony order-checking by fast PCR.

8, wheat taUreGsequential analysis

DNAMAN software is used to analyze sequencing result, with taUreG-F1with taUreG-R1the wheat of primer amplification taUreGcDNA sequence as shown in SEQ ID NO:1, its cDNA sequence 945 bp, comprise one long be the open reading frame of 855 bp, 284 amino acid of encoding, aminoacid sequence is as shown in SEQ ID NO: 3.With taUreG-F1with taUreG-R1the wheat of primer amplification taUreGgDNA sequence as shown in SEQ ID NO:2, its gDNA sequence 2546 bp, comprises 7 exons and 6 introns.From 5 ' end, the length of exon is followed successively by 77 bp, 204 bp, 61 bp, 202 bp, 122 bp, 105 bp, 84 bp, the length of intron is followed successively by 86 bp, 600 bp, 336 bp, 345 bp, 129 bp, 105 bp, and gene structure is see Fig. 1.

Embodiment 2 Drought-resistance in Wheat gene taUreGchromosomal localization

With the genomic dna of the nulli-tetrasomes based material of a set of Common Wheat Varieties China spring for template, with taActinfor internal reference (primer taActin-F1: 5 '-GTTCCAATCTATGAGGGATACACGC-3 ' (see SEQ ID NO:6), taActin-R1: 5 '-GAACTTCCACTGAGAACAACATTACC-3 ' (see SEQ ID NO:7).With taUreGspecial primer taUreG-F1with taUreG-R1carry out pcr amplification.Referring to Fig. 2, result shows, taUreGin the nulli-tetrasomes system of deletion 1A, 2.5 this band of Kb specific amplified disappearances, prove that this assignment of genes gene mapping is on wheat 1A karyomit(e).

Embodiment 3 real-time fluorescence quantitative PCR (Real-time PCR) analysis verification taUreGeffect in the ABA signal path that Wheat Drought is correlated with

Experimental technique:

(1) material is cultivated and ABA process: section's agriculture 9204 wheat seed is seeded in the culture dish being covered with 2 metafiltration paper, dH ₂o soaks, and sprout 4 days, remove endosperm, Hogland is cultured to the 10th day, and select the consistent seedling of growing way and carry out 100 μMs of ABA process, 0 h, 1 h, 3 h, 6 h sample respectively ,-80 DEG C of preservations after liquid nitrogen freezing;

(2) utilize Trizol to extract total serum IgE, concrete steps are see the 2nd step of embodiment 1;

(3) PrimeScriptTM RT reagent Kit with gDNA Eraser(TaKaRa, RR047A is utilized) carry out the first chain cDNA and synthesize;

A, removal genomic dna, reaction system: 5 × gDNA Eraser Buffer 2 μ l, gDNA Eraser 1 μ l, Total RNA 5 μ l(200 ng/ μ l), RNase Free dH ₂o 2 μ l; Response procedures: hatch 2 min for 42 DEG C, 4 DEG C of preservations.

B, reverse transcription, the reaction solution 10 μ l of reaction system: step a, PrimeScript RT Enzyme Mix I 1 μ l, RT Primer Mix 1 μ l, 5 × PrimeScript Buffer 24 μ l, RNase Free dH ₂o 4 μ l; Response procedures: hatch 15 min for 37 DEG C, 85 DEG C of 5 s termination reaction, 4 DEG C of preservations.

(4) SYBR is used ^?premix Ex Taq II(TaKaRa, RR820A) test kit carries out real-time fluorescence quantitative PCR analysis, concrete steps:

Mix following reaction system, be then sub-packed in 96 hole optics versions, and cover upper blooming, the instrument that amplification uses is ABI PRISM 7500 real-time PCR instrument;

PCR response procedures: 95 DEG C of denaturation 30 s; 95 DEG C of sex change 5 s, anneal and extend 34 s for 60 DEG C, 40 circulations;

20 μ l PCR reaction systems are formulated as follows: SYBR ^?premix Ex Taq II(2 ×) 10 μ l, Forward Primer(10 μM) 0.8 μ l, Reverse Primer(10 μM) 0.8 μ l, ROX Reference Dye II(50 ×) 0.4 μ l, cDNA template 2 μ l, ddH ₂o 6 μ l;

Primer sequence is:

Upstream primer taUreG-F2: 5 '-GCCGATTTGCTGCTCTGT GA-3 ' (SEQ ID NO:8),

Downstream primer taUreG-R2: 5 '-GCCTGTTCTTGGTATCTTGTCCC-3 ' (SEQ ID NO:9);

Internal reference taActinprimer taActin-F2: 5 '-TGCTATCCTTCGTTTG GACCTT-3 ' (SEQ ID NO:10),

Internal reference taActinprimer taActin-R2: 5 '-AGCGGTTGTTGTGAGGGAGT-3 ' (SEQ ID NO:11).

(5) experimental result: as shown in Figure 3, along with the passing in treatment time, taUreGexpression in wheat leaf blade and root all regulates by ABA, presents the downward trend again that first rises.Within 1 hour in blade, reach climax, within 3 hours in root, arrive climax, expression amount declines afterwards, explanation taUreGtake part in ABA signal transduction pathway; taUreGshould be that the signal transduction pathway relied on by ABA take part in the response of wheat to drought stress.

Embodiment 4 real-time fluorescence quantitative PCR (Real-time PCR) analysis verification taUreGeffect in the ROS signal path that Wheat Drought is correlated with

Experimental technique:

(1) material is cultivated and H ₂o ₂process: section's agriculture 9204 wheat seed is seeded in the culture dish being covered with 2 metafiltration paper, dH ₂o soaks, and sprout 4 days, remove endosperm, Hogland is cultured to the 10th day, selects the consistent seedling of growing way and carries out 10 mM H ₂o ₂process, 0 h, 1 h, 3 h, 6 h sample respectively ,-80 DEG C of preservations after liquid nitrogen freezing;

(3) PrimeScriptTM RT reagent Kit with gDNA Eraser(TaKaRa, RR047A is utilized) to carry out the first chain cDNA and synthesize, concrete steps are see the 3rd step of embodiment 3;

(4) SYBR is used ^?premix Ex Taq II(TaKaRa, RR820A) test kit carries out real-time fluorescence quantitative PCR analysis, and concrete steps are see the 4th step of embodiment 3;

(5) experimental result: as shown in Figure 4, along with the passing in treatment time, taUreGexpression in wheat leaf blade is by H ₂o ₂regulate, present and first decline, rear rising and then downward trend.But, taUreGexpression in wheat root then not by H ₂o ₂regulate.The explanation of real-time fluorescence quantitative PCR result taUreGtake part in the signal transduction pathway of the ROS level rising that drought stress causes. taUreGthe excavation participating in Drought-resistance in Wheat mechanism is that the utilization of this gene in Drought-resistance in Wheat breeding provides more reliable evidence.Process LAN in wheat taUreG, or suppress taUreGexpression, by affecting the adaptive faculty of wheat to drought stress, cultivate the transgenic wheat with high drought resistance, to raising wheat yield be significant.

Embodiment 5

Experimental technique:

(1) material is cultivated and PEG-6000 manual simulation Osmotic treatment: section's agriculture 9204 wheat seed is seeded in the culture dish being covered with 2 metafiltration paper, dH ₂o soaks, and sprouts 4 days, removes endosperm, Hogland is cultured to the 10th day, select the consistent seedling of growing way and carry out the PEG-6000 process that mass percent concentration is 20%, 0 h, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h sample respectively ,-80 DEG C of preservations after liquid nitrogen freezing;

(5) experimental result: as shown in Figure 5, along with the passing in treatment time, taUreGexpression in wheat leaf blade and root, all by drought-induced obvious rise, reaches climax in 12 hours in blade, and within 6 hours in root, arrive climax, expression amount declines afterwards, explanation taUreGadapt to play an important role in drought stress mechanism at wheat.Process LAN in wheat taUreG, or suppress taUreGexpression, by affecting the adaptive faculty of wheat to drought stress, cultivate the transgenic wheat with high drought resistance, to raising wheat yield be significant.Show thus, contain taUreGthe wheat plant of gene has stronger drought-resistant ability, the gene relevant to Drought-resistance in Wheat taUreGcan be applied in seed selection Drought resistant Wheat kind; The screening of described method and seed selection can be adopted to contain the Drought resistant Wheat kind of this gene, certainly, also can by the gene of described nucleotide sequence as shown in SEQ ID NO:1 taUreGproceed to Wheat Tissue and cultivate the transgenic wheat with high drought resistance.

Foregoing description only proposes, not as the single restricted condition to its technical scheme itself as the enforceable technical scheme of the present invention.

SEQUENCE LISTING

<110> Inst. of Genetics and Development Biology, CAS

The gene TaUreG that <120> is relevant to Drought-resistance in Wheat and application thereof

<130>

<160> 11

<170> PatentIn version 3.3

<210> 1

<211> 945

<212> DNA

<213> wheat TaUreG gene cDNA

<400> 1

ccttccactt ccaagtctgg attccgtcca tggcgtccca ggatcaccac caccaccacg 60

gcggccactc ccacgacgac gaccaccatc accgccacca tcacggggat gccgccggga 120

agggggcggg ggcggggtcg tgggtcggcg aggacgggcg cgtgtggcac tcccacgacg 180

gcctggcgcc gcactcccac gagcccatct actccgccgg ggacttctcc aagcgcgcgc 240

cgccgctcga ctcccgcagc ttcgccgacc gcgccttcac cgtcggcatc ggcggccccg 300

tcggcaccgg gaagactgcc ctgatgttag cactctgcac ttgcctccgt gacaaatata 360

gtcttgcagc ggttacaaat gatatattca caaaagagga tggagaattc ttggtcaagc 420

atggagctct gcctgaagag cgcatacgtg ctgtcgaaac tggaggctgc cctcatgccg 480

ctatacgtga ggacatcagc ataaatctgg gccctctgga ggagctatcc aacttgtaca 540

aggccgattt gctgctctgt gaatctggag gagataacct ggcagccaac ttcagcaggg 600

agctagcaga ctacataatc tacatcatcg acgtgtccgg tggggacaag ataccaagaa 660

caggcggccc tgggataacc caagcagatc tcttggtcat aaacaagaca gaccttgcct 720

ccgcggttgg agccgaccta gccgtgatgg agcgagacgc ccttcggatg cgggaaggag 780

ggcccttcgt gttcgcccag gtgaaacacg gggttggcgt ggaggagatt gtggaccacg 840

tgctgcgggc ctgggagatc gccaccggca acaggcgccg atagagaggc tctctcacgc 900

gaccgaaacg ccggcgagta attttcgggc atgtgttcac gtatg 945

<210> 2

<211> 2546

<212> DNA

<213> wheat TaUreG gene gDNA

<400> 2

ccttccactt ccaagtctgg attccgtcca tggcgtccca ggatcaccac caccaccacg 60

gcggccactc ccacgacgac gaccaccatc accgccacca tcacgggtga gtctacccgc 120

ctccgcctcg ccatccttct cgccgtccgc gcagccgact caaccagcac ctgccctgct 180

ttgcgcttgc agggatgccg ccgggaaggg ggcgggggcg gggtcgtggg tcggcgagga 240

cgggcgcgtg tggcactccc acgacggcct ggcgccgcac tcccacgagc ccatctactc 300

cgccggggac ttctccaagc gcgcgccgcc gctcgactcc cgcagcttcg ccgaccgcgc 360

cttcaccgtc ggcatcggcg gccccgtcgg caccgggtac gcccagcttt tctcctgcgg 420

ttgcttaggt gccggactgg ccgttatgct gtgttgtccg gcgagatagg agccgtctct 480

gctgcatttt ccctctgaat ctgcctgtgt ttcccgcgta gtgtcggtgc agatgtcagc 540

tatagccttt tgatctttgg agataactgt ggatgcatgt actacacaca ttttagccgc 600

tgaaaaatgt tgctttgacc acaaatctgt tgtgaaatat tggagttatt ctcgagcaga 660

atagacatta tatgcactgt gattactgat tagtgaacac tgtcatatgc ttagggattg 720

tctgcctccc ttgctgtgct tacagccata cactattttg gtgttcttgc tgttgcggtt 780

atgtttccct gccacagtat aacatcctag cattttggaa ttcagaagcg agttctgggg 840

gtctgggcaa ttcgaagaaa tttgtgcaca gcctttttgt gttgacgaaa taacctcatg 900

tggtgaagca agggcttcta atttcatatc ataacaacta tggaatgacg aagtgagatg 960

acccccacca tcttctctct gttttgcctt atgcaggaag actgccctga tgttagcact 1020

ctgcacttgc ctccgtgaca aatatagtct tgcagcggta tgtggcatgg ttctatcctg 1080

tgcttctctt aagattgctt aagataccat ctacttctcc tgattaaact acaccgcttg 1140

ctgcatcttt gggcattgga ctttcatcat aacatttgaa gatatatttg gttggtcctg 1200

tttctgaact tcaaaacagt tgattcatat aacaggtcaa tttttgtctg tgggggccag 1260

cattgaaatc ttactacttc taatattctt gtgatatttt cttaaacaca aatacctaat 1320

agtctccacc ctgtatgctt gaattatatt acacccttag gatttctctt tctttatttt 1380

ttattatgtg caggttacaa atgatatatt cacaaaagag gatggagaat tcttggtcaa 1440

gcatggagct ctgcctgaag agcgcatacg tgctgtcgaa actggaggct gccctcatgc 1500

cgctatacgt gaggacatca gcataaatct gggccctctg gaggagctat ccaacttgta 1560

caaggccgat ttgctgctct gtgaatctgg aggaggtatt gtccctgttg aatcccacat 1620

tggaaggttt atcatcgtgt atgtttagac cggcttagtg ttgtcagttg actgtttcga 1680

aaatgtcctg tagctttttg ttttgcgatg aaacctgtat ccttttccgt aatattgttt 1740

aggaatatgc agtgtgcact actggttttt ctgaagctgt tttgttgttt cttgggcatt 1800

agttgaacat ctcgattgtc cctctgcttc tggataatgg gtcactgaag agcatcatca 1860

cggccccagc ttaagccatt ttgtgattaa gtccatgacc aggcactaag aaagctactt 1920

tttgtgcatc ttgtgtgcag ataacctggc agccaacttc agcagggagc tagcagacta 1980

cataatctac atcatcgacg tgtccggtgg ggacaagata ccaagaacag gcggccctgg 2040

gataacccaa gcagatctct tggtgcgtca ccgcaccttc taagccattc aactagactg 2100

caaacctgta tcacgatgct gatttatctt ctcaccttgc tgagacaagt tcaccgtttt 2160

cactaactcg gaccatccgt cccaaactca ggtcataaac aagacagacc ttgcctccgc 2220

ggttggagcc gacctagccg tgatggagcg agacgccctt cggatgcggg aaggagggcc 2280

cttcgtgttc gcccaggttc aagatcaaac catgcacaca catgcacaca tgcgcagttc 2340

tctattcctt gtaataatcg taatgccatg aactcattct gctctgacat cgctggtgca 2400

ggtgaaacac ggggttggcg tggaggagat tgtggaccac gtgctgcggg cctgggagat 2460

cgccaccggc aacaggcgcc gatagagagg ctctctcacg cgaccgaaac gccggcgagt 2520

aattttcggg catgtgttca cgtatg 2546

<210> 3

<211> 284

<212> PRT

<213> wheat TaUreG

<400> 3

Met Ala Ser Gln Asp His His His His His Gly Gly His Ser His Asp

1 5 10 15

Asp Asp His His His Arg His His His Gly Asp Ala Ala Gly Lys Gly

20 25 30

Ala Gly Ala Gly Ser Trp Val Gly Glu Asp Gly Arg Val Trp His Ser

35 40 45

His Asp Gly Leu Ala Pro His Ser His Glu Pro Ile Tyr Ser Ala Gly

50 55 60

Asp Phe Ser Lys Arg Ala Pro Pro Leu Asp Ser Arg Ser Phe Ala Asp

65 70 75 80

Arg Ala Phe Thr Val Gly Ile Gly Gly Pro Val Gly Thr Gly Lys Thr

85 90 95

Ala Leu Met Leu Ala Leu Cys Thr Cys Leu Arg Asp Lys Tyr Ser Leu

100 105 110

Ala Ala Val Thr Asn Asp Ile Phe Thr Lys Glu Asp Gly Glu Phe Leu

115 120 125

Val Lys His Gly Ala Leu Pro Glu Glu Arg Ile Arg Ala Val Glu Thr

130 135 140

Gly Gly Cys Pro His Ala Ala Ile Arg Glu Asp Ile Ser Ile Asn Leu

145 150 155 160

Gly Pro Leu Glu Glu Leu Ser Asn Leu Tyr Lys Ala Asp Leu Leu Leu

165 170 175

Cys Glu Ser Gly Gly Asp Asn Leu Ala Ala Asn Phe Ser Arg Glu Leu

180 185 190

Ala Asp Tyr Ile Ile Tyr Ile Ile Asp Val Ser Gly Gly Asp Lys Ile

195 200 205

Pro Arg Thr Gly Gly Pro Gly Ile Thr Gln Ala Asp Leu Leu Val Ile

210 215 220

Asn Lys Thr Asp Leu Ala Ser Ala Val Gly Ala Asp Leu Ala Val Met

225 230 235 240

Glu Arg Asp Ala Leu Arg Met Arg Glu Gly Gly Pro Phe Val Phe Ala

245 250 255

Gln Val Lys His Gly Val Gly Val Glu Glu Ile Val Asp His Val Leu

260 265 270

Arg Ala Trp Glu Ile Ala Thr Gly Asn Arg Arg Arg

275 280

<210> 4

<211> 20

<212> DNA

<213> TaUreG-F1

<400> 4

ccttccactt ccaagtctgg 20

<210> 5

<211> 20

<212> DNA

<213> TaUreG-R1

<400> 5

catacgtgaa cacatgcccg 20

<210> 6

<211> 25

<212> DNA

<213> TaActin-F1

<400> 6

gttccaatct atgagggata cacgc 25

<210> 7

<211> 26

<212> DNA

<213> TaActin-R1

<400> 7

gaacttccac tgagaacaac attacc 26

<210> 8

<211> 20

<212> DNA

<213> TaUreG-F2

<400> 8

gccgatttgc tgctctgtga 20

<210> 9

<211> 23

<212> DNA

<213> TaUreG-R2

<400> 9

gcctgttctt ggtatcttgt ccc 23

<210> 10

<211> 22

<212> DNA

<213> TaActin-F2

<400> 10

tgctatcctt cgtttggacc tt 22

<210> 11

<211> 20

<212> DNA

<213> TaActin-R2

<400> 11

agcggttgtt gtgagggagt 20

Claims

1. a gene relevant to Drought-resistance in Wheat taUreG, it is characterized in that, it has the cDNA nucleotide sequence shown in SEQ ID NO: 1 or the gDNA nucleotide sequence shown in SEQ ID NO: 2.

2. the gene relevant to Drought-resistance in Wheat according to claim 1 taUreG, it is characterized in that, described gDNA contains 7 exons and 6 introns.

3. a gene relevant to Drought-resistance in Wheat taUreG, it is characterized in that, the proteins encoded of this gene is as shown in SEQ ID NO: 3.

4. the gene relevant to Drought-resistance in Wheat according to claim 1 taUreG,it is characterized in that, clone described gene taUreGupstream primer taUreG-F1nucleotide sequence as shown in SEQ ID NO: 4, downstream primer taUreG-R1nucleotide sequence as shown in SEQ ID NO: 5.

5. the gene relevant to Drought-resistance in Wheat according to claim 1 taUreG,it is characterized in that, described gene is positioned on wheat 1A karyomit(e).

6. the gene relevant to Drought-resistance in Wheat according to claim 1 taUreG,it is characterized in that, when adopting real-time PCR detection, its upstream primer taUreG-F2nucleotide sequence as shown in SEQ ID NO: 8, downstream primer taUreG-R2nucleotide sequence as shown in SEQ ID NO: 9; Internal reference upstream primer taActin-F2nucleotide sequence as shown in SEQ ID NO: 10, internal reference downstream primer taActin-R2nucleotide sequence as shown in SEQ ID NO: 11.