CN104762305B - The gene TaUreG related to Drought-resistance in Wheat and its application - Google Patents
The gene TaUreG related to Drought-resistance in Wheat and its application Download PDFInfo
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- CN104762305B CN104762305B CN201510207339.3A CN201510207339A CN104762305B CN 104762305 B CN104762305 B CN 104762305B CN 201510207339 A CN201510207339 A CN 201510207339A CN 104762305 B CN104762305 B CN 104762305B
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Abstract
The invention discloses a kind of gene related to Drought-resistance in WheatTaUreG, it has SEQ ID NO:CDNA nucleotide sequences or SEQ ID NO shown in 1:GDNA nucleotide sequences shown in 2.Invention applies plant gene engineering technology, has cloned the gene associated with Drought-resistance in Wheat from wheat firstTaUreG, detected by real-time fluorescence quantitative PCR, the results showed that, the geneTaUreGExpression adjusted by drought stress, can wheat adapt to drought stress early stage just play an important role；And the geneTaUreGExpression equally by ABA and H2O2Regulation, explanationTaUreGResponse of the wheat to drought stress should be take part in by the ABA signal transduction pathways relied on, and take part in the horizontal elevated signal transduction pathways of ROS caused by drought stress.Therefore, when cultivating wheat breed, the wheat plant containing the gene has stronger drought-resistant ability, and this is to screening Drought resistant Wheat kind and cultivates the transgenic wheat with high drought resistance, improves plantation yield of the wheat under drought environment and is significant.
Description
Technical field
The present invention relates to technical field of biological genetic engineering, specifically a kind of and Drought-resistance in Wheat related geneTaUreG
And its application.
Background technology
At present, global climate, which warms, causes soil drought degree increasingly severe, and this forms direct prestige to grain-production
The side of body.Wheat is one of most important cereal crops, and the second largest cereal crops in China in the world.Plant is in reply drought stress
When can produce a series of protection mechanism, study the molecular mechanism of Drought-resistance in Wheat, culture Drought resistant Wheat new varieties are to improve wheat
One main path of yield, for ensureing that national food security and Water resources security are significant.
Plant produces a series of basic reaction, including growth and the suppression of metaboilic level, gene table under drought stress
Up to horizontal change, the change of hormone in vivo level, induction and suppress the generation of signal transduction pathway, soluble permeation protective agent
Accumulation, reactive oxygen species（ROS）Plasma membrane oxidation etc. caused by level rise.Drought signal mainly passes through abscisic acid（ABA）Rely on
And ABA independent of DREB transcription factors mediation signal transduction pathway regulation downstream gene expression（Yoshida et
al. ABA-dependent and ABA-independent signaling in response to osmotic stress
in plants. 2014）.Research shows that plant can produce a series of protection mechanism when tackling drought stress；Drought stress
The expression of many plant genes can be induced, the expression product of some genes can directly strengthen stress tolerance, some can sense and
Transduction stress signals-modulating gene expression.In recent years, with the development of genomics and molecular biology, Chinese and overseas scholars is from wheat
It is middle to have cloned the related gene of a collection of drought resisting, the Expression of a wheat MYB of such as 2011 Liu et al. reports
gene in transgenic tobacco enhances resistance to Ralstonia solanacearum, and
to drought and salt stresses；The Wheat WRKY genes of Niu et al. reports in 2012TaWRKY2
and TaWRKY19 regulate abiotic stress tolerance in transgenic Arabidopsis
plants；The Over-expression of of Qin et al. reports in 2012TaMYB33 encoding a novel
wheat MYB transcription factor increases salt and drought tolerance in
Arabidopsis；Molecular characterization of novel disclosed in Tang et al. in 2012 TaNAC
genes in wheat and overexpression of TaNAC2a confers drought tolerance in
tobacco；These, which are screening Drought resistant Wheat kind, improve the wheat planting yield under drought environment has established solid base
Plinth.
The content of the invention
It is an object of the invention to provide a kind of and Drought-resistance in Wheat related geneTaUreGAnd its application.
A kind of gene related to Drought-resistance in WheatTaUreG, it has SEQ ID NO:CDNA nucleotides sequences shown in 1
Row or SEQ ID NO:GDNA nucleotide sequences shown in 2.
GDNA of the present invention includes 7 extrons and 6 intrones.
A kind of gene related to Drought-resistance in WheatTaUreG, the encoding proteins such as SEQ ID NO of the gene:Shown in 3.
The present invention clones the geneTaUreGSense primerTaUreG-F1Nucleotide sequence such as SEQ ID NO:
4 is shown, anti-sense primerTaUreG-R1Nucleotide sequence such as SEQ ID NO:Shown in 5.
Gene of the present invention is located on wheat 1A chromosomes.
Gene source provided by the invention, according to sequence structure feature, is named as in wheat section agriculture 9204TaUreG, its
The bp of cDNA sequence total length 945, a length of 855 bp ORFs is included, encode 284 amino acid；GDAN sequences are complete
Long 2546 bp, are made up of 7 extrons and 6 intrones.Since 5' ends, the length of extron is followed successively by 77 bp, 204
Bp, 61 bp, 202 bp, 122 bp, 105 bp, 84 bp, the length of introne are followed successively by 86 bp, 600 bp, 336 bp, 345
Bp, 129 bp, 105 bp, gene structure are shown in Fig. 1.
The related gene of drought resisting disclosed by the inventionTaUreGWhen being analyzed by real-time PCR detection, its sense primerTaUreG-F2Nucleotide sequence such as SEQ ID NO:8 is shown, anti-sense primerTaUreG-R2Nucleotide sequence such as SEQ ID
NO :Shown in 9；Internal reference upstream primerTaActin-F2Nucleotide sequence such as SEQ ID NO:10 is shown, internal reference downstream primerTaActin-R2Nucleotide sequence such as SEQ ID NO:Shown in 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, ddH2O 6 μl；20 μ l altogether；Its PCR response procedures：
95 DEG C of s of pre-degeneration 30；95 DEG C of 5 s of denaturation, 60 DEG C of 34 s that anneal and extend, 40 circulate.
Section's agriculture 9204 used in the present invention is Approved variety, is made a joint checkup in 2002 by Hebei province Crop breed audit committee member
It is fixed；By national variety certification, variety certification numbering is that state examines wheat 2003037 within 2003.
Due to gene disclosed by the inventionTaUreGWith drought resistance, therefore can be by the nucleotide sequence such as SEQ ID NO
:1 or gene of its encoding proteins matter as shown in SEQ ID NO.3TaUreGWheat Tissue is transferred to, it is small so as to cultivate into drought tolerance
Wheat.
Invention applies plant gene engineering technology, has cloned the gene related to Drought-resistance in Wheat from wheat firstTaUreG, detected by real-time fluorescence quantitative PCR, its result shows, the geneTaUreGExpression by drought stress
Regulation, can just it be played an important role early stage wheat adapts to drought stress；And the geneTaUreGExpression it is same
By ABA and H2O2Regulation, explanationTaUreGIt is that wheat take part in drought stress by the ABA signal transduction pathways relied on
Response, and take part in the horizontal elevated signal transduction pathways of ROS caused by drought stress；TaUreGParticipate in Drought-resistance in Wheat mechanism
The utilization for the gene in Drought-resistance in Wheat breeding of excavating provide more structurally sound evidence.It is indicated above that containTaUreG
The wheat plant of gene has stronger drought-resistant ability, the gene related to Drought-resistance in WheatTaUreGIn seed selection Drought resistant Wheat kind
In can be applied；Especially can be by the nucleotide sequence such as SEQ ID NO:Gene shown in 1TaUreGIt is transferred to small
Wheat tissue cultivating goes out the transgenic wheat with high drought resistance.
Brief description of the drawings
Fig. 1 isTaUreGGene structure figure.Square frame represents extron, and lines represent introne.ATG and TAG are respectively
Beginning codon and terminator codon.
Fig. 2 is to utilize China spring nulli-tetrasomes system pairTaUreGCarry out the result of chromosome mapping.CS represents China spring；M
Represent marker；WithTaActin（Amplifiable A, B, D groups）DNA integrality is proved for internal reference.
Fig. 3 is real-time fluorescence quantitative PCR pairTaUreGThe analysis result of expression characteristic in the case where ABA handles different time.WithTaActinFor internal reference.
Fig. 4 is real-time fluorescence quantitative PCR pairTaUreGIn H2O2Handle the analysis result of expression characteristic under different time.WithTaActinFor internal reference.
Fig. 5 is real-time fluorescence quantitative PCR pairTaUreGExpressed under PEG-6000 manual simulation's Osmotic treatment different times
The analysis result of feature.WithTaActinFor internal reference.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.
Experimental method in following embodiments, it is conventional method unless otherwise specified.Experiment material used in embodiment
Material, reagent etc., unless otherwise specified, commercially obtain.Quantitative test in following examples, is respectively provided with and weighs three times
Multiple experiment, results averaged.
The Drought-resistance in Wheat related gene of embodiment 1TaUreGClone
1st, wheatTaUreGCDNA sequence electronic cloning
（1）The rice submitted with NCBIOsUreG（HM369059）Gene order is probe, searches for wheat est database,
Obtained sequence DNAMAN softwares are spliced, to ORFs（ORF）It is predicted, to obtain complete wheatTaUreG
CDNA sequence；
（2）A pair of special primers with Primer5 Software for Design and are filtered out according to obtained full length cDNA sequenceTaUreG- F1WithTaUreG-R1, it is respectively used to wheatTaUreGCDNA and gDNA gene clonings.Thereon, anti-sense primer is respectively：
Sense primerTaUreG-F1：5’-CCTTCCACTTCCAAGTCTGG-3’（SEQ ID NO：4）
Anti-sense primerTaUreG-R1：5’-CATACGTGAACACATGCCCG-3’（SEQ ID NO：5）.
2nd, Trizol is utilized（Invitrogen）Total serum IgE is extracted, is comprised the following steps that：
（1）Liquid nitrogen grinding 50-100 mg wheat leaf blades, it is transferred in the EP pipes containing 1 ml Trizol reagents, fills after grinding
Divide and mix, be stored at room temperature 5 min；（2）0.2 ml chloroforms are added, fully mixes, is stored at room temperature 2-3 min；At 4 DEG C, 12000
× g centrifuges 15 min；（3）The colourless aqueous phase in upper strata is transferred in a 1.5 clean ml EP pipes, it is different to add 0.5 ml
Propyl alcohol, room temperature places 10 min after mixing；At 4 DEG C, 12000 × g centrifuges 10 min；（4）Supernatant is removed, will be precipitated with 1 ml
75% ethanol cleans, and at 4 DEG C, 7500 × g centrifuges 5 min, afterwards air-dries precipitation；（5）Precipitation is dissolved in appropriate RNase-
In free deionized water, 60 DEG C of min of dissolution 10；Produce required total serum IgE；Can micro-spectrophotometer NanoDrop ND-
2000 Spectrotometer are quantitatively detected, electrophoretic analysis RNA integrality.
3rd, plant genome DNA extracts kit is utilized（Tiangeng, DP305）DNA is extracted, is comprised the following steps that：
（1）The mg of wheat leaf agreement that contracts a film or TV play to an actor or actress 100 is taken, liquid nitrogen is added and fully mills；（2）Ground powder is quickly transferred to pre-
In centrifuge tube first equipped with 700 μ l, 65 DEG C of preheating buffer solution GP1（Mercaptoethanol is added in the GP1 of preheating before experiment, makes it
Final concentration of 0.1%）, after rapid reverse mixing, centrifuge tube is placed on 65 DEG C of min of water-bath 20, centrifuge tube is overturned during water-bath
With biased sample for several times；（3）700 μ l chloroforms are added, are fully mixed, 12000 rpm centrifuge 5 min；（4）Carefully by upper one
Step gained upper strata aqueous phase is transferred in a new centrifuge tube, is added 700 μ l buffer solution GP2, is fully mixed；（5）By the liquid of mixing
Body is transferred in adsorption column CB3, and 12000 rpm centrifuge 30 s, discard waste liquid；（6）500 μ l buffer solutions are added into adsorption column CB3
GD, 12000 rpm centrifuge 30 s, outwell waste liquid, adsorption column CB3 is put into collecting pipe；（7）600 are added into adsorption column CB3
μ l rinsing liquids PW, 12000 rpm 30 s of centrifugation, outwell waste liquid, adsorption column CB3 are put into collecting pipe；（8）Repeat step
（7）；（9）Adsorption column CB3 is put back in collecting pipe, 12000 rpm centrifuge 2 min, outwell waste liquid, adsorption column CB3 is placed in into room
Temperature is placed several minutes, thoroughly to dry rinsing liquid remaining in sorbing material；（10）By adsorption column CB3 be transferred to one it is clean from
In heart pipe, 50-200 μ l elution buffer TE are vacantly added dropwise to the middle part of adsorbed film, room temperature places 2-5 min, and 12000
Rpm centrifuges 2 min, solution is collected into centrifuge tube, as required DNA.
4th, GoldScript cDNA synthetic agent box is utilized（Invitrogen, c81401190）Carry out the first chain cDNA conjunctions
Into comprising the following steps that：
（1）The μ l of total serum IgE 5 of step 2 extraction are sequentially added in 0.2 ml centrifuge tubes（200 ng/µl）、10 mM
The μ l of dNTP mix 1, μ l of Oligo dT Primer 1, the μ l of DEPC water 3；（2）Above-mentioned RNA/ primer mixtures are placed on 65
DEG C be incubated 5 min, be then placed within 1-2 min on ice；（3）In another pipe, 2 × reaction mixture is prepared, is sequentially added into
Following component：10 × RT buffer solutions 2 μ l, 25 mM MgCl242 μ l of μ l, 0.1 M DTT, recombinate RNase inhibitor
（40U/ µl）1 µl；（4）To above-mentioned（2）9 μ 2 × reaction premixed liquids of l are added in each RNA/ primers premixed liquid of step, gently
Soft mixing, 7500 rpm centrifuge 30 s；（5）42 DEG C of 2 min of incubation；1 μ l GoldScript RT are added in each pipe；42℃
It is incubated 50 min；（6）70 DEG C of 15 min of incubation, terminating reaction, cooled on ice；（8）7500 rpm centrifuge 30 s, into each pipe
1 μ l RNaseH are added, 37 DEG C of 20 min of incubation, -20 DEG C preserve.
5th, PCR（PCR）Reaction carries out sequence amplification
WithTaUreG-F1WithTaUreG-R1Primer enters performing PCR amplification to wheat cDNA and gDNA respectively；
Its PCR amplification system is 50 μ l, including：5 × PrimeSTAR GXL Buffer 10 μ l, dNTP Mixture
4 μ l, sense primerTaUreG-F1（10 μM）2.5 μ l, anti-sense primerTaUreG-R1（10 μM）2.5 μ l, template cDNA or
μ l, the PrimeSTAR GXL DNA Polymerase of gDNA 2（TaKaRa, R050Q）0.5 μ l, ddH2O 28.5 µl。
Its pcr amplification reaction program：98 DEG C of min of pre-degeneration 2；98 DEG C of 10 s of denaturation, 58 DEG C of 15 s of annealing, 68 DEG C extend 3
Min, 30 circulations；68 DEG C of 10 min of extension；4 DEG C of preservations.
6th, pcr amplification product is purified and reclaimed, use Ago-Gel QIAquick Gel Extraction Kit（Tiangeng, DP209）, specific steps
It is as follows：
（1）Column equilibration step：Into adsorption column CA2（Adsorption column is put into collecting pipe）500 μ l equilibrium liquid BL are added,
12000 rpm centrifuge 1 min, outwell the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe；（2）By single mesh
DNA bands cut and be put into clean centrifuge tube from Ago-Gel, weigh weight；（3）Isoploid is added into blob of viscose
Product solution PN, 50 DEG C of water-baths are placed, and constantly centrifuge tube are leniently spun upside down therebetween, to ensure that blob of viscose fully dissolves；（4）Will be upper
One step resulting solution is added in an adsorption column CA2（Adsorption column is put into collecting pipe）, room temperature place 2 min, 12000 rpm from
Heart 30-60 s, outwell the waste liquid in collecting pipe, and adsorption column CA2 is put into collecting pipe；（5）600 are added into adsorption column CA2
μ l rinsing liquids PW, 12000 rpm centrifugation 30-60 s, outwell the waste liquid in collecting pipe, adsorption column CA2 are put into collecting pipe；Weight
The step is operated again；（6）Adsorption column CA2 is put back in collecting pipe, 12000 rpm centrifuge 2 min, eliminate rinsing liquid as far as possible；Will
Adsorption column CA2 is placed in room temperature and placed several minutes, thoroughly dries；（7）Adsorption column CA2 is put into a clean centrifuge tube, to
Appropriate elution buffer EB is vacantly added dropwise in adsorbed film centre position, and room temperature places 2 min；12000 rpm centrifuge 2 min and collected
DNA solution.
7th, gene cloning
The product after 4 μ l PCR purifying recovery is taken, adds 1 μ l pEASY-Blunt Cloning carriers（Quan Shijin,
CB101-01）, it is gently mixed, reacts 5 min at being 20 DEG C -37 DEG C in temperature, complete connection；Connection product is converted into large intestine bar
Bacterium DH5 α bacterial strains, 8 μ l IPTG are scribbled on surface（500 mM）, 40 μ l X-gal（20 mg/ml）Kanamycins（50 μg/
ml）The 37 DEG C of growths of LB flat boards are overnight；White colony is selected, positive colony sequencing is selected by fast PCR.
8th, wheatTaUreGSequence analysis
Sequencing result is analyzed with DNAMAN softwares, withTaUreG-F1WithTaUreG-R1The wheat of primer amplificationTaUreGCDNA sequence such as SEQ ID NO：Shown in 1, its cDNA sequence 945 bp, comprising a length of 855 bp
ORFs, encode 284 amino acid, amino acid sequence such as SEQ ID NO:Shown in 3.WithTaUreG-F1WithTaUreG- R1The wheat of primer amplificationTaUreGGDNA sequences such as SEQ ID NO：Shown in 2, its gDNA sequence 2546 bp, comprising
7 extrons and 6 intrones.Since holding 5 ', the length of extron be followed successively by 77 bp, 204 bp, 61 bp, 202 bp,
122 bp, 105 bp, 84 bp, the length of introne are followed successively by 86 bp, 600 bp, 336 bp, 345 bp, 129 bp, 105
Bp, gene structure is referring to Fig. 1.
The Drought-resistance in Wheat gene of embodiment 2TaUreGChromosome mapping
Using the genomic DNA of the nulli-tetrasomes based material of a set of Common Wheat Varieties China spring as template, withTaActin
For internal reference（PrimerTaActin-F1：5’-GTTCCAATCTATGAGGGATACACGC-3’（See SEQ ID NO：6）,TaActin- R1：5’-GAACTTCCACTGAGAACAACATTACC-3’（See SEQ ID NO：7）.WithTaUreGSpecial primerTaUreG-F1
WithTaUreG-R1Enter performing PCR amplification.Referring to Fig. 2, as a result show,TaUreGIn deletion 1A nulli-tetrasomes system
2.5 Kb specific amplifieds band missings, it was demonstrated that the assignment of genes gene mapping is on wheat 1A chromosomes.
The real-time fluorescence quantitative PCR of embodiment 3（Real-time PCR）Analysis checkingTaUreGRelated in Wheat Drought
Effect in ABA signal paths
Experimental method：
（1）Material culture and ABA processing：The wheat seed of section's agriculture 9204, which is seeded in, to be covered with the culture dish of 2 layers of filter paper, dH2O
Soak, sprout 4 days, remove endosperm, Hogland, which cultivates, to the 10th day, to be selected at the consistent 100 μM of ABA of seedling progress of growing way
Reason, 0 h, 1 h, 3 h, 6 h are separately sampled, -80 DEG C of preservations after liquid nitrogen frozen；
（2）Using Trizol extract total serum IgE, specific steps referring to embodiment 1 the 2nd step；
（3）Utilize PrimeScriptTM RT reagent Kit with gDNA Eraser（TaKaRa, RR047A）Enter
The first chain of row cDNA is synthesized；
A, genomic DNA, reaction system are removed：The μ l of 5 × gDNA Eraser Buffer, 2 μ l, gDNA Eraser 1,
Total RNA 5 µl（200 ng/µl）, RNase Free dH2O 2 µl；Response procedures：42 DEG C of 2 min of incubation, 4 DEG C of guarantors
Deposit.
B, reverse transcription, reaction system：The step a μ l of 10 μ l, PrimeScript RT Enzyme Mix I of reaction solution 1,
μ l, the RNase Free dH of 1 μ l, 5 × PrimeScript Buffer of RT Primer Mix 242O 4 µl；Reaction interval
Sequence：37 DEG C of 15 min of incubation, 85 DEG C of 5 s terminating reaction, 4 DEG C of preservations.
（4）Use SYBR®Premix Ex Taq II（TaKaRa, RR820A）Kit carries out real-time fluorescence quantitative PCR
Analysis, specific steps：
Following reaction system is mixed, is then sub-packed in 96 hole optics versions, and covers upper optical film, expands the instrument used
For the real-time PCR instruments of ABI PRISM 7500；
PCR response procedures：95 DEG C of s of pre-degeneration 30；95 DEG C of 5 s of denaturation, 60 DEG C of 34 s that anneal and extend, 40 circulate；
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, ddH2O 6 μl；
Primer sequence is：
Sense primerTaUreG-F2：5’-GCCGATTTGCTGCTCTGT GA-3’（SEQ ID NO：8）,
Anti-sense primerTaUreG-R2：5’-GCCTGTTCTTGGTATCTTGTCCC-3’（SEQ ID NO：9）；
Internal referenceTaActinPrimerTaActin-F2：5’-TGCTATCCTTCGTTTG GACCTT-3’（SEQ ID NO：
10）,
Internal referenceTaActinPrimerTaActin-R2：5’-AGCGGTTGTTGTGAGGGAGT-3’（SEQ ID NO：11）.
（5）Experimental result：As shown in figure 3, with the passage of processing time,TaUreGExpression in wheat leaf blade and root
Adjusted by ABA, presentation first rises downward trend again.Reach top within 1 hour in blade, reach most within 3 hours in root
Peak, afterwards expression quantity decline, explanationTaUreGIt take part in ABA signal transduction pathways；TaUreGShould be relied on by ABA
Signal transduction pathway take part in response of the wheat to drought stress.
The real-time fluorescence quantitative PCR of embodiment 4（Real-time PCR）Analysis checkingTaUreGRelated in Wheat Drought
Effect in ROS signal paths
Experimental method：
（1）Material culture and H2O2Processing：The wheat seed of section's agriculture 9204, which is seeded in, to be covered with the culture dish of 2 layers of filter paper, dH2O
Soak, sprout 4 days, remove endosperm, Hogland, which cultivates, to the 10th day, selects the consistent 10 mM H of seedling progress of growing way2O2Processing, 0
H, 1 h, 3 h, 6 h are separately sampled, -80 DEG C of preservations after liquid nitrogen frozen；
（2）Using Trizol extract total serum IgE, specific steps referring to embodiment 1 the 2nd step；
（3）Utilize PrimeScriptTM RT reagent Kit with gDNA Eraser（TaKaRa, RR047A）Enter
The first chain of row cDNA synthesize, specific steps referring to embodiment 3 the 3rd step；
（4）Use SYBR®Premix Ex Taq II（TaKaRa, RR820A）Kit carries out real-time fluorescence quantitative PCR
Analysis, specific steps referring to embodiment 3 the 4th step；
（5）Experimental result：As shown in figure 4, with the passage of processing time,TaUreGExpression in wheat leaf blade by
H2O2Regulation, presentation are fallen before, rear to rise then downward trend again.ButTaUreGExpression in wheat root then not by
H2O2Regulation.Real-time fluorescence quantitative PCR result explanationTaUreGThe horizontal elevated signals of ROS caused by drought stress are take part in turn
Guiding path.TaUreGUtilization of the excavation of participation Drought-resistance in Wheat mechanism for the gene in Drought-resistance in Wheat breeding, which provides, more may be used
The evidence leaned on.It is overexpressed in wheatTaUreG, or suppressTaUreGExpression, adaptation energy of the wheat to drought stress will be influenceed
Power, the transgenic wheat with high drought resistance is cultivated, be significant to improving wheat yield.
Embodiment 5
Experimental method：
（1）Material culture and PEG-6000 manual simulation's Osmotic treatments：The wheat seed of section's agriculture 9204, which is seeded in, is covered with 2 layers of filter
In the culture dish of paper, dH2O soaks, and sprouts 4 days, removes endosperm, Hogland was cultivated to the 10th day, is selected the consistent seedling of growing way and is entered
Row mass percent concentration is 20% PEG-6000 processing, and 0 h, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h are separately sampled, liquid
- 80 DEG C of preservations after chilled nitrogen；
（2）Using Trizol extract total serum IgE, specific steps referring to embodiment 1 the 2nd step；
（3）Utilize PrimeScriptTM RT reagent Kit with gDNA Eraser（TaKaRa, RR047A）Enter
The first chain of row cDNA synthesize, specific steps referring to embodiment 3 the 3rd step；
（4）Use SYBR®Premix Ex Taq II（TaKaRa, RR820A）Kit carries out real-time fluorescence quantitative PCR
Analysis, specific steps referring to embodiment 3 the 4th step；
（5）Experimental result：As shown in figure 5, with the passage of processing time,TaUreGExpression in wheat leaf blade and root
By drought-induced obvious up-regulation, reach top within 12 hours in blade, reach top within 6 hours in root, express afterwards
Amount declines, explanationTaUreGAdapt to play an important role in drought stress mechanism in wheat.It is overexpressed in wheatTaUreG, or suppression
SystemTaUreGExpression, wheat will be influenceed to the adaptability of drought stress, cultivate the transgenic wheat with high drought resistance,
It is significant to improving wheat yield.It is indicated above that containTaUreGThe wheat plant of gene has stronger drought resisting
Ability, the gene related to Drought-resistance in WheatTaUreGIt can be applied in seed selection Drought resistant Wheat kind；It can use described
Method is screened and seed selection contains the Drought resistant Wheat kind of the gene, it is of course also possible to by the nucleotide sequence such as SEQ ID
NO:Gene shown in 1TaUreGIt is transferred to Wheat Tissue and cultivates the transgenic wheat with high drought resistance.
Foregoing description only proposes as the enforceable technical scheme of the present invention, not as to its technical scheme single in itself
Restrictive condition.
SEQUENCE LISTING
<110>Inst. of Genetics and Development Biology, CAS
<120>The gene TaUreG related to Drought-resistance in Wheat and its application
<130>
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 945
<212> DNA
<213>Wheat TaUreG gene cDNAs
<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 genes 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 (6)
- A kind of 1. gene related to Drought-resistance in WheatTaUreG, it is characterised in that it has SEQ ID NO:CDNA shown in 1 Nucleotide sequence or SEQ ID NO:GDNA nucleotide sequences shown in 2.
- 2. the gene related to Drought-resistance in Wheat according to claim 1TaUreG, it is characterised in that described gDNA contains 7 extrons and 6 intrones.
- A kind of 3. gene related to Drought-resistance in WheatTaUreG, it is characterised in that the encoding proteins of the gene such as SEQ ID NO: Shown in 3.
- 4. the gene related to Drought-resistance in Wheat according to claim 1TaUreG,Characterized in that, clone the geneTaUreGSense primerTaUreG-F1Nucleotide sequence such as SEQ ID NO:4 is shown, anti-sense primerTaUreG-R1's Nucleotide sequence such as SEQ ID NO:Shown in 5.
- 5. the gene related to Drought-resistance in Wheat according to claim 1TaUreG,Characterized in that, the gene is positioned at small On wheat 1A chromosomes.
- 6. the gene related to Drought-resistance in Wheat according to claim 1TaUreG,Characterized in that, using real-time fluorescence When PCR is detected, its sense primerTaUreG-F2Nucleotide sequence such as SEQ ID NO:8 is shown, anti-sense primerTaUreG-R2 Nucleotide sequence such as SEQ ID NO:Shown in 9；Internal reference upstream primerTaActin-F2Nucleotide sequence such as SEQ ID NO :10 is shown, internal reference downstream primerTaActin-R2Nucleotide sequence such as SEQ ID NO:Shown in 11.
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