CN108559753A

CN108559753A - The breeding method of application and Rust resistance bacterium wheat of the wheat stripe rust PSTG_17694 genes in stripe rust prevention

Info

Publication number: CN108559753A
Application number: CN201810108056.7A
Authority: CN
Inventors: 吴佳洁; 黄德华; 张会飞; 刘强; 倪飞; 付道林
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2018-02-02
Filing date: 2018-02-02
Publication date: 2018-09-21
Anticipated expiration: 2038-02-02
Also published as: CN108559753B

Abstract

The present invention provides application of the wheat stripe rust PSTG_17694 genes in stripe rust of wheat prevention, and the sequence of the PSTG_17694 genes is as shown in Seq ID No.1；The PSTG_17694 genes can effectively regulate and control the growth and breeding of wheat stripe rust, using PSTG_17694 genes as the molecular target of transcriptional control or as the gene target that protein function inhibits, have the function that inhibit wheat stripe rust growth and breeding by PSTG_17694 genes described in silence.The wheat that the method provided by the invention for cultivating Rust resistance bacterium wheat using the gene is obtained has significant wheat stripe rust resistance.

Description

Application and Rust resistance of the wheat stripe rust PSTG_17694 genes in stripe rust prevention The breeding method of bacterium wheat

Technical field

The invention belongs to genetic engineerings and crop molecular breeding technology field, and in particular to wheat stripe rust PSTG_ The breeding method of application and Rust resistance bacterium wheat of 17694 genes in stripe rust prevention.

Background technology

Wheat is the second largest cereal crops in China, and average annual sown area is about 3.6 hundred million mu.The stabilization of wheat yield and raising It involves the interests of the state and the people.However for many years, fungal disease constitutes a serious threat to Wheat Production, as rust, powdery mildew, head blight, The generation of the diseases such as banded sclerotial blight seriously affects the yield and quality of wheat.Wherein, stripe rust of wheat is commonly called as " jaundice ", is by special Property parasitic strip rust bacteria (Puccinia striiformis Westend.f.sp.tritici) cause.In China, wheat item rust Morbidity is rampant, and long-term injured area is up to 6,000~8,0,000,000 mu of (Ministry of Agriculture's files：Nong Nongfa 2006-9)；It falls ill serious year Part causes the substantially underproduction (20~30%), threatens grain security.There are zoogamy for strip rust bacteria, and Toxicity Variation is fast, newly in recent years Dominant races persistently occur, cause stripe rust control difficulty increase.The stripe rust of the wheat major production areas Huang-Huai-Hai area of wheat occurs Area has the tendency that gradually increasing, and it is more than 4,000 to count 7 provinces and cities such as Henan, Shandong, Anhui in May, 2017 to add up occurring area Ten thousand mu (Agricultural Science ＆ Technology Extensions, 2017).Therefore, effectively prevention stripe rust is of great significance for China's grain security.

Disease-resistant variety is cultivated and planted, is the prevention economical and effective important measures of stripe rust of wheat.Currently, China saves more Wheat breed authorization whether have stripe rust resistance be classified as key index.It improves resistance level, cultivate durable resistance Have become the important goal of new variety of wheat cultivation.The cultivation of disease-resistant variety mainly passes through the excavation of Stripe Rust Resistance Gene and profit With, however due to the quick variation of strip rust bacteria biological strain toxicity, disease-resistant gene large area may lose in a short time after utilizing Lose disease resistance.Such as once widely used green ant No.1, " Lip river class ", " the anti-source such as numerous 6 " Derivative line, to working as on wheat breeding Preceding Epidemic Races lose resistance.501 parts of main cultivations current to China and the detection of standby kind show the kind less than 30% to working as Preceding Epidemic Races performance is disease-resistant, and its anti-source is concentrated mainly on a small number of genes such as Yr26/Yr24 (Han Dejun etc., northwest-China North-the middle and lower reach of Yangtze River current wheat breed of stripe rust Epidemic Flora (is) that stripe rust resisting is evaluated, Scientia Agricultura Sinica, 2010 (43)).Therefore, new stripe rust control strategy is developed in the anti-source for widening wheat stripe rust resisting disease, big for reducing stripe rust of wheat The risk of area prevalence is of great significance.

For wheat stripe rust due to its biotroph and obligatory parasitism characteristic, genetic transformation is difficult；And wheat itself is same Sample is difficult to carry out genetic transformation, therefore the genetics research difficulty of wheat-strip rust bacteria interaction is very big, and progress is slower.At present small Identified in wheat strip rust bacteria infect or the key gene negligible amounts that cause a disease, and there has been no gene, to be efficiently applied to wheat anti-at present Sick breeding.

Invention content

In view of this, the purpose of the present invention is to provide a kind of wheat stripe rust gene as molecular target in wheat crops Breeding or the application in stripe rust prevention and a kind of method for cultivating Rust resistance bacterium wheat using the gene.

In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：Wheat stripe rust PSTG_17694 genes Application in stripe rust of wheat prevention, the sequence of the PSTG_17694 genes is as shown in Seq ID No.1.

Preferably, the PSTG_17694 genes inhibit as the molecular target of Transcription inhibition or as protein function Gene target, by PSTG_17694 genes described in silence have the function that inhibit wheat stripe rust growth and breeding.

Preferably, the application is in the silence expression vector importing wheat crops for will carry the PSTG_17694 genes Obtain the wheat crops of Rust resistance bacterium.

Preferably, the application is to be inhibited by spraying the transcription inhibitor of the PSTG_17694 genes to wheat leaf blade The growth and breeding of strip rust bacteria.

Preferably, the transcription inhibitor of the PSTG_17694 genes is that the PSTG_17694 genes can be inhibited to turn The dsRNA solution of record.

Preferably, the application is the activity inhibitor of the coding albumen by spraying the PSTG_17694 genes to small Wheat blade inhibits the growth and breeding of strip rust bacteria.

The present invention also provides a kind of breeding methods of Rust resistance bacterium wheat, include the following steps：1) to infect strip rust bacteria The wheat leaf blade cDNA to fall ill afterwards is that template carries out PCR amplification acquisition PSTG_17694 genetic fragments；2) utilize step 1) described PSTG_17694 genetic fragments build wheat stripe rust PSTG_17694 gene silencing expression vectors；The PSTG_17694 The structure of gene silencing expression vector uses GATEWAY clone technologies；3) use Biolistic mediated transformation method by the wheat item Rest fungus PSTG_17694 gene silencing expression vectors are transferred to acquisition Rust resistance bacterium wheat in wheat.

Preferably, PCR amplification described in step 1) is CQM17694-F2 and CQM17694-R2 primers with primer；It is described The sequence of CQM17694-F2 is as shown in Seq ID No.3；The sequence of the CQM17694-R2 primers such as Seq ID No.4 institutes Show.

Preferably, the gene silencing expression vector of wheat stripe rust PSTG_17694 described in step 2) includes that hygromycin is anti- The positive sequence and PSTG_17694 genetic fragments of property gene Hyg, herbicide resistance gene Bar, PSTG_17694 genetic fragment Reverse sequence expression cassette.

Preferably, the wheat stripe rust PSTG_17694 gene silencings expression vector from T-DNALeft Boarder to The sequence of RightBoarder sections is as shown in sequence table SeqID No.6.

Beneficial effects of the present invention：Present invention firstly discovers that the PSTG_17694 genes can effectively regulate and control wheat The growth and breeding of strip rust bacteria enriches the key gene for infecting or causing a disease in wheat stripe rust；By the PSTG_17694 genes Reached by PSTG_17694 genes described in silence in being prevented applied to stripe rust of wheat and inhibits wheat stripe rust growth and breeding Effect.The wheat that the method provided by the invention for cultivating Rust resistance bacterium wheat using the gene is obtained has significant wheat item Rust resistance.

Description of the drawings

Fig. 1 is the positive transgenic weeding for wheat agent the selection result figure that silence expresses PSTG_17694 in embodiment 1；

Fig. 2 is 1 moderate resistance strip rust bacteria wheat PCR selective mechanisms results of embodiment；

Fig. 3 is that 2 moderate resistance strip rust bacteria wheat greenhouse of embodiment is inoculated with strip rust bacteria rear blade incidence figure；

Fig. 4 is that 2 moderate resistance strip rust bacteria wheat growth cabinet of embodiment is inoculated with strip rust bacteria rear blade incidence graph；

Fig. 5 is the fluorescent quantitation expression point that 3 moderate resistance strip rust bacteria wheat of embodiment is inoculated with PSTG_17694 genes after strip rust bacteria Analyse result figure；

Fig. 6 is that PSTG_17694 is inoculated with strip rust bacteria rear blade tissue staining observation chart in embodiment 3.

Specific implementation mode

The present invention provides application of the wheat stripe rust PSTG_17694 genes in stripe rust of wheat prevention.In the present invention Described in PSTG_17694 genes GenBank accession number be KNE88861.1, the sequence of the PSTG_17694 genes is such as Shown in Seq ID No.1, gene order length is 972bp, and the PSTG_17694 gene coding amino acids sequence has CE4_ MrCDA_Like structural domains (cd10952), particular sequence is as shown in Seq ID No.2.

In the present invention, the PSTG_17694 genes are preferably as the molecular target of transcriptional control or as albumen The gene target of matter function inhibitio reaches the work for inhibiting wheat stripe rust growth and breeding by PSTG_17694 genes described in silence With.In specific implementation process of the present invention, carry out silence PSTG_17694 genes preferably through following three kinds of methods, to realize State application of the PSTG_17694 genes in stripe rust of wheat prevention：(1) the silence table of the PSTG_17694 genes will be carried Up in vector introduction wheat crops, the wheat crops of Rust resistance bacterium are obtained；(2) by spraying the PSTG_17694 genes Transcription inhibitor inhibits the growth and breeding of strip rust bacteria to wheat leaf blade；The transcription inhibitor can preferably inhibit described The dsRNA solution of PSTG_17694 genetic transcriptions；(3) pressed down by spraying the activity of the PSTG_17694 gene coded proteins Preparation inhibits the growth and breeding of strip rust bacteria to wheat leaf blade.

The present invention is preferably designed PCR amplification primer before expanding PSTG_17694 genetic fragments.In the present invention In, it is preferably reference with the whole genome sequence of wheat stripe rust, to assume functional protein gene PS TG_17694 (GenBank:KNE88861.1 coding region sequence) is stencil design PCR primer；It is more preferably small with wheat stripe rust physiology Kind PST-78 whole genome sequences are to refer to (GenBank accession number AJIL00000000.1 or BROAD download links ftp:// ftp.broadinstitute.org/pub/annotation/fungi/puccinia/genomes/puccinia_striifo rmis_pst-78/).In the present invention, the method for the PCR amplification design of primers uses this field conventional method, specifically Primer-design software can be used to realize.In the present invention the PCR amplification primer be preferably CQM17694-F2 and CQM17694-R2 primers；The sequence of the CQM17694-F2 is as shown in Seq ID No.3；The CQM17694-R2 primers Sequence is as shown in Seq ID No.4.

The present invention preferably tests the amplification section of the PCR amplification primer after design obtains PCR amplification primer Card.The verification is specifically the sequence and wheat stripe rust PST-78 full-length genomes data and ncbi database with amplification section In wheat stripe rust sequence be compared, check whether the sequence of the amplification section being capable of specific target PSTG_17694 Gene.In the present invention the amplification sector sequence of above-mentioned PCR amplification primer and wheat stripe rust PST-78 full-length genomes data and After wheat stripe rust sequence alignment in ncbi database, by after sequence alignment find PSTG_17694 First Exons with PSTG_19439 homologys are higher, in addition to this do not find that there are length to be more than with other genes for the sequence for expanding section 100% concensus sequence of 20bp, showing can special target PSTG_ using the sequence of CQM17694-F2/R2 primer amplification sections 17694 genes.

In the present invention, PCR amplification acquisition PSTG_ is carried out as template to infect the wheat leaf blade cDNA to fall ill after strip rust bacteria 17694 genetic fragments.In the present invention, it is preferred to using the wheat leaf blade system fallen ill after infection strip rust bacteria biological strain CRY32 Standby cDNA；The method for preparing cDNA preferably prepares purifying RNA using routine TRIZOL methods, and reverse transcription obtains cDNA. In the present invention, it is described prepare purifying RNA and reverse transcription and obtain cDNA RNA purification kits and Reverse Transcription is respectively adopted Box.In specific implementation process of the present invention, the purifying RNA for preparing preferably uses Tiangeng biochemical technology (Beijing) limited public affairs The kit of department, article No. DP405 is realized；The reverse transcription obtains cDNA and preferably uses the complete limited public affairs of formula gold biotechnology in Beijing Take charge of reverse transcription reagent box, article No. AT311-03.

The present invention is after obtaining the wheat leaf blade cDNA for infecting and falling ill after strip rust bacteria, using the cDNA as template, carries out PCR Amplification obtains PSTG_17694 genetic fragments.The enzyme that the PCR amplification uses in the present invention is preferably high fidelity enzyme, more excellent Choosing is Phusion high fidelity enzymes (Thermo Scientific companies, article No. F-530S)；The system of the PCR amplification and Program uses the PCR amplification systems and program of this field routine, specifically in implementation process of the present invention, the PCR bodies System is：5X Phusion HF Buffer:10 μ l, Phusion DNAPolymerase:0.5 μ l, CQM17694-F2:1.5 μ l, CQM17694-R2:1.5 μ l, 10mM dNTPs:1 μ l, Template DNA:1 μ l, ddH₂O:34.5μl；The PCR programs are： 98℃ 30s；98 DEG C of 10s, 60 DEG C of 20s, 72 DEG C of 30s 35 are recycled；72℃ 10min.

The present invention is preferably detached and is sequenced to amplified production after obtaining amplified production, heretofore described separation Using the separation method of this field routine；The sequencing commission sequencing company is completed；Heretofore described amplified production is surveyed For the sequence that sequence obtains as shown in Seq ID No.5, the sequence similarity 99% with reference gene PSTG_17694 has 1 SNP The site (Single Nucleotide Polymorphism).

The present invention builds wheat stripe rust after obtaining PSTG_17694 genetic fragments, using GATEWAY clone technologies PSTG_17694 gene silencing expression vectors.In the present invention, the wheat stripe rust PSTG_17694 gene silencings expression carries The construction method of body is referring to bibliography (clone of Xiu-Li Han's barley salicylic acid synthetic genes ICS and PAL and the mountains analysis [D] Eastern agriculture university, 2013.), specifically include the following steps：(A) the PSTG_17694 genetic fragments and entry vector are connected It is transferred to competent escherichia coli cell after connecing and obtains positive colony plasmid；(B) by the Plasmid DNA and RNAi of the positive colony Silence expression vector skeleton PC336 carries out LR reactions, at the same by the forward direction of PSTG_17694 genetic fragments, reverse sequence recombinate to Carrier PC336 obtains RNAi silence expression vectors.

In the present invention, it is preferred to the PSTG_17694 genetic fragments are connect with entry vector；The entry vector The preferably PC414C of laboratory transformation, compared with conventional entry vector pENTR-D-TOPO, PC414C inserts polyclonal Site can realize directed cloning by the method for digestion, connection.The method of specific connection PC414C and target fragment is referring to reference Document (clone of Xiu-Li Han's barley salicylic acid synthetic genes ICS and PAL and analysis [D] Shandong Agricultural University, 2013.).This Invention is preferably to increase protection base and digestion on CQM17694-F2/R2 primer sequences using GATEWAY clone technologies Primer after site is improved.The preferred protection base is connected to 5 ' end of primer sequence；The number of the protection base Preferably 2~4；In specific implementation process of the present invention, the protection base on the CQM17694-F2 is preferably CAA It is preferably TCA with the protection base on CQM17694-R2.In the present invention, the restriction enzyme site is preferably NotI digestions Site and AscI restriction enzyme sites, the CQM17694-F2 primer sequences connect NotI restriction enzyme sites, and the CQM17694-R2 draws Object sequence connects AscI restriction enzyme sites.

In specific implementation process of the present invention, before the PSTG_17694 genetic fragments are connect with entry vector, preferably By primer amplification after above-mentioned improvement obtain include PSTG_17694 genetic fragments pcr amplification product and entry vector PC414C into Row NotI, AscI double digestion obtains digestion products, then utilizes T4-DNA ligases by digestion products (i.e. PSTG_ after purification 17694 genetic fragments and entry vector) it is attached acquisition connection product.The present invention preferably will after obtaining connection product The connection product is converted to competent escherichia coli cell, and screening obtains positive colony plasmid DNA after purification.In the present invention In, the competent escherichia coli cell after screening conversion obtains the positive colony plasmid.The method of heretofore described screening Preferably bacterium colony PCR；The primer of the bacterium colony PCR is preferably CQM17694-F2/R2；The purifying positive colony plasmid DNA preferably uses Plasmid DNA kit to complete (TIANGEN Biotech (Beijing) Co., Ltd., article No. DP103).The present invention exists After the Plasmid DNA for obtaining positive colony, Plasmid DNA and the RNAi silence expression vector skeleton PC336 (Korea Spro of positive colony are utilized The clone of beautiful barleys salicylic acid synthetic gene ICS and PAL and analysis [D] Shandong Agricultural University, 2013.) LR reactions are carried out, The forward direction of PSTG_17694 genetic fragments, reverse sequence are recombinated to carrier PC336 and obtain RNAi silence expression vectors.The present invention In, the vector construction can obtain RNAi using the carrier construction method and program of this field routine without other particular/special requirements Silence expression vector.

The wheat stripe rust PSTG_17694 gene silencing expression vectors number obtained in the present invention is PC897, described small From T-DNALB (Left Boarder) to RB (RightBoarder) on wheat strip rust bacteria PSTG_17694 gene silencing expression vectors For the sequence of section preferably as shown in Seq IDNo.6, the T-DNALB (LeftBoarder) arrives RB (RightBoarder) The sequence of section is to include hygromycin gene Hyg connected in sequence, herbicide resistance gene Bar and PSTG_17694 gene The expression cassette of the positive sequence of segment and the reverse sequence of PSTG_17694 genetic fragments.

The present invention is after obtaining wheat stripe rust PSTG_17694 gene silencing expression vectors, using Biolistic mediated transformation The wheat stripe rust PSTG_17694 gene silencing expression vectors are transferred to acquisition Rust resistance bacterium wheat in wheat by method.The present invention Middle acceptor material is preferably the strain CB037 of high sense stripe rust of wheat.The particular technique flow of heretofore described conversion is such as Described in Wang Shu rue Master's thesis (Wang Shu rue wheats source regenerates foundation [D] the Shandong Agricultural University with transformation system, 2012.).Details are not described herein.

Material after conversion is preferably carried out callus by the present invention after the completion of conversion on luring more culture medium The differentiation culture of induction, callus obtains vegetative seedling, and soil can be transplanted to by being obtained after the vegetative seedling culture of rootage In wheat seedling.Culture obtains the specific steps of wheat seedling after the conversion in the present invention and parameter reference Wang Shuyun master discusses (Wang Shu rue wheats source regenerates and foundation [D] the Shandong Agricultural University of transformation system, 2012.) described in text.

With reference to embodiment to wheat stripe rust PSTG_17694 genes provided by the invention stripe rust prevention in It is described in detail using the breeding method with Rust resistance bacterium wheat, but they cannot be interpreted as protecting model to the present invention The restriction enclosed.

Embodiment 1

(1) PCR amplification obtains PSTG_17694 genetic fragments

It is to refer to (GenBank accession number with Physiologic Races of Wheat Stripe Rust PST-78 whole genome sequences AJIL00000000.1 or BROAD download links ftp://ftp.broadinstitute.org/pub/annotation/ Fungi/puccinia/genomes/puccinia_striifo rmis_pst-78/), to assume functional protein (hypothetical protein) gene PS TG_17694 (GenBank:KNE88861.1 coding region sequence) sets for template Count PCR primer.

Forward primer sequence C QM17694-F2, as shown in Seq ID No.3：

5`-caagcggccgcTCATTCGACCAAAGAAAGGC-3`；

Reverse primer sequences CQM17694-R2, as shown in SeqID No.4：

5`-tcaggcgcgccGATCACCTCGTTTCCATGCT-3`。

Expand section length 490bp.Utilize the sequence alignment wheat stripe rust PST-78 full-length genome data of amplification section (https://genome.jgi.doe.gov/Pucst_PST78_1/Pucst_PST78_1.home.html) and NCBI data Library wheat stripe rust sequence (Pucciniastriiformis f.sp.tritici, taxid:168172), in the present invention The amplification sector sequence for stating PCR amplification primer, by finding PSTG_17694 First Exons and PSTG_ after sequence alignment 19439 homologys are higher, and in addition to this not finding the sequence for expanding section, there are length more than 20bp's with other genes 100% concensus sequence, showing can special target PSTG_17694 using the sequence of CQM17694-F2/R2 primer amplification sections Gene.

The wheat leaf blade fallen ill after materials infection strip rust bacteria biological strain CRY32, using conventional TRIZOL methods, (Tiangeng is given birth to Change scientific and technological (Beijing) Co., Ltd, article No. DP405) purifying RNA, and prepare the (Beijing Quanshijin Biotechnology Co., Ltd cDNA Reverse transcription reagent box, article No. AT311-03).Use Phusion high fidelity enzymes (Thermo Scientific companies, article No. F- 530S) primer CQM17694-F2 and CQM17694-R2 is used to carry out PCR amplification (PCR system：5X Phusion HF Buffer:10ul,Phusion DNA Polymerase:0.5ul,CQM17694-F2:1.5ul,CQM17694-R2:1.5ul, 10mM dNTPs:1ul,Template DNA:1ul,ddH₂O:34.5ul；PCR programs：98℃ 30s；98 DEG C of 10 s, 60 DEG C 20s, 72 DEG C of 30s 35 are recycled；72 DEG C of 10min), it detaches PSTG_17694 genetic fragments and carries out sequence verification.

The gene fragment order isolated from CRY32 is as shown in Seq ID No.5, with reference gene PSTG_17694 Sequence similarity 99%, have the site 1 SNP (Single Nucleotide Polymorphism).

(2) structure of wheat stripe rust gene PS TG_17694 silence expression vectors

To build gene silencing expression vector, CQM17694-F2/R2 primers both ends difference using GATEWAY clone technologies Increase by 3 protection bases (CAA, TCA) and NotI restriction enzyme sites (GCGGCCGC) or AscI restriction enzyme sites (GGCGCGCC), with It is connected into improved entry vector PC414C (clone of Xiu-Li Han's barley salicylic acid synthetic genes ICS and PAL and analysis [D] Shandong Agricultural University, 2013.).Pcr amplification product and carrier PC414C carry out NotI, AscI double digestion first, and digestion products are pure It is attached using T4-DNA ligases after change.Connection product is converted to competent escherichia coli cell (the full formula gold biology in Beijing Technology Co., Ltd., article No. CD201).Bacterium colony PCR, which is carried out, using primer CQM17694-F2/R2 carries target gene piece to screen The positive colony of section, and plasmid DNA purification (TIANGEN Biotech (Beijing) Co., Ltd., article No. DP103).

Plasmid DNA using positive colony and RNAi silence expression vector skeletons PC336 (Xiu-Li Han's barley salicylic acids conjunctions At clone and analysis [D] the Shandong Agricultural University of gene ICS and PAL, 2013.) LR reactions are carried out, while by PSTG_17694 The forward direction of genetic fragment, reverse sequence are recombinated to carrier PC336 to build RNAi silence expression vectors.

LR reaction systems：1 μ l of purpose carrier (150ng/ μ l)；Entry vector PC414C (50-150ng) 1 μ l, ddH₂O7μ l；LR Clonase^TM2 μ l of II Enzyme Mix (Invitrogen companies, article No. 11791)；25 DEG C of reaction 1h；1 μ l are added Proteinase K, 37 DEG C of inactivation 10min.Reaction solution converts Trans5 α competent cells, and (the full formula gold biotechnology in Beijing has Limit company, article No. CD201), picking colony plasmid DNA purification carries out bacterium colony PCR screenings using CQM17694-F2/R2 primers (PCR systems：2xBuffer 10ul, CQM17694-F20.5ul, CQM17694-R20.5ul, ddH₂O 9ul, single bacterium colony； PCR programs：94℃ 5min；94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s 35 are recycled；72℃ 10min).

From T-DNA LB (Left Boarder) to RB on wheat stripe rust gene PS TG_17694 silence expression vectors (RightBoarder) sequence of section such as sequence table Seq ID No.6.Wherein include hygromycin gene (Hyg), weeding The reverse sequence of agent resistant gene (Bar), the positive sequence of PSTG_17694 genetic fragments and PSTG_17694 genetic fragments Expression cassette.

(3) genetic transformation of common wheat and offspring's screening

Efficiency of Wheat Transformation uses Biolistic mediated transformation method, acceptor material to select the strain of high sense stripe rust of wheat CB037.(Wang Shu rue wheats source regenerates the mountains foundation [D] with transformation system to techniqueflow as described in Wang Shu rue Master's thesis Eastern agriculture university, 2012.).15 days or so after wheat pollination, the rataria clip tassel stripping of a diameter of 1.5mm sizes of rataria is selected It takes seed, strips rataria after surface sterilization sterilizing and be positioned on hypertonic culture medium and use biolistic bombardment, then on luring more culture medium 23 DEG C of light culture surroundings, subculture is primary every two weeks.From luring more culture medium to go to differential medium after four weeks, from differential medium Callus starts to be placed on illumination cultivation (23 DEG C, 16hlight, 8hdark).Differentiation culture is returned again to from differential medium after two weeks On base, until regenerating seedling.The seedling taking-up differentiated is placed on root media, waits within about 3-4 weeks that root long is strong It is transplanted in soil.

Herbicide preliminary screening positive transgenic offspring is smeared by blade.Herbicide is AgrEvo Products Finale, article No. F30617006 use a concentration of 0.3%.The sensitivity that position is smeared according to blade after 3-5 days screens sun Property transgenic progeny.

As shown in Figure 1, wherein CB037 is transgene receptor, strain 5678-2A, 5726-1 is negative transgenosis single plant； 2079-5A, 5758-1A, 5769-3A are positive transgenic single plant.The blade of negative transgenic line smears the apparent yellow in position Or withered necrosis, and the blade of positive transgenic material smears position slightly yellow, the apparent withered necrosis of nothing.

Meanwhile the blade for transgenic wheat of drawing materials prepares DNA and cDNA, and PCR is carried out using primer CQM17694-F2/R2 Amplification carries out screening confirmation to transgenic line.As shown in Figure 2 A, A, B are using gDNA as the PCR results of template；A figures PCR draws Object is CQM17694-F2/R2； H₂O is negative control, and P is Plasmid DNA positive control；B figure PCR primers are LB306F/ CQM17694-R2, wherein primer LB306F are the primer sequence of GUS linker segments in matching vector frame, and position is located at Among CQM17694 forward directions, reversed segment, the primer sequence of LB306F is as shown in Seq ID No.11, and specially 5 '- GACCTCGCAAGGCATATTG-3'；H₂O is negative control, and P is Plasmid DNA positive control.Most samples can generate and the positive (carrier DNA) identical amplified band is compareed, shows to come from positive transgenic material.

2 Rust resistance bacterium wheat stripe rust resistance of embodiment is identified

The breeding of wheat stripe rust Fresh spores：It is red that susceptible variety Huixian is planted in greenhouse, a leaf one heart stage syringe Injection spore aqueous solution connects bacterium, and then being sprayed water with watering can and putting up plastic film carries out moisturizing.To ensure that fully morbidity is repeatable Connect bacterium 2-3 times, every minor tick 1 week.The preparation of spore aqueous solution：Spore that is dry, freezing (- 80 DEG C) is taken, with appropriate tap water It is suspended into pale orange, is placed in shaking table concussion mixing 30min (180rpm) at room temperature, then injection connects bacterium.Connecing about 20 days can after bacterium See fragmentary morbidity, visible a large amount of blade morbidities, collect the inoculation that a large amount of Fresh spores are used for transgenic line after about 30 days Identification.

Utilize T_1:2(seed of first generation transgenic line in harvest embodiment 1) seed is planted in greenhouse from generation to generation And it is inoculated with wheat stripe rust, identify its stripe rust resistance.Plantation the last week greenhouse irrigates, and dries one week or so.Select 15 The full uniform program request of transgenic seed of grain health, line-spacing 25cm, the long 1m of row.2 row transgene receptor kinds are planted every ten rows CB037.To be grown to take Fresh spores to carry out connecing bacterium to one heart stage an of leaf or so, watering after inoculation and putting up plastic cloth keeps high Wet environment.In order to ensure to be inoculated with successfully, repeated inoculation is carried out after primary vaccination week about, in triplicate, every plant is at least inoculated with Three tillers.After the onset of wild type CB037 is abundant, disease-resistant sex investigation is carried out to transgenic line and is recorded, is repeated week about Identification is primary, in triplicate.

The disease-resistant material identified using greenhouse harvests T_2:3After generation seed, repetitive identified is carried out in growth cabinet. Take clean 9cm culture dishes, built-in 3 layers of circular filter paper that 4mL deionized waters are added, filter paper is made to soak.Place the full kind of health Sub 15~20/ware covers culture ware lid and is sealed with sealed membrane；It is protected from light in low-temperature treatment 2 in 4 DEG C of refrigerators with aluminium-foil paper package ~3 days, break seed dormancy.It is transplanted into soil after cultivating 3~5d in 23 DEG C of illumination boxs, chooses the side of length of side 15cm Shape small flower plants 4 per basin.Plant to be planted was grown to one heart stage an of leaf, and the strip rust bacteria mixing spore bred using greenhouse is connect Kind.Spore and talcum powder are pressed about 1:After 10 ratios mix well, dips spore with small brushes and carry out blade and connect bacterium.After inoculation First dark processing for 24 hours, 11 DEG C of temperature, humidity 100%；Then normal illumination culture, 22 DEG C of illumination 16h/ temperature, dark are carried out 15 DEG C of 8h/ temperature periodically humidifies, and keeps blade tip to have water droplet, until control material blade fully falls ill (10-12 days).

Transgenic wheat greenhouse is inoculated with strip rust bacteria rear blade incidence as shown in figure 3, wherein A is wild type CB037, B For PC897 transgenic lines.Chamber planting T_1:2Material repeats after connecing bacterium three times, and wild type CB037 morbidities are abundant, blade table There are a large amount of sorus in face；And transgenic line blade surface has no that sorus generates, stripe rust resistance is with obvious effects.

It is as shown in Figure 4 that transgenic wheat growth cabinet is inoculated with strip rust bacteria rear blade incidence：A is wild type CB037； B is PC897 transgenic lines.T_2:3After bacterium is inscribed 12 days in incubator, wild type CB037's material fully falls ill, blade surface cloth Full spore；And PC897 transgenic lines only have slight chlorisis, blade to there are no spore generation, show PSTG_17694 genes Silence expression is remarkably improved resistance of the wheat to strip rust bacteria.

The expression identification of PSTG_17694 genes in 3 Rust resistance bacterium wheat of embodiment

After Wheat Seedling blade connects bacterium 12 days, wild type CB037 blade surfaces are covered with a large amount of sorus.It is different after connecing bacterium Time point takes CB037 and transgenic line blade about 100mg, and RNA is extracted using Trizol methods, prepares cDNA for glimmering in real time Fluorescent Quantitative PCR.Real-time fluorescence quantitative PCR primer sequence is CQM_17694-F3 and CQM_17694-R3, and specific primer sequence is such as Shown in sequence table Seq ID No.7, Seq ID No.8.Using wheat stripe rust α microtubule protein genes as internal reference (Huang Xue's tinkling of pieces of jade etc., agriculture Industry biotechnology journal, 2012,20 (2)：181-187), primer TUBA-F/R sequences such as sequence table Seq ID No.9, Seq Shown in ID No.10.

PCR system is：5 μ l 2 × SYBR GreenMaster, positive and reverse primer (10 μM) each 1 μ l, 1 μ l CDNA, 2 μ l ddH2O, total system are 10 μ l.PCR reactions use two-step method：95 DEG C of 10min, 95 DEG C of 15s of 40 cycles, 60 ℃1min。

Utilize 2^{- △ △ CT}The expression of each gene of Algorithm Analysis is mapped using SigmaPlot12.5 softwares.

The fluorescent quantitation expression analysis result of gene of the present invention is as shown in Fig. 5 after transgenic wheat inoculation strip rust bacteria： Wherein PC897 is selected positive transgenic wheat, and CB037 is wheat transgenic receptor.Wheat leaf blade inoculation strip rust bacteria difference takes Material detects gene expression dose.Quantification PCR primer is CQM17694-F3/R3.

Transgenic wheat is inoculated with the observation of strip rust bacteria rear blade tissue staining, and the results are shown in Figure 6, and A is PC897 transgenosis materials Material, B are wild type CB037.The 12nd day after connecing bacterium, with WGA-FITC fluorescent dyeings.Mycelia in wild type CB037 blades It is intensive, the visible sorus in surface (B)；And transgenic line only observes secondary colony in blade interior and hyphae length is few and short (A)；Show compared with wild type CB037, in rotaring gene plant blade the expression of wheat stripe rust PSTG_17694 genes by It is apparent to inhibit, illustrate to express RNAi silent carrier PC897 in wheat, can effectively reduce the target in the strip rust bacteria for infecting wheat The expression of gene.

By above-described embodiment it is found that PSTG_17694 genes provided by the invention can effectively regulate and control wheat stripe rust The PSTG_17694 genes are applied in stripe rust of wheat prevention, pass through PSTG_17694 bases described in silence by growth and breeding Because having the function that inhibit wheat stripe rust growth and breeding.The side provided by the invention that Rust resistance bacterium wheat is cultivated using the gene The wheat that method is obtained has significant wheat stripe rust resistance.

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Sequence table

<110>Shandong Agricultural University

<120>The cultivation side of application and Rust resistance bacterium wheat of the wheat stripe rust PSTG_17694 genes in stripe rust prevention Method

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 972

<212> DNA

<213> Puccinia striiformis PST-78

<400> 1

atggactcat tcgaccaaag aaaggctctc aggaatggaa aatgctctac ccaccttcca 60

aaccagctgg acctctacct ctaccaaact ggatcaaaag atacaattat cttcgatcaa 120

tctcttctgc tattaagatt gactctcccc ctctctacca cttggactac tcctcaagct 180

aaactatcca tgcaatccgg actggctgaa tatccggagg ggacgatctt cacctctgat 240

cagaatggga tctgtagtta tcgtcgtgct agttgtttga gaggtgaaaa agaggatcgc 300

gggttgggtc tacgagatat ttggttgggt aaaaataata cttggtcgat taactttgat 360

gatggtccgc tcccgccgag tagctcactg tacaaatttc tggacgaaca agacaagaca 420

gcgactcact tctgggtagg agccaatgtg cgtgattatc cagaactagc gcttcaagca 480

tggaaacgag gtgatcatct tgctgttcat acctggacac atgctcacct taccacgtta 540

agtgatctcg aaatcctcgg tgaattggga tggacaattc aaatcattca cgatctgact 600

gggatggtcc cattatacta ccggccacca tacggagatg ttgataaccg agtgagagca 660

cttgcaaagc atgtattcgg cttaacaacg acattgtgga attgcgattc atcagattgg 720

tcgttaaatc agacttacgc cttgggggat ttcttagacc cccccatgaa cggctttgga 780

gttcaagaat cggtgggaat gattaatgga catatcaatc aacccgataa gtcggtcggt 840

aaaatcatcc tcgaacatga attgagcttc gaatccgtcg aggctttcaa actcactttc 900

ccaaatctga tccggaactc ttggtcaact tgtaatctgg ctgattgctt ggatctcaaa 960

tggtatcaat ga 972

<210> 2

<211> 323

<212> PRT

<213> Puccinia striiformis PST-78

<400> 2

Met Asp Ser Phe Asp Gln Arg Lys Ala Leu Arg Asn Gly Lys Cys Ser

1 5 10 15

Thr His Leu Pro Asn Gln Leu Asp Leu Tyr Leu Tyr Gln Thr Gly Ser

20 25 30

Lys Asp Thr Ile Ile Phe Asp Gln Ser Leu Leu Leu Leu Arg Leu Thr

35 40 45

Leu Pro Leu Ser Thr Thr Trp Thr Thr Pro Gln Ala Lys Leu Ser Met

50 55 60

Gln Ser Gly Leu Ala Glu Tyr Pro Glu Gly Thr Ile Phe Thr Ser Asp

65 70 75 80

Gln Asn Gly Ile Cys Ser Tyr Arg Arg Ala Ser Cys Leu Arg Gly Glu

85 90 95

Lys Glu Asp Arg Gly Leu Gly Leu Arg Asp Ile Trp Leu Gly Lys Asn

100 105 110

Asn Thr Trp Ser Ile Asn Phe Asp Asp Gly Pro Leu Pro Pro Ser Ser

115 120 125

Ser Leu Tyr Lys Phe Leu Asp Glu Gln Asp Lys Thr Ala Thr His Phe

130 135 140

Trp Val Gly Ala Asn Val Arg Asp Tyr Pro Glu Leu Ala Leu Gln Ala

145 150 155 160

Trp Lys Arg Gly Asp His Leu Ala Val His Thr Trp Thr His Ala His

165 170 175

Leu Thr Thr Leu Ser Asp Leu Glu Ile Leu Gly Glu Leu Gly Trp Thr

180 185 190

Ile Gln Ile Ile His Asp Leu Thr Gly Met Val Pro Leu Tyr Tyr Arg

195 200 205

Pro Pro Tyr Gly Asp Val Asp Asn Arg Val Arg Ala Leu Ala Lys His

210 215 220

Val Phe Gly Leu Thr Thr Thr Leu Trp Asn Cys Asp Ser Ser Asp Trp

225 230 235 240

Ser Leu Asn Gln Thr Tyr Ala Leu Gly Asp Phe Leu Asp Pro Pro Met

245 250 255

Asn Gly Phe Gly Val Gln Glu Ser Val Gly Met Ile Asn Gly His Ile

260 265 270

Asn Gln Pro Asp Lys Ser Val Gly Lys Ile Ile Leu Glu His Glu Leu

275 280 285

Ser Phe Glu Ser Val Glu Ala Phe Lys Leu Thr Phe Pro Asn Leu Ile

290 295 300

Arg Asn Ser Trp Ser Thr Cys Asn Leu Ala Asp Cys Leu Asp Leu Lys

305 310 315 320

Trp Tyr Gln

<210> 3

<211> 31

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 3

caagcggccg ctcattcgac caaagaaagg c 31

<210> 4

<211> 31

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 4

tcaggcgcgc cgatcacctc gtttccatgc t 31

<210> 5

<211> 490

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 5

ctcattcgac caaagaaagg ctctcaggaa tggaaaatgc tctacccacc ttccaaacca 60

gctggacctc tacctctacc aaactggatc aaaagataca attatcttcg atcaatctct 120

tctgctatta agattgactc tcccctctct accacttgga ctactcctca agctaaacta 180

tccatgcaat ccggactggc tgaatatccg gaggggacga tcttcacctc tgatcagaat 240

gggatctgta gttatcgtcg tgctagttgt ttgagaggtg aaaaagagga tcgcgggttg 300

ggtctacgag atatttggtt gggtaaaaat aatacttggt cgattaactt tgatgatggt 360

ccgctcccgc cgagtagctc actgtacaaa tttctggacg aacaagacaa gacagcgact 420

cacttctggg taggagccaa tgtgcgtgat tatccagaac tagcgcttca agcatggaaa 480

cgaggtgatc 490

<210> 6

<211> 8699

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 6

tggcaggata tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg 60

gacgttttta atgtactgaa ttaacgccga attaattcgg gggatctgga ttttagtact 120

ggattttggt tttaggaatt agaaatttta ttgatagaag tattttacaa atacaaatac 180

atactaaggg tttcttatat gctcaacaca tgagcgaaac cctataggaa ccctaattcc 240

cttatctggg aactactcac acattattat ggagaaactc gagcttgtcg atcgacagat 300

cccggtcggc atctactcta tttctttgcc ctcggacgag tgctggggcg tcggtttcca 360

ctatcggcga gtacttctac acagccatcg gtccagacgg ccgcgcttct gcgggcgatt 420

tgtgtacgcc cgacagtccc ggctccggat cggacgattg cgtcgcatcg accctgcgcc 480

caagctgcat catcgaaatt gccgtcaacc aagctctgat agagttggtc aagaccaatg 540

cggagcatat acgcccggag tcgtggcgat cctgcaagct ccggatgcct ccgctcgaag 600

tagcgcgtct gctgctccat acaagccaac cacggcctcc agaagaagat gttggcgacc 660

tcgtattggg aatccccgaa catcgcctcg ctccagtcaa tgaccgctgt tatgcggcca 720

ttgtccgtca ggacattgtt ggagccgaaa tccgcgtgca cgaggtgccg gacttcgggg 780

cagtcctcgg cccaaagcat cagctcatcg agagcctgcg cgacggacgc actgacggtg 840

tcgtccatca cagtttgcca gtgatacaca tggggatcag caatcgcgca tatgaaatca 900

cgccatgtag tgtattgacc gattccttgc ggtccgaatg ggccgaaccc gctcgtctgg 960

ctaagatcgg ccgcagcgat cgcatccata gcctccgcga ccggttgtag aacagcgggc 1020

agttcggttt caggcaggtc ttgcaacgtg acaccctgtg cacggcggga gatgcaatag 1080

gtcaggctct cgctaaactc cccaatgtca agcacttccg gaatcgggag cgcggccgat 1140

gcaaagtgcc gataaacata acgatctttg tagaaaccat cggcgcagct atttacccgc 1200

aggacatatc cacgccctcc tacatcgaag ctgaaagcac gagattcttc gccctccgag 1260

agctgcatca ggtcggagac gctgtcgaac ttttcgatca gaaacttctc gacagacgtc 1320

gcggtgagtt caggcttttt catatctcat tgccccccgg gatctgcgaa agctcgagag 1380

agatagattt gtagagagag actggtgatt tcagcgtgtc ctctccaaat gaaatgaact 1440

tccttatata gaggaaggtc ttgcgaagga tagtgggatt gtgcgtcatc ccttacgtca 1500

gtggagatat cacatcaatc cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc 1560

acgatgctcc tcgtgggtgg gggtccatct ttgggaccac tgtcggcaga ggcatcttga 1620

acgatagcct ttcctttatc gcaatgatgg catttgtagg tgccaccttc cttttctact 1680

gtccttttga tgaagtgaca gatagctggg caatggaatc cgaggaggtt tcccgatatt 1740

accctttgtt gaaaagtctc aatagccctt tggtcttctg agactgtatc tttgatattc 1800

ttggagtaga cgagagtgtc gtgctccacc atgttatcac atcaatccac ttgctttgaa 1860

gacgtggttg gaacgtcttc tttttccacg atgctcctcg tgggtggggg tccatctttg 1920

ggaccactgt cggcagaggc atcttgaacg atagcctttc ctttatcgca atgatggcat 1980

ttgtaggtgc caccttcctt ttctactgtc cttttgatga agtgacagat agctgggcaa 2040

tggaatccga ggaggtttcc cgatattacc ctttgttgaa aagtctcaat agccctttgg 2100

tcttctgaga ctgtatcttt gatattcttg gagtagacga gagtgtcgtg ctccaccatg 2160

ttggcaagct gctctagcca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 2220

aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta 2280

atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta 2340

tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt 2400

acgaattccc gatctagtaa catagatgac accgcgcgcg ataatttatc ctagtttgcg 2460

cgctatattt tgttttctat cgcgtattaa atgtataatt gcgggactct aatcataaaa 2520

acccatctca taaataacgt catgcattac atgttaatta ttacatgctt aacgtaattc 2580

aacagaaatt atatgataat catcgcaaga ccggcaacag gattcaatct taagaaactt 2640

tattgccaaa tgtttgaacg atcggggaaa ttcgggtcat cagatctcgg tgacgggcag 2700

gaccggacgg ggcggtaccg gcaggctgaa gtccagctgc cagaaaccca cgtcatgcca 2760

gttcccgtgc ttgaagccgg ccgcccgcag catgccgcgg ggggcatatc cgagcgcctc 2820

gtgcatgcgc acgctcgggt cgttgggcag cccgatgaca gcgaccacgc tcttgaagcc 2880

ctgtgcctcc agggacttca gcaggtgggt gtagagcgtg gagcccagtc ccgtccgctg 2940

gtggcggggg gagacgtaca cggtcgactc ggccgtccag tcgtaggcgt tgcgtgcctt 3000

ccaggggccc gcgtaggcga tgccggcgac ctcgccgtcc acctcggcga cgagccaggg 3060

atagcgctcc cgcagacgga cgaggtcgtc cgtccactcc tgcggttcct gcggctcggt 3120

acggaagttg accgtgcttg tctcgatgta gtggttgacg atggtgcaga ccgccggcat 3180

gtccgcctcg gtggcacggc ggatgtcggc cgggcgtcgt tctgggctca tggtagatcc 3240

ccggggatcc tctagagtcc cccgtgttct ctccaaatga aatgaacttc cttttccact 3300

atcttcacaa taaagtgaca gatagctggg caatggaatc cgaggaggtt tccggatatt 3360

accctttgtt gaaaagtctc aattgccctt tggtcttctg agactgtatc tttgatattt 3420

ttggagtaga caagtgtgtc gtgctccacc atgttgacga agattttctt cttgtcattg 3480

agtcgtaaga gactctgtat gaactgttcg ccagtcttta cggcgagttc tgttaggtcc 3540

tctatttgaa tctttgactc catggccttt gattcagtgg gaactacctt tttagagact 3600

ccaatctcta ttacttgcct tggtttgtga agcaagcctt gaatcgtcca tactggaata 3660

gtacttctga tcttgagaaa tatatctttc tctgtgttct tgatgcagtt agtcctgaat 3720

cttttgactg catctttaac cttcttggga aggtatttga tttcctggag attattgctc 3780

gggtagatcg tcttgatgag acctgctgcg taagcctctc taaccatctg tgggttagca 3840

ttctttctga aattgaaaag gctaatctgg ggacctgcag gcatgcaagc ttgcatgcct 3900

gcagtgcagc gtgacccggt cgtgcccctc tctagagata atgagcattg catgtctaag 3960

ttataaaaaa ttaccacata ttttttttgt cacacttgtt tgaagtgcag tttatctatc 4020

tttatacata tatttaaact ttactctacg aataatataa tctatagtac tacaataata 4080

tcagtgtttt agagaatcat ataaatgaac agttagacat ggtctaaagg acaattgagt 4140

attttgacaa caggactcta cagttttatc tttttagtgt gcatgtgttc tccttttttt 4200

ttgcaaatag cttcacctat ataatacttc atccatttta ttagtacatc catttagggt 4260

ttagggttaa tggtttttat agactaattt ttttagtaca tctattttat tctattttag 4320

cctctaaatt aagaaaacta aaactctatt ttagtttttt tatttaataa tttagatata 4380

aaatagaata aaataaagtg actaaaaatt aaacaaatac cctttaagaa attaaaaaaa 4440

ctaaggaaac atttttcttg tttcgagtag ataatgccag cctgttaaac gccgtcgacg 4500

agtctaacgg acaccaacca gcgaaccagc agcgtcgcgt cgggccaagc gaagcagacg 4560

gcacggcatc tctgtcgctg cctctggacc cctctcgaga gttccgctcc accgttggac 4620

ttgctccgct gtcggcatcc agaaattgcg tggcggagcg gcagacgtga gccggcacgg 4680

caggcggcct cctcctcctc tcacggcacg gcagctacgg gggattcctt tcccaccgct 4740

ccttcgcttt cccttcctcg cccgccgtaa taaatagaca ccccctccac accctctttc 4800

cccaacctcg tgttgttcgg agcgcacaca cacacaacca gatctccccc aaatccaccc 4860

gtcggcacct ccgcttcaag gtacgccgct cgtcctcccc ccccccccct ctctaccttc 4920

tctagatcgg cgttccggtc catggttagg gcccggtagt tctacttctg ttcatgtttg 4980

tgttagatcc gtgtttgtgt tagatccgtg ctgctagcgt tcgtacacgg atgcgacctg 5040

tacgtcagac acgttctgat tgctaacttg ccagtgtttc tctttgggga atcctgggat 5100

ggctctagcc gttccgcaga cgggatcgat ttcatgattt tttttgtttc gttgcatagg 5160

gtttggtttg cccttttcct ttatttcaat atatgccgtg cacttgtttg tcgggtcatc 5220

ttttcatgct tttttttgtc ttggttgtga tgatgtggtc tggttgggcg gtcgttctag 5280

atcggagtag aattctgttt caaactacct ggtggattta ttaattttgg atctgtatgt 5340

gtgtgccata catattcata gttacgaatt gaagatgatg gatggaaata tcgatctagg 5400

ataggtatac atgttgatgc gggttttact gatgcatata cagagatgct ttttgttcgc 5460

ttggttgtga tgatgtggtg tggttgggcg gtcgttcatt cgttctagat cggagtagaa 5520

tactgtttca aactacctgg tgtatttatt aattttggaa ctgtatgtgt gtgtcataca 5580

tcttcatagt tacgagttta agatggatgg aaatatcgat ctaggatagg tatacatgtt 5640

gatgtgggtt ttactgatgc atatacatga tggcatatgc agcatctatt catatgctct 5700

aaccttgagt acctatctat tataataaac aagtatgttt tataattatt ttgatcttga 5760

tatacttgga tgatggcata tgcagcagct atatgtggat ttttttagcc ctgccttcat 5820

acgctattta tttgcttggt actgtttctt ttgtcgatgc tcaccctgtt gtttggtgtt 5880

acttctgcag gtcgactcta gaggatcccc cgggggtacc gggccccccc tcgaggtcat 5940

caccactttg tacaagaaag ctgggtcggc gcgccgatca cctcgtttcc atgcttgaag 6000

cgctagttct ggataatcac gcacattggc tcctacccag aagtgagtcg ctgtcttgtc 6060

ttgttcgtcc agaaatttgt acagtgagct actcggcggg agcggaccat catcaaagtt 6120

aatcgaccaa gtattatttt tacccaacca aatatctcgt agacccaacc cgcgatcctc 6180

tttttcacct ctcaaacaac tagcacgacg ataactacag atcccattct gatcagaggt 6240

gaagatcgtc ccctccggat attcagccag tccggattgc atggatagtt tagcttgagg 6300

agtagtccaa gtggtagaga ggggagagtc aatcttaata gcagaagaga ttgatcgaag 6360

ataattgtat cttttgatcc agtttggtag aggtagaggt ccagctggtt tggaaggtgg 6420

gtagagcatt ttccattcct gagagccttt ctttggtcga atgagcggcc gcggagcctg 6480

cttttttgta caaacttgtg atgacggtat cgataagctt gatatctacc cgcttcgcgt 6540

cggcatccgg tcagtggcag tgaagggcga acagttcctg attaaccaca aaccgttcta 6600

ctttactggc tttggtcgtc atgaagatgc ggacttgcgt ggcaaaggat tcgataacgt 6660

gctgatggtg cacgaccacg cattaatgga ctggattggg gccaactcct accgtacctc 6720

gcattaccct tacgctgaag agatgctcga ctgggcagat gaacatggca tcgtggtgat 6780

tgatgaaact gctgctgtcg gctttaacct ctctttaggc attggtttcg aagcgggcaa 6840

caagccgaaa gaactgtaca gcgaagaggc agtcaacggg gaaactcagc aagcgcactt 6900

acaggcgatt aaagagctga tagcgcgtga caaaaaccac ccaagcgtgg tgatgtggag 6960

tattgccaac gaaccggata cccgtccgca aggtgcacgg gaatatttcg cgccactggc 7020

ggaagcaacg cgtaaactcg acccgacgcg tccgatcacc tgcgtcaatg taatgttctg 7080

cgacgctcac accgatacca tcagcgatct ctttgatgtg ctgtgcctga accgttatta 7140

cggatggtat gtccaaagcg gcgatttgga aacggcagag aaggtactgg aaaaagaact 7200

tctggcctgg caggagaaac tgcatcagcc gattatcatc accgaatacg gcgtggatac 7260

gttagccggg ctgcactcaa tgtacaccga catgtggagt gaagagtatc agtgtgcatg 7320

gctggatatg tatcaccgcg tctttgatcg cgtcagcgcc gtcgtcggtg aacaggtatg 7380

gaatttcgcc gattttgcga cctcgcaagg catattgcgc gttggcggta acaagaaagg 7440

gatcttcact cgatcgaatt cctgcagccc gggggatcca ctagatgcat gctcgagcgg 7500

ccgccagtgt gatggatatc tgcagaattc gcccttatca caagtttgta caaaaaagca 7560

ggctccgcgg ccgctcattc gaccaaagaa aggctctcag gaatggaaaa tgctctaccc 7620

accttccaaa ccagctggac ctctacctct accaaactgg atcaaaagat acaattatct 7680

tcgatcaatc tcttctgcta ttaagattga ctctcccctc tctaccactt ggactactcc 7740

tcaagctaaa ctatccatgc aatccggact ggctgaatat ccggagggga cgatcttcac 7800

ctctgatcag aatgggatct gtagttatcg tcgtgctagt tgtttgagag gtgaaaaaga 7860

ggatcgcggg ttgggtctac gagatatttg gttgggtaaa aataatactt ggtcgattaa 7920

ctttgatgat ggtccgctcc cgccgagtag ctcactgtac aaatttctgg acgaacaaga 7980

caagacagcg actcacttct gggtaggagc caatgtgcgt gattatccag aactagcgct 8040

tcaagcatgg aaacgaggtg atcggcgcgc cgacccagct ttcttgtaca aagtggtgat 8100

aagggcgaat tccagcacac tggcggccgt tactagtgga tccgagctcg aatttccccg 8160

atcgttcaaa catttggcaa taaagtttct taagattgaa tcctgttgcc ggtcttgcga 8220

tgattatcat ataatttctg ttgaattacg ttaagcatgt aataattaac atgtaatgca 8280

tgacgttatt tatgagatgg gtttttatga ttagagtccc gcaattatac atttaatacg 8340

cgatagaaaa caaaatatag cgcgcaaact aggataaatt atcgcgcgcg gtgtcatcta 8400

tgttactaga tcgggaattc gatatcaagc ttggcactgg ccgtcgtttt acaacgtcgt 8460

gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc 8520

agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 8580

aatggcgaat gctagagcag cttgagcttg gatcagattg tcgtttcccg ccttcagttt 8640

aaactatcag tgtttgacag gatatattgg cgggtaaacc taagagaaaa gagcgttta 8699

<210> 7

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 7

tcatcttgct gttcatacct gg 22

<210> 8

<211> 21

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 8

gatgatttta ccgaccgact t 21

<210> 9

<211> 24

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 9

aaggacccac gctgccaata acta 24

<210> 10

<211> 24

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 10

tggagtcccg aacaattatc cgct 24

<210> 11

<211> 19

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 11

gacctcgcaa ggcatattg 19

Claims

1. application of the wheat stripe rust PSTG_17694 genes in stripe rust of wheat prevention, which is characterized in that the PSTG_ The sequence of 17694 genes is as shown in Seq ID No.1.

2. application according to claim 1, which is characterized in that molecule of the PSTG_17694 genes as Transcription inhibition Target or the gene target inhibited as protein function inhibit wheat stripe rust by PSTG_17694 genes described in silence Growth and breeding.

3. application according to claim 1 or 2, which is characterized in that the application is that will carry the PSTG_17694 bases The silence expression vector of cause imports in wheat crops the wheat crops for obtaining Rust resistance bacterium.

4. application according to claim 2, which is characterized in that the application is by spraying the PSTG_17694 genes Transcription inhibitor to wheat leaf blade inhibit strip rust bacteria growth and breeding.

5. application according to claim 4, which is characterized in that the transcription inhibitor is that can inhibit the PSTG_ The dsRNA solution of 17694 genetic transcriptions.

6. application according to claim 2, which is characterized in that the application is by spraying the PSTG_17694 genes Coding protein active inhibitor to wheat leaf blade inhibit strip rust bacteria growth and breeding.

7. a kind of breeding method of Rust resistance bacterium wheat, includes the following steps：

1) PCR amplification acquisition PSTG_17694 gene pieces are carried out as template to infect the wheat leaf blade cDNA to fall ill after strip rust bacteria Section；

2) the PSTG_17694 genetic fragments structure wheat stripe rust PSTG_17694 gene silencings described in step 1) is utilized to express Carrier；The structure of the PSTG_17694 gene silencings expression vector uses GATEWAY clone technologies；

3) the wheat stripe rust PSTG_17694 gene silencing expression vectors are transferred to by wheat using Biolistic mediated transformation method Middle acquisition Rust resistance bacterium wheat.

8. breeding method according to claim 7, which is characterized in that PCR amplification described in step 1) is with primer CQM17694-F2 and CQM17694-R2 primers；The sequence of the CQM17694-F2 primers is as shown in Seq ID No.3；It is described The sequence of CQM17694-R2 primers is as shown in Seq ID No.4.

9. breeding method according to claim 7, which is characterized in that wheat stripe rust PSTG_17694 described in step 2) Gene silencing expression vector includes hygromycin gene Hyg, herbicide resistance gene Bar, PSTG_17694 genetic fragment Positive sequence and PSTG_17694 genetic fragments reverse sequence expression cassette.

10. breeding method according to claim 9, which is characterized in that the wheat stripe rust PSTG_17694 genes are heavy Silent sequence such as sequence table Seq ID No.6 institute of the expression vector from T-DNALeft Boarder to Right Boarder sections Show.