CN108559753B

CN108559753B - Application of wheat stripe rust PSTG _17694 gene in stripe rust prevention and treatment and stripe rust resistant wheat cultivation method

Info

Publication number: CN108559753B
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: 2020-08-11
Anticipated expiration: 2038-02-02
Also published as: CN108559753A

Abstract

The invention provides application of a wheat stripe rust PSTG _17694 gene in wheat stripe rust prevention and treatment, wherein the sequence of the PSTG _17694 gene is shown as Seq ID No. 1; the PSTG _17694 gene can effectively regulate the growth and the propagation of wheat stripe rust, the PSTG _17694 gene is used as a molecular target for transcriptional regulation or a gene target for protein function inhibition, and the PSTG _17694 gene is silenced to achieve the effect of inhibiting the growth and the propagation of wheat stripe rust. The wheat obtained by the method for cultivating the wheat with the stripe rust resistance by using the gene has obvious wheat stripe rust resistance.

Description

Application of wheat stripe rust PSTG _17694 gene in stripe rust prevention and treatment and stripe rust resistant wheat cultivation method

Technical Field

The invention belongs to the technical field of genetic engineering and crop molecular breeding, and particularly relates to application of a wheat stripe rust PSTG _17694 gene in stripe rust prevention and treatment and a cultivation method of stripe rust resistant wheat.

Background

Wheat is the second crop of China, and the annual average sowing area is about 3.6 hundred million acres. The stability and the improvement of the wheat yield are related to the national family. However, for years, fungal diseases pose serious threats to wheat production, such as rust disease, powdery mildew disease, gibberellic disease, banded sclerotial blight and other diseases, and seriously affect the yield and quality of wheat. Among them, wheat stripe rust is commonly called "jaundice" and is caused by obligate parasitic stripe rust (Puccinia striiformis west. In China, the wheat stripe rust is rampant, and the annual damaged area reaches 6,000-8,000 ten thousand mu (the document of Ministry of agriculture: Nongnong 2006-9); the serious years of disease cause great yield reduction (20-30 percent) and threaten the grain safety. The rust stripe has sexual reproduction and rapid toxicity variation, and new dominant race continuously appears in recent years, which leads to the increase of difficulty in controlling the rust stripe. The stripe rust occurrence area of the Huang-Huai-Hai wheat area in the main wheat production area tends to increase gradually, and the cumulative occurrence area of 7 provinces and cities such as Henan, Shandong, Anhui and the like in 5 months in 2017 exceeds 4,000 ten thousand mu (popularization of Chinese agriculture science and technology, 2017). Therefore, the effective control of the stripe rust is of great significance to the food safety in China.

The cultivation and planting of disease-resistant varieties are economic and effective important measures for preventing and treating the wheat stripe rust. Currently, the wheat varieties of various provinces in China are examined and judged to have stripe rust resistance as key indexes. Improving resistance level and cultivating durable resistance also become important targets for cultivating new wheat varieties. The breeding of the disease-resistant variety is mainly realized by excavating and utilizing the rust-resistant gene, but the disease resistance of the rust-resistant gene can be lost in a short period after the large-area utilization of the rust-resistant gene due to the rapid variation of the toxicity of the physiological race of the rust. For example, resistance sources such as Bimantha I, "Luo" and "mu 6" derived lines, which have been widely used in wheat breeding, all have lost resistance to the current epidemic race. The detection of 501 main cultivars and backup cultivars in China currently shows that less than 30% of cultivars have disease resistance to currently popular races, and the resistance sources of the cultivars are mainly concentrated on a few genes such as Yr26/Yr24 (Korean Jun et al, evaluation on stripe rust resistance of current wheat cultivars (lines) in northwest-North China-Yangtze river middle and downstream stripe rust epidemic areas, Chinese agricultural science, 2010 (43)). Therefore, the method widens the resistance source of wheat stripe rust resistance, develops a new stripe rust prevention strategy, and has important significance for reducing the risk of large-area spread of wheat stripe rust.

The wheat stripe rust fungus is difficult to be genetically transformed due to the living body nutrition type and the obligate parasitic characteristic; moreover, wheat itself is also difficult to carry out genetic transformation, so the genetic research of wheat-stripe rust interaction is very difficult and the progress is slow. At present, the number of infection or pathogenic key genes identified in the wheat stripe rust is small, and no gene is effectively applied to wheat disease-resistant breeding.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of a wheat stripe rust gene as a molecular target in wheat crop breeding or stripe rust prevention and treatment, and a method for cultivating stripe rust resistant wheat by using the gene.

In order to achieve the above object, the present invention provides the following technical solutions: the application of the wheat stripe rust PSTG _17694 gene in the prevention and treatment of wheat stripe rust is disclosed, wherein the sequence of the PSTG _17694 gene is shown in Seq ID No. 1.

Preferably, the PSTG _17694 gene is used as a molecular target for transcription inhibition or a gene target for protein function inhibition, and the PSTG _17694 gene is silenced to achieve the effect of inhibiting the growth and reproduction of wheat stripe rust.

Preferably, the application is to introduce the silencing expression vector carrying the PSTG _17694 gene into wheat crops to obtain the wheat crops with rust resistance.

Preferably, the application is to inhibit the growth and the propagation of the puccinia striiformis by spraying the transcription inhibitor of the PSTG _17694 gene to wheat leaf blades.

Preferably, the transcription inhibitor of the PSTG _17694 gene is a dsRNA solution capable of inhibiting the transcription of the PSTG _17694 gene.

Preferably, the application is to inhibit the growth and the propagation of the puccinia striiformis by spraying an activity inhibitor of the protein coded by the PSTG _17694 gene to wheat leaves.

The invention also provides a cultivation method of the wheat resisting the stripe rust fungus, which comprises the following steps: 1) carrying out PCR amplification by taking wheat leaf cDNA infected with rust as a template to obtain a PSTG _17694 gene segment; 2) constructing a wheat stripe rust PSTG _17694 gene silencing expression vector by using the PSTG _17694 gene segment in the step 1); the construction of the PSTG _17694 gene silencing expression vector adopts GATEWAY cloning technology; 3) and transferring the wheat stripe rust PSTG _17694 gene silencing expression vector into wheat by adopting a gene gun mediated transformation method to obtain the stripe rust resistant wheat.

Preferably, the primers for PCR amplification in the step 1) are CQM17694-F2 and CQM17694-R2 primers; the sequence of the CQM17694-F2 is shown as Seq ID No. 3; the sequence of the CQM17694-R2 primer is shown in Seq ID No. 4.

Preferably, the wheat stripe rust PSTG _17694 gene silencing expression vector in the step 2) comprises an expression cassette of a hygromycin resistance gene Hyg, a herbicide resistance gene Bar, a forward sequence of a PSTG _17694 gene fragment and a reverse sequence of a PSTG _17694 gene fragment.

Preferably, the sequence of the gene silencing expression vector of the wheat stripe rust PSTG _17694 from T-DNALeft Border to Right Border is shown in a sequence table SeqID No. 6.

The invention has the beneficial effects that: the PSTG _17694 gene is found to be capable of effectively regulating and controlling the growth and propagation of wheat stripe rust for the first time, and key genes for infection or pathogenicity in wheat stripe rust are enriched; the PSTG _17694 gene is applied to the control of wheat stripe rust, and the effect of inhibiting the growth and the propagation of wheat stripe rust is achieved by silencing the PSTG _17694 gene. The wheat obtained by the method for cultivating the wheat with the stripe rust resistance by using the gene has obvious wheat stripe rust resistance.

Drawings

FIG. 1 is a diagram showing the screening results of a positive transgenic wheat herbicide which silence expression of PSTG _17694 in example 1;

FIG. 2 shows the results of PCR screening test of Puccinia striiformis resistant wheat in example 1;

FIG. 3 is a graph showing the leaf morbidity of rust-resistant wheat in example 2 after greenhouse inoculation with rust in a greenhouse;

FIG. 4 is a leaf pathogenesis chart of puccinia striiformis inoculated in the anti-puccinia striiformis wheat phytotron in example 2;

FIG. 5 is a graph showing the results of fluorescent quantitative expression analysis of PSTG _17694 gene after rust stripe inoculation of rust stripe resistant wheat in example 3;

FIG. 6 is a photograph showing the staining of the leaf tissue after inoculation of P.chrysosporium PSTG _17694 in example 3.

Detailed Description

The invention provides application of a wheat stripe rust PSTG _17694 gene in wheat stripe rust prevention and control. The GenBank accession number of the PSTG _17694 gene is KNE88861.1, the sequence of the PSTG _17694 gene is shown as SeqID No.1, the length of the gene sequence is 972bp, the PSTG _17694 gene coding amino acid sequence has a CE4_ MrCDA _ Like structural domain (cd10952), and the specific sequence is shown as Seq ID No. 2.

In the invention, the PSTG _17694 gene is preferably used as a molecular target for transcriptional regulation or a gene target for protein function inhibition, and the PSTG _17694 gene is silenced to achieve the effect of inhibiting the growth and the propagation of wheat stripe rust. In the specific implementation process of the invention, the PSTG _17694 gene is preferably silenced by the following three methods to realize the application of the PSTG _17694 gene in the control of wheat stripe rust: introducing a silencing expression vector carrying the PSTG _17694 gene into a wheat crop to obtain the rust stripe resistant wheat crop; secondly, the transcription inhibitor of the PSTG _17694 gene is sprayed to the wheat leaf to inhibit the growth and the propagation of the puccinia striiformis; the transcription inhibitor is preferably dsRNA solution capable of inhibiting the transcription of the PSTG _17694 gene; and (III) spraying an activity inhibitor of the protein coded by the PSTG _17694 gene to the wheat leaf to inhibit the growth and the propagation of the puccinia striiformis.

In the invention, PCR amplification primers are preferably designed before amplification of the PSTG _17694 gene fragment. In the present invention, it is preferable to design PCR primers using the whole genome sequence of Puccinia striiformis as a reference and the coding region sequence of the putative functional protein gene PSTG _17694(GenBank: KNE88861.1) as a template; more preferably, the whole genome sequence of the P.cereus strain PST-78 is used as a reference (GenBank accession number AJIL00000000.1 or BROAD download link ftp:// ftp. Broadantintitute. org/pub/annotation/fungi/puccinia/genes/puccinia _ strifiduciflors _ PST-78 /). In the invention, the method for designing the PCR amplification primer can be realized by adopting a conventional method in the field, and can be specifically realized by adopting primer design software. The PCR amplification primers in the present invention are preferably CQM17694-F2 and CQM17694-R2 primers; the sequence of the CQM17694-F2 is shown as Seq ID No. 3; the sequence of the CQM17694-R2 primer is shown in Seq ID No. 4.

After the PCR amplification primers are designed and obtained, the amplification section of the PCR amplification primers is preferably verified. The verification specifically comprises the steps of comparing the sequence of the amplification segment with wheat stripe rust PST-78 whole genome data and wheat stripe rust sequences in an NCBI database, and checking whether the sequence of the amplification segment can specifically target a PSTG _17694 gene. After the sequence of the amplified segment of the PCR amplification primer is compared with the whole genome data of wheat stripe rust PST-78 and the sequence of wheat stripe rust in NCBI database, the homology of the first exon of PSTG _17694 and PSTG _19439 is found to be higher after sequence comparison, and besides, no 100% identical sequence with the length of more than 20bp exists between the sequence of the amplified segment and other genes, which shows that the sequence of the segment amplified by using the CQM17694-F2/R2 primer can specifically target the PSTG _17694 gene.

In the invention, PCR amplification is carried out by taking wheat leaf cDNA infected with stripe rust as a template to obtain a PSTG _17694 gene fragment. In the invention, preferably, the wheat leaves infected with the puccinia striiformis physiological race CRY32 are used for preparing cDNA; the method for preparing cDNA preferably employs a conventional TRIZOL method to prepare purified RNA and reverse transcribe the RNA to obtain cDNA. In the present invention, the preparation of purified RNA and the reverse transcription to obtain cDNA are carried out using an RNA purification kit and a reverse transcription kit, respectively. In the specific implementation process of the invention, the preparation of the purified RNA is preferably realized by a kit of the Purchase number DP405 of Tiangen Biochemical technology (Beijing) Ltd; the reverse transcription to obtain cDNA preferably adopts a reverse transcription kit of Beijing Quanji Biotechnology Limited company, Cat number AT 311-03.

After obtaining wheat leaf cDNA infected with stripe rust fungus to cause disease, the invention uses the cDNA asAnd carrying out PCR amplification on the template to obtain a PSTG _17694 gene fragment. The enzyme used for the PCR amplification in the present invention is preferably a high fidelity enzyme, more preferably Phusion high fidelity enzyme (Thermo Scientific Co., Ltd., cat # F-530S); the PCR amplification system and the PCR amplification program can be realized by adopting the conventional PCR amplification system and program in the field, and specifically in the implementation process of the invention, the PCR system comprises the following steps: 10. mu.l of 5X Phusion HF Buffer, 0.5. mu.l of Phusion DNApolymerase, 1.5. mu.l of CQM17694-F2, 1.5. mu.l of CQM17694-R2, 1. mu.l of 10mM dNTPs, 1. mu.l of Template DNA, and 1. mu.l of ddH₂34.5 mul of O; the PCR program is as follows: 30s at 98 ℃; circulating at 98 deg.C for 10s, 60 deg.C for 20s, and 72 deg.C for 30s 35; 10min at 72 ℃.

In the invention, after obtaining the amplification product, the amplification product is preferably separated and sequenced, and the separation in the invention can be realized by adopting a separation method which is conventional in the field; the sequencing is entrusted to a sequencing company to complete; the sequence obtained by sequencing the amplification product is shown as Seq ID No.5, has 99 percent of sequence similarity with a reference gene PSTG _17694, and has 1 SNP (single Nucleotide polymorphism) site.

After obtaining the PSTG _17694 gene fragment, the invention constructs a wheat stripe rust PSTG _17694 gene silencing expression vector by using a GATEWAY cloning technology. In the invention, a method for constructing the wheat stripe rust PSTG _17694 gene silencing expression vector is disclosed in a reference (Korean, cloning and analysis of barley salicylic acid synthesis genes ICS and PAL [ D ]. Shandong agricultural university, 2013.), and specifically comprises the following steps: (A) connecting the PSTG _17694 gene segment with an entry vector, and transferring the gene segment into escherichia coli competent cells to obtain positive cloning plasmids; (B) and carrying out LR reaction on the positively cloned plasmid DNA and an RNAi silencing expression vector framework PC336, and recombining the forward sequence and the reverse sequence of the PSTG _17694 gene fragment to the vector PC336 to obtain the RNAi silencing expression vector.

In the present invention, it is preferable to link the PSTG _17694 gene fragment with an entry vector; the entry vector is preferably PC414C modified in a laboratory, compared with the conventional entry vector pENTR-D-TOPO, the PC414C is inserted into a multiple cloning site, and directional cloning can be realized by a method of enzyme digestion and connection. Specific methods for linking PC414C with the target fragment are described in the literature references (korean beauty. cloning and analysis of the barley salicylic acid synthesis genes ICS and PAL [ D ]. Shandong university of agriculture, 2013.). The invention relates to an improved primer obtained by adding protective base and enzyme cutting site on CQM17694-F2/R2 primer sequence preferably by using GATEWAY cloning technology. Preferably the protective base is attached to the 5' end of the primer sequence; the number of the protected bases is preferably 2-4; in the practice of the present invention, the protected base on CQM17694-F2 is preferably CAA and the protected base on CQM17694-R2 is preferably TCA. In the invention, the enzyme cutting sites are preferably NotI enzyme cutting sites and AscI enzyme cutting sites, the CQM17694-F2 primer sequence is connected with the NotI enzyme cutting sites, and the CQM17694-R2 primer sequence is connected with the AscI enzyme cutting sites.

In the specific implementation process of the invention, before the PSTG _17694 gene fragment is ligated to the entry vector, preferably, the PCR amplification product of the PSTG _17694 gene fragment obtained by the improved primer amplification and the entry vector PC414C are subjected to NotI and AscI double digestion to obtain a digestion product, and then the purified digestion product (i.e., the PSTG _17694 gene fragment and the entry vector) is ligated by using T4-DNA ligase to obtain a ligation product. After obtaining the ligation product, the invention preferably transforms the ligation product into an escherichia coli competent cell, and obtains positive clone plasmid DNA after screening and purifying. In the present invention, the transformed E.coli competent cells are screened to obtain the positive cloning plasmid. The screening method of the present invention is preferably colony PCR; the colony PCR primer is preferably CQM 17694-F2/R2; the purification of the positive clone plasmid DNA is preferably accomplished using a plasmid DNA kit (Tiangen Biochemical technology (Beijing) Ltd., cat # DP 103). After plasmid DNA of positive clone is obtained, the plasmid DNA of the positive clone and an RNAi silencing expression vector skeleton PC336 (Korean, barley salicylic acid synthesis genes ICS and PAL are cloned and analyzed [ D ]. Shandong agricultural university, 2013.) are used for LR reaction, and the forward sequence and the reverse sequence of a PSTG _17694 gene fragment are recombined to the vector PC336 to obtain the RNAi silencing expression vector. In the invention, the vector construction adopts the conventional vector construction method and program in the field, has no other special requirements, and can obtain the RNAi silencing expression vector.

The number of the wheat stripe rust PSTG _17694 gene silencing expression vector obtained in the invention is PC897, the sequence of a T-DNALB (left Border) to RB (right Border) segment on the wheat stripe rust PSTG _17694 gene silencing expression vector is preferably shown as Seq ID No.6, and the sequence of the T-DNALB (left Border) to RB (right Border) segment is an expression cassette comprising a hygromycin resistance gene Hyg, a herbicide resistance gene Bar and a forward sequence of a PSTG _17694 gene segment and a reverse sequence of a PSTG _17694 gene segment which are connected in sequence.

After obtaining the wheat stripe rust PSTG _17694 gene silencing expression vector, the invention adopts a gene gun mediated transformation method to transfer the wheat stripe rust PSTG _17694 gene silencing expression vector into wheat to obtain the stripe rust resistant wheat. The preferred recipient material of the present invention is the line CB037 of high-grade wheat stripe rust. The specific technical process of transformation described in the present invention is described in the Wang Tree Yun Master thesis (Wang Tree Yun. establishment of wheat germ source regeneration and transformation system [ D ]. Shandong agriculture university, 2012.). And will not be described in detail herein.

After the transformation is finished, preferably, the transformed material is subjected to callus induction on a callus induction culture medium, the callus is differentiated and cultured to obtain regenerated seedlings, and the regenerated seedlings are subjected to rooting culture to obtain wheat seedlings which can be transplanted into soil. The specific steps and parameters for obtaining wheat seedlings by post-transformation culture are described in the royal tree cisco thesis (royal tree cisco. establishment of wheat germ source regeneration and transformation system [ D ]. Shandong agriculture university, 2012.).

The application of the wheat stripe rust PSTG — 17694 gene in stripe rust prevention and control and the cultivation method of stripe rust resistant wheat provided by the invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the invention.

Example 1

(I) PCR amplification to obtain PSTG _17694 gene fragment

PCR primers were designed using the coding region sequence of the putative functional protein gene PSTG _17694(GenBank: KNE88861.1) as a template with reference to the whole genome sequence of P.cereus physiological race PST-78 of Puccinia striiformis (GenBank accession number AJIL00000000.1 or BROAD download Link ftp:// ftp. electromagnetically induced. org/pub/mutation/fungi/puccinia/genes/puccinia _ striiformis _ PST-78 /).

The forward primer sequence CQM17694-F2, as shown in Seq ID No. 3:

5`-caagcggccgcTCATTCGACCAAAGAAAGGC-3`；

the reverse primer sequence CQM17694-R2, as shown in SeqID No. 4:

5`-tcaggcgcgccGATCACCTCGTTTCCATGCT-3`。

the length of the amplified segment is 490 bp. Alignment of puccinia striiformis PST-78 genome data by sequence alignment of amplified segments (https://genome.jgi.doe.gov/Pucst_PST78_1/Pucst_PST78_1.home.html) And NCBI database wheat stripe rust fungus sequence (puccinitia striiformis f.sp.tritici, taxi: 168172), the sequence of the amplified segment of the PCR amplification primer in the invention is found to have higher homology with PSTG _19439 in the first exon of PSTG _17694 after sequence alignment, and in addition, no 100% identical sequence with the length of other genes exists in the sequence of the amplified segment and other genes, which indicates that the sequence of the segment amplified by using the CQM17694-F2/R2 primer can specifically target the PSTG _17694 gene.

Wheat leaves infected with the rust fungus physiological race CRY32 and having a disease are taken, RNA is purified by a conventional TRIZOL method (Tiangen Biotechnology technology (Beijing) Co., Ltd., product No. DP405), and cDNA (reverse transcription kit of Beijing Quanyujin Biotechnology Co., Ltd., product No. AT311-03) is prepared. PCR amplification Using Phusion Hi-Fi enzyme (Thermo Scientific Co., cat # F-530S) Using primers CQM17694-F2 and CQM17694-R2 (PCR system: 5X Phusion HFbuffer:10ul, Phusion DNA Polymerase:0.5ul, CQM 94-F2:1.5ul, CQM17694-R2:1.5ul,10mM dNTPs:1ul, Template DNA:1ul, ddH 176₂O is 34.5 ul; PCR procedure: 30s at 98 ℃; circulating at 98 deg.C for 10s, 60 deg.C for 20s, and 72 deg.C for 30s 35; 72 ℃ for 10min), the PSTG _17694 gene fragment is separated and the sequencing verification is carried out.

The sequence of the gene fragment isolated from CRY32 is shown in Seq ID No.5, and has 99% sequence similarity with the reference gene PSTG _17694, and has 1 SNP (single Nucleotide polymorphism) site.

Construction of wheat stripe rust gene PSTG _17694 silent expression vector

In order to construct a gene silencing expression vector by using a GATEWAY cloning technology, 3 protective bases (CAA and TCA) and a NotI enzyme cutting site (GCGGCCGC) or an AscI enzyme cutting site (GGCGCGCC) are respectively added at two ends of a CQM17694-F2/R2 primer so as to be connected into a modified entry vector PC414C (Korean, the cloning and analysis of a barley salicylic acid synthesis gene ICS and PAL [ D ]. Shandong agricultural university, 2013.). The PCR amplification product and the vector PC414C are firstly subjected to NotI and AscI double enzyme digestion, and the enzyme digestion product is purified and then is connected by T4-DNA ligase. The ligation product was transformed into E.coli competent cells (Beijing Quanjin Biotechnology Ltd., cat # CD 201). Colony PCR was performed using primers CQM17694-F2/R2 to screen positive clones carrying the gene fragment of interest, and plasmid DNA was purified (Tiangen Biochemical technology (Beijing) Co., Ltd., cat # DP 103).

Plasmid DNA of positive clone was used to perform LR reaction with RNAi silencing expression vector backbone PC336 (Korean, barley salicylic acid synthesis genes ICS and PAL clone and analysis [ D ]. Shandong agricultural university, 2013.), and simultaneously forward and reverse sequences of PSTG _17694 gene fragment were recombined to vector PC336 to construct RNAi silencing expression vector.

LR reaction system: 1 μ l of the target vector (150 ng/. mu.l); portal vector PC414C (50-150ng) 1. mu.l, ddH₂O7μl；LR Clonase^TMII Enzyme Mix (Invitrogen corporation, cat 11791) 2. mu.l, reaction at 25 ℃ for 1h, adding 1. mu.l of protease K, inactivating at 37 ℃ for 10min, transforming Trans5 α competent cells (Beijing Quanjin Biotechnology Co., Ltd., cat # CD201) with the reaction solution, picking up colonies, purifying plasmid DNA, and performing colony PCR screening using CQM17694-F2/R2 primers (PCR system: 2xBuffer 10. mu.l, CQM17694-F20.5ul, CQM17694-R20.5ul, ddH)₂O9 ul, single colony; PCR procedure: 5min at 94 ℃; 30s at 94 ℃, 30s at 58 ℃ and 30s at 72 ℃ for 35 cycles; 72 ℃ for 10 min).

The sequence of the T-DNA LB (Left Border) to RB (Right Border) segment on the wheat stripe rust gene PSTG _17694 silent expression vector is shown as the sequence table Seq ID No. 6. The expression cassette comprises hygromycin resistance gene (Hyg), herbicide resistance gene (Bar), forward sequence of PSTG _17694 gene fragment and reverse sequence of PSTG _17694 gene fragment.

(III) genetic transformation and progeny screening of common wheat

The genetic transformation of wheat adopts a gene gun mediated transformation method, and the receptor material is a strain CB037 of high-sensitivity wheat stripe rust. The technical process is described in the Wang Shu Shuoshi paper (Wang Shu Yun. establishment of wheat germ regeneration and transformation system [ D ]. Shandong university of agriculture, 2012.). Selecting young embryos with the diameter of 1.5mm after wheat pollination for about 15 days, shearing ears to strip seeds, carrying out surface disinfection and sterilization, then stripping the young embryos, placing the young embryos on a hypertonic culture medium, bombarding the young embryos by using a gene gun, and carrying out dark culture on the young embryos on a healing induction culture medium at 23 ℃ for four weeks, and carrying out subculture once every two weeks. Four weeks later, the callus induction medium was changed to a differentiation medium, and the callus was cultured in light (23 ℃, 16hlight, 8hdark) from the differentiation medium. After two weeks, the medium was transferred from the differentiation medium to the differentiation medium again until the seedlings were regenerated. Taking out the differentiated seedling and placing the seedling on a rooting culture medium, and transplanting the seedling into soil after the root grows strong for about 3-4 weeks.

The positive transgenic offspring is primarily screened by coating herbicide on the leaves. The herbicide was Finale, product of AgrEvo, Cat # F30617006, used at a concentration of 0.3%. And 3-5 days later, positive transgenic offspring is screened according to the sensitivity of the smearing part of the leaves.

As shown in figure 1, wherein CB037 is a transgenic receptor, lines 5678-2A and 5726-1 are negative transgenic individuals; 2079-5A, 5758-1A and 5769-3A are positive transgenic individuals. The smearing part of the negative transgenic material leaves obviously yellows or withers and necroses, while the smearing part of the positive transgenic material leaves slightly yellows and does not have obvious withers and necroses.

Meanwhile, DNA and cDNA are prepared by taking leaves of transgenic wheat, and PCR amplification is carried out by using primers CQM17694-F2/R2 to screen and confirm the transgenic material. As shown in FIG. 2A, A, B shows the result of PCR using gDNA as a template; the PCR primer in panel A is CQM 17694-F2/R2; h₂O is negative control, P is positive control of plasmid DNA; the PCR primers in panel B are LB306F/CQM17694-R2, wherein the primer LB306F is a matched carrier frameworkThe primer sequence of the GUS linker fragment is positioned between the forward and reverse fragments of CQM17694, and the primer sequence of LB306F is shown as Seq ID No.11, specifically 5'-GACCTCGCAAGGCATATTG-3'; h₂O is negative control, P is positive control of plasmid DNA. Most samples produced the same amplified band as the positive control (vector DNA), indicating that they were derived from positive transgenic material.

Example 2 resistance identification of wheat stripe rust against stripe rust

Propagation of fresh spores of wheat stripe rust: planting susceptible variety Huizhou red in a greenhouse, inoculating bacteria by injecting spore water solution with an injector in one-leaf and one-heart period, spraying water with a spray can, and covering a plastic film for moisturizing. The inoculation can be repeated for 2-3 times with 1 week interval to ensure sufficient morbidity. Preparation of aqueous spore solution: taking the dried and frozen spore at (-80 ℃) to be suspended to light orange by using a proper amount of tap water, placing the suspension in a shaking table at room temperature, shaking and mixing the suspension for 30min (180rpm), and then injecting and inoculating the strain. Sporadic morbidity can be seen about 20 days after inoculation, a large amount of leaf morbidity can be seen about 30 days after inoculation, and a large amount of fresh spores can be collected for inoculation identification of transgenic materials.

By T_1:2Generation (harvesting of seeds of the first generation transgenic material in example 1) seeds were grown in a greenhouse and inoculated with puccinia striiformis f.sp.tritici, and their puccinia striiformis resistance was identified. Watering the greenhouse thoroughly a week before planting, and airing for about a week. And selecting 15 healthy and plump transgenic seeds, and uniformly dibbling the seeds, wherein the row spacing is 25cm, and the row length is 1 m. 2 rows of transgenic receptor variety CB037 were planted every ten rows. And (3) when the spores grow to about one-leaf and one-heart periods, inoculating the fresh spores, watering the inoculated spores and covering plastic cloth to keep a high-humidity environment. In order to ensure successful inoculation, repeated inoculation is carried out every other week after primary inoculation, and the repeated inoculation is carried out three times, and each plant is inoculated with at least three tillers. After full disease of wild type CB037, the transgenic material was disease resistance investigated and recorded, and the identification was repeated three times every other week.

Harvesting T from the greenhouse-identified disease-resistant material_2:3After the generation of seeds, the identification is repeated in an artificial climate box. A clean 9cm petri dish was filled with 3 layers of round filter paper, and 4mL of deionized water was added to soak the filter paper. PlacingCovering 15-20 healthy and plump seeds per dish, covering a culture dish cover, and sealing the culture dish cover by using a sealing film; and wrapping the seeds with aluminum foil paper, and keeping the seeds away from light, treating the seeds in a refrigerator at 4 ℃ for 2-3 days at low temperature to break seed dormancy. Culturing in a 23 ℃ illumination incubator for 3-5 days, transplanting into soil, selecting small square flowerpots with the side length of 15cm, and planting 4 plants in each pot. When the plants grow to the stage of one leaf and one heart, the mixed spores of the rust streak bred in the greenhouse are used for inoculation. And (3) fully and uniformly mixing the spores and the talcum powder according to the proportion of about 1:10, and dipping the spores by using a hairbrush for inoculating the leaves. After inoculation, the mixture is firstly treated in the dark for 24 hours at the temperature of 11 ℃ and the humidity of 100 percent; then normal light culture is carried out, the light is 16 h/temperature 22 ℃, the dark is 8 h/temperature 15 ℃, humidification is carried out regularly, and the water drops on the leaf tips are kept until the control material leaves are fully attacked (10-12 days).

The leaf morbidity of transgenic wheat after greenhouse inoculation of puccinia striiformis is shown in fig. 3, wherein A is wild type CB037, and B is a PC897 transgenic material. Greenhouse planting T_1:2After the material is inoculated repeatedly for three times, the wild CB037 is fully developed, and a large number of sporophytes are arranged on the surface of a leaf; and no sporophyte is generated on the surface of the transgenic material leaf, so that the stripe rust resistance effect is obvious.

The leaf morbidity of transgenic wheat after the artificial climate box is inoculated with the puccinia striiformis is shown in figure 4: a is wild type CB 037; b is PC897 transgenic material. T is_2:3After the material is inoculated with bacteria in an incubator for 12 days, the wild CB037 fully attacks the disease, and spores are fully distributed on the surfaces of leaves; and the PC897 transgenic material is only slightly green, and no spores are generated on leaves, which shows that the silent expression of the PSTG _17694 gene can obviously improve the resistance of wheat to the stripe rust.

Example 3 expression identification of PSTG _17694 Gene in Puccinia striiformis resistant wheat

After 12 days of inoculation of wheat seedling stage leaves, the surfaces of wild type CB037 leaves are full of a large number of sporophytes. CB037 and about 100mg of transgenic material leaves are taken at different time points after inoculation, RNA is extracted by using a Trizol method, and cDNA is prepared for real-time fluorescence quantitative PCR. The real-time fluorescent quantitative PCR primer sequences are CQM _17694-F3 and CQM _17694-R3, and the specific primer sequences are shown in sequence tables Seq ID No.7 and Seq ID No. 8. The primer TUBA-F/R sequence is shown in sequence tables Seq ID No.9 and Seq ID No.10 by taking the wheat stripe rust alpha tubulin gene as an internal reference (Diels et al, J. xue et al, Proc. agrobiological science, 2012, 20 (2): 181-.

The PCR system is as follows: mu.l of 2 XSSYBR GreenMaster, 1. mu.l each of forward and reverse primers (10. mu.M), 1. mu.l of cDNA, 2. mu.l of ddH2O, total 10. mu.l. The PCR reaction adopts a two-step method: 95 ℃ for 10min, 40 cycles of 95 ℃ for 15s, 60 ℃ for 1 min.

By use of 2^－△△CTThe algorithm analyzed the expression of each gene and mapped using the sigmaplot12.5 software.

The fluorescence quantitative expression analysis result of the gene of the invention after the transgenic wheat is inoculated with the rust streak is shown in figure 5: wherein PC897 is selected positive transgenic wheat, and CB037 is a wheat transgenic receptor. The wheat leaf is inoculated with puccinia striiformis and different materials are taken to detect the gene expression level. The quantitative PCR primer is CQM 17694-F3/R3.

The observation result of leaf tissue staining after transgenic wheat is inoculated with puccinia striiformis is shown in fig. 6, wherein A is a PC897 transgenic material, and B is wild type CB 037. On day 12 after inoculation, staining was performed with WGA-FITC fluorescent dye. Hyphae in wild type CB037 leaves are dense, and sporophyte (B) can be seen on the surfaces; while the transgenic material only observed secondary colonies inside the leaves and had small and short hyphae (A); the expression of the wheat stripe rust PSTG _17694 gene in the transgenic plant leaf is obviously inhibited compared with that of wild CB037, and the expression of the RNAi silencing vector PC897 in wheat can effectively reduce the expression level of the target gene in the wheat stripe rust.

The embodiment shows that the PSTG _17694 gene provided by the invention can effectively regulate the growth and the propagation of wheat stripe rust, and the PSTG _17694 gene is applied to the control of wheat stripe rust, so that the effect of inhibiting the growth and the propagation of wheat stripe rust is achieved by silencing the PSTG _17694 gene. The wheat obtained by the method for cultivating the wheat with the stripe rust resistance by using the gene has obvious wheat stripe rust resistance.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Shandong university of agriculture

Application of <120> wheat stripe rust PSTG _17694 gene in stripe rust prevention and treatment and cultivation method of stripe rust resistant wheat

<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 gaagcagacg4560

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. The application of the wheat stripe rust PSTG _17694 gene in the control of wheat stripe rust is characterized in that the sequence of the PSTG _17694 gene is shown as Seq ID No. 1; the PSTG _17694 gene is used as a molecular target for transcriptional inhibition, and the growth and propagation of the puccinia striiformis is inhibited by silencing the PSTG _17694 gene.

2. The use of claim 1, wherein the use is to introduce a silencing expression vector carrying the PSTG _17694 gene into wheat to obtain the wheat with stripe rust resistance.

3. A cultivation method of wheat with stripe rust resistance comprises the following steps:

1) carrying out PCR amplification by taking wheat leaf cDNA infected with rust as a template to obtain a PSTG _17694 gene segment;

2) constructing a wheat stripe rust PSTG _17694 gene silencing expression vector by using the PSTG _17694 gene segment in the step 1); the construction of the PSTG _17694 gene silencing expression vector adopts GATEWAY cloning technology;

3) transferring the wheat stripe rust PSTG _17694 gene silencing expression vector into wheat by adopting a gene gun mediated transformation method to obtain stripe rust resistant wheat;

the sequence of the PSTG _17694 gene is shown as Seq ID No. 1;

the primers for PCR amplification in the step 1) are CQM17694-F2 and CQM17694-R2 primers; the sequence of the CQM17694-F2 primer is shown as Seq ID No. 3; the sequence of the CQM17694-R2 primer is shown as Seq ID No. 4;

the wheat stripe rust PSTG _17694 gene silencing expression vector in the step 2) comprises expression cassettes of a hygromycin resistance gene Hyg, a herbicide resistance gene Bar, a forward sequence of a PSTG _17694 gene fragment and a reverse sequence of a PSTG _17694 gene fragment.

4. The cultivation method as claimed in claim 3, wherein the sequence of the T-DNAleft Border to Right Border segment of the wheat stripe rust PSTG _17694 gene silencing expression vector is shown in sequence table Seq ID No. 6.