CN111205357B - Wheat stripe rust resistance related protein TaWLT14.2, coding gene and application thereof - Google Patents

Abstract

The invention discloses a wheat stripe rust resistance related protein TaWLT14.2, and a coding gene and application thereof. The invention provides TaWLT14.2 protein which is TaWLT14.2-A1 protein (SEQ ID No.1), TaWLT14.2-B1 protein (SEQ ID No.2) and/or TaWLT14.2-D1 protein (SEQ ID No. 3). The TaWLT14.2 gene can regulate the resistance of wheat to stripe rust. The method has important significance for cultivating wheat varieties with high stripe rust resistance.

Description

Wheat stripe rust resistance related protein TaWLT14.2 and coding gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a wheat stripe rust resistance related protein TaWLT14.2 and a coding gene and application thereof.

Background

Wheat stripe rust is a worldwide wheat fungal disease caused by wheat stripe rust infection. Puccinia striiformis f.sp.tritici, a living parasitic fungus, belongs to the puccinia striiformis f.sp.tritici.

Stripe rust is a serious disease that harms wheat production in China and the world. Although chemical agents can effectively control the economic losses caused by wheat diseases, the development and promotion of disease-resistant varieties is still considered to be the most fundamental, economical and safe approach from the viewpoint of environmental safety and human health (Line and Chen, 1995; Xu et al, 2013). The cloning and function research of the wheat stripe rust resistance related gene has very important significance for the research of the wheat stripe rust resistance molecular mechanism and the molecular disease resistance breeding.

LT14(Low-Temperature-Induced Protein 14) is a family of Low-Temperature response proteins specific to cereal plants and has been reported in cereal crops such as barley, rye and wheat (Dunn et al, 1990; Gana et al, 1997; Zhang et al, 1993). The genes encoding these proteins are strongly induced to be expressed at low temperatures (2 ℃ C. -6 ℃ C.) or when subjected to frost (Babben et al, 2018), and the N-terminus of the encoded protein has a 20 amino acid signal peptide that facilitates secretion outside the cell (Ohno and Takumi, 2015). Gana (1997) and the like clone a TaCR7 gene of LT14 family in wheat, and find that the expression of the TaCR7 gene is specifically induced by low temperature and is insensitive to ABA, salt, high temperature and other abiotic stresses. At present, no report is found about the function research of Wheat Low Temperature induction Gene WLT14(Wheat Low-Temperature-Induced Gene 14) in biotic stress.

Disclosure of Invention

The invention aims to provide a wheat stripe rust resistant related protein TaWLT14.2, a coding gene and application thereof.

In a first aspect, the invention claims a protein, named TaWLT14.2 protein, in particular TaWLT14.2-A1 protein, TaWLT14.2-B1 protein and/or TaWLT14.2-D1 protein.

The TaWLT14.2-A1 protein can be any one of the following proteins:

(A1) a protein having an amino acid sequence of SEQ ID No. 1;

(A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.1 and has the same function;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

The TaWLT14.2-B1 protein can be any one of the following proteins:

(B1) a protein having an amino acid sequence of SEQ ID No. 2;

(B2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.2 and has the same function;

(B3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (B1) to (B2) and having the same function;

(B4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (B1) to (B3).

The TaWLT14.2-D1 protein can be any one of the following proteins:

(D1) a protein having an amino acid sequence of SEQ ID No. 3;

(D2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.3 and has the same function;

(D3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with the amino acid sequence defined in any one of (D1) to (D2) and having the same function;

(D4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (D1) to (D3).

In the above protein, the tag is a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracking and/or purification of the target protein. The tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In a second aspect, the invention claims nucleic acid molecules encoding the proteins described hereinbefore (TaWLT14.2 proteins).

Further, the nucleic acid molecule encoding the tawlt14.2-a1 protein may be a DNA molecule as described in any one of (a1) - (a3) below:

(a1) a DNA molecule shown as SEQ ID No. 4;

(a2) a DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (a1) and encodes said tawlt14.2-a1 protein;

(a3) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the DNA sequence limited by (a1) or (a2) and encodes the TaWLT14.2-A1 protein.

Further, the nucleic acid molecule encoding the tawlt14.2-B1 protein may be a DNA molecule as described in any one of (B1) - (B3) below:

(b1) DNA molecule shown in SEQ ID No. 5;

(b2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) and encodes said tawlt14.2-B1 protein;

(b3) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the DNA sequence limited by (B1) or (B2) and encodes the TaWLT14.2-B1 protein.

Further, the nucleic acid molecule encoding the tawlt14.2-D1 protein may be a DNA molecule as described in any one of (D1) - (D3) below:

(d1) a DNA molecule shown as SEQ ID No. 6;

(d2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (D1) and encodes the tawlt14.2-D1 protein;

(d3) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of homology with the DNA sequence limited by (D1) or (D2) and encodes the TaWLT14.2-D1 protein.

In the above genes, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M Na ₃ PO ₄ Hybridization with a mixed solution of 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; it can also be: 50 ℃ in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing at 50 ℃ in1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with a mixed solution of 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: in a solution of 6 XSSC, 0.5% SDS at 65 ℃ and then washed once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In a third aspect, the invention claims expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines containing the nucleic acid molecules.

The recombinant expression vector can be constructed by using the existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like, such as pLGY-02, pCAMBIA-1300-221, pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other derivative plant expression vectors. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can direct the addition of poly A to the 3' end of the mRNA precursor. When the gene is used for constructing a recombinant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added in front of the transcription initiation nucleotide, such as a cauliflower mosaic virus (CAMV)35S promoter, a Ubiquitin gene Ubiquitin promoter (pUbi), a stress inducible promoter rd29A and the like, and the promoter can be used alone or combined with other plant promoters; in addition, when the gene of the present invention is used to construct a recombinant expression vector, enhancers, including translational enhancers or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codons or initiation codons of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the recombinant expression vectors used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change, antibiotic markers having resistance or chemical resistance marker genes, etc., which are expressed in plants. Or directly screening the transformed plants in a stress environment without adding any selective marker gene.

In a fourth aspect, the present invention claims the use of the protein or the nucleic acid molecule or the expression cassette or the recombinant vector or the recombinant bacterium as described above for modulating a plant's resistance to stripe rust.

In said use, the activity and/or the expression level of the protein described hereinbefore (TaWLT14.2 protein) or of a gene coding therefor in said plant is reduced, and the plant is less resistant to stripe rust; the activity and/or the expression level of the protein (TaWLT14.2 protein) or the gene coding for it in the plant is increased, and the plant resistance to stripe rust is increased.

In a fifth aspect, the invention claims a method of growing a plant variety with altered resistance to stripe rust by method I or method II:

the method I comprises the following steps: a method of breeding a plant variety with increased resistance to stripe rust disease, comprising the step of increasing the expression level and/or activity of a protein as described hereinbefore (tawlt14.2 protein) in a recipient plant.

In general, the method will have a phenotype of increased resistance to stripe rust if the expression level and/or activity of one of the TaWLT14.2 proteins (i.e.TaWLT14.2-A1 protein, TaWLT14.2-B1 protein or TaWLT14.2-D1 protein) is increased in the recipient plant.

Method II: a method of breeding a plant variety with reduced resistance to stripe rust, comprising the step of reducing the expression level and/or activity of a protein as described hereinbefore (tawlt14.2 protein) in a recipient plant.

In said method, it is generally necessary to simultaneously reduce the expression and/or activity of three proteins tawlt14.2 (i.e. the proteins tawlt14.2-a1, tawlt14.2-B1 and tawlt 14.2-D1) in a recipient plant, which has a phenotype of reduced resistance to stripe rust.

In a sixth aspect, the invention claims a method of breeding transgenic plants with altered resistance to stripe rust, method III or method IV:

method III: a method of breeding a transgenic plant with increased resistance to stripe rust, comprising the steps of: introducing into a recipient plant a nucleic acid molecule capable of expressing the protein described hereinbefore (TaWLT14.2 protein) to obtain a transgenic plant; the transgenic plant has increased resistance to stripe rust compared to the recipient plant.

In the method, generally, if one coding gene of TaWLT14.2 protein (namely TaWLT14.2-A1 protein, TaWLT14.2-B1 protein or TaWLT14.2-D1 protein) is over-expressed in a receptor plant, the obtained transgenic plant has a phenotype of improved stripe rust resistance.

Wherein the introduction into said recipient plant of a nucleic acid molecule capable of expressing the protein described above (TaWLT14.2 protein) is achieved by any means capable of achieving this object.

In a particular embodiment of the invention, this is achieved in particular by introducing into said recipient plant a recombinant vector containing a nucleic acid molecule capable of producing the protein described hereinbefore (TaWLT14.2 protein). The recombinant vector is specifically a recombinant plasmid obtained by inserting a nucleic acid molecule capable of expressing the protein (TaWLT14.2 protein) into a pLGY-02 vector.

Method IV: a method of breeding a transgenic plant with reduced resistance to stripe rust can comprise the steps of: inhibiting and expressing the nucleic acid molecule capable of expressing the protein (TaWLT14.2 protein) in the receptor plant to obtain a transgenic plant; the transgenic plant has reduced resistance to stripe rust as compared to the recipient plant.

In the method, generally, the phenotype of reduced stripe rust resistance is generated only by simultaneously inhibiting and expressing coding genes of three TaWLT14.2 proteins (namely, TaWLT14.2-A1 protein, TaWLT14.2-B1 protein and TaWLT14.2-D1 protein) in a recipient plant.

Wherein the suppression of the expression of the nucleic acid molecule capable of expressing the protein described hereinbefore (TaWLT14.2 protein) in said recipient plant is achieved by any means which is capable of achieving this object.

In one embodiment of the present invention, specifically by introducing into the recipient plant a BSMV viral vector α, a BSMV viral vector β and a recombinant BSMV viral vector γ; the recombinant BSMV viral vector gamma contains DNA molecules shown in the 9 th to 219 th positions from the 5 'end of SEQ ID No.6 or DNA molecules shown in the 10 th to 113 th positions from the 5' end of SEQ ID No. 6.

Wherein, the DNA molecule shown in the 9 th to 219 th positions from the 5 'end of SEQ ID No.6 or the DNA molecule containing the 10 th to 113 th positions from the 5' end of SEQ ID No.6 has homologous fragments continuously exceeding 21 bases compared with the corresponding fragments of SEQ ID No.4 and SEQ ID No.5, which can interfere the stabilization of the sequences of SEQ ID No.4 and SEQ ID No. 5. Therefore, after the BSMV viral vector alpha, the BSMV viral vector beta and the recombinant BSMV viral vector gamma are introduced into the receptor plant, the expression of coding genes of three proteins, namely TaWLT14.2-A1, TaWLT14.2-B1 and TaWLT14.2-D1 in the receptor plant can be inhibited.

In a particular embodiment of the invention, this is achieved in particular by the crisp sper/Cas9 technique; the 5' -N identity in the genomic fragment of the gene coding for the protein described previously (TaWLT14.2 protein) _X -NGG-3 'or 5' -CCN-N _X -a fragment with a regular 3' sequence arrangement is the target sequence; n represents any one of A, G, C and T, 14 ≦ X ≦ 30, and X is an integer, N _X Represents X consecutive deoxyribonucleotides. More specifically, in one embodiment of the present invention, the target sequence is specifically 5'-CTTGAATGGTGCTGGTACCGGTAA-3' (position 176-195 of SEQ ID Nos. 4-6).

In each of the above aspects, the increased resistance to stripe rust may be embodied as: after increasing the expression level and/or activity of the protein (TaWLT14.2 protein) described above in the recipient plant, the number of yellow rust spores on the plant is increased.

In each of the above aspects, the reduced resistance to stripe rust may be embodied as: after reducing the expression level and/or activity of the protein (TaWLT14.2 protein) described above in the recipient plant, the number of spores of Puccinia striiformis on the plant is reduced.

In each of the above aspects, the stripe rust may be stripe rust caused by the species CYR17 and/or CYR33 of the species pythium strigae (blumeria sp. tritici). The stripe rust bacteria can be stripe rust bacteria (Blumeeriagriminis f.sp.tritici) physiological race CYR17 and/or CYR 33.

In each of the above aspects, the plant may be a monocot. Further, the monocotyledon may be a gramineae plant. Further, the gramineous plant may be wheat.

In a particular embodiment of the invention, the wheat is in particular the wheat variety hydrophyten 11 (variety of anti-rust strain CYR17) or the wheat variety Fielder (variety of rust-sensitive strain CYR 33).

Experiments prove that the expression of the TaWLT14.2 gene in wheat leaves is reduced through virus-induced gene silencing or a CRISPR/Cas9 technology, so that the infection of the wheat variety, namely the water source 11, to the avirulent rust streak race CYR17 is changed from disease resistance to infection. Therefore, the TaWLT14.2 gene of wheat is proved to be involved in the resistance regulation of wheat to stripe rust. Furthermore, the TaWLT14.2 gene is over-expressed in the wheat variety Fielder, the expression level of the TaWLT14.2 gene in the wheat is improved, and the infection of the wheat variety Fielder to the virulent rust stripe race CYR33 is changed from infection to disease resistance. It can be seen that the TaWLT14.2 gene can regulate the resistance of wheat to stripe rust. The method has important significance for cultivating the wheat variety with high stripe rust resistance.

Drawings

FIG. 1 shows the relative expression of TaWLT14.2 gene in 11 leaves of common wheat variety jellyfish after BSMV-VIGS down-regulates the expression of TaWLT14.2 gene. Both silencing fragments TaWLT14.2-1 and TaWLT14.2-2 were shown to significantly down-regulate the expression of the TaWLT14.2 gene, ". x" indicates that P.ltoreq.0.01. Mock: simulating inoculated blank control plants; BSMV GFP: transferring GFP gene negative control plant; BSMV TaWLT14.2-1 and BSMV TaWLT14.2-2 represent plants which utilize silent segments TaWLT14.2-1 and TaWLT14.2-2 to down-regulate TaWLT14.2 gene expression, respectively.

FIG. 2 shows leaf phenotype 14 days after inoculation of physiological race CYR17 of avirulent Puccinia triticina to Sauropus cereus 11 plants with BSMV-VIGS down-regulated TaWLT14.2 gene expression. This indicates that the aquagen 11, which down-regulated the expression of the tawlt14.2 gene, lost resistance to CYR17 and fell to a high sensation (response type 3-4). Mock: simulating inoculated blank control plants; BSMV GFP: transferring GFP gene negative control plant; BSMV TaWLT14.2-1 and BSMV TaWLT14.2-2 represent plants which utilize silent segments TaWLT14.2-1 and TaWLT14.2-2 to down-regulate TaWLT14.2 gene expression, respectively.

Fig. 3 is sgRNA activity assay for tawlt14.2 gene in CRISPR/Cas9 system. wlt 14.2.2 denotes sgRNA of the target tawlt14.2 gene. + AgeI/BshTI represents TaWLT14.2 gene fragment obtained by PCR amplification using protoplast DNA transformed by pTaU6-WLT14.2-sgRNA vector plasmid (WLT 14.2.2) as template and Wild Type (WT) protoplast DNA as template, and AgeI/BshTI enzyme is added for enzyme digestion treatment. The red arrow indicates the mutation band.

Fig. 4 is a schematic diagram of the gene editing types of the three watersogen 11 transgenic lines with CRISPR/Cas9 editing tawlt14.2 genes. The red line indicates the PAM site, the orange line indicates the leader sequence (WLT14.2-sg) used for WLT14.2 gene editing, the "+" or "-" indicates the insertion or deletion event, the number indicates the number of inserted or deleted bases, and the "… …" indicates the omitted bases. Su11 represents the acceptor wheat variety jellyfish 11 wild type sequence; t1-171-1.2, T1-173-1.9 and T1-671-1 are sequences of T1 generation wheat transgenic lines after TaWLT14.2 genes are edited by CRISPR/Cas 9.

Figure 5 is the leaf phenotype of the CRISPR/Cas9 edited an aquatic 11 plant of the tawlt14.2 gene after inoculation with CYR 17. Shows that the water source 11 edited by the TaWLT14.2 gene loses the resistance to the non-toxic yellow rust physiological race CYR17, the response type is 3-4 grade, and the disease belongs to the susceptible phenotype. Meanwhile, the leaves of a control plant which is not subjected to gene editing have no sporophyte, and are shown to be immune or nearly immune, and the response type is 0-0; and (4) stage. Su 11: a recipient wheat variety, jellyfish 11; t1-171-1.2, T1-173-1.9 and T1-671-1 are T1 generation wheat transgenic lines after the TaWLT14.2 gene is edited by CRISPR/Cas 9.

FIG. 6 shows the relative expression of TaWLT14.2 gene in transgenic wheat with overexpression. Shows that the expression level of TaWLT14.2 in the overexpression transgenic plants is obviously improved compared with that of the control plants. OEWLT14.2-L4, OEWLT14.2-L6 and OEWLT14.2-L10 represent 3 different transgenic lines; null is a transgenic negative line; WT stands for recipient wheat variety Fielder.

FIG. 7 is the phenotype of the leaves of the diseased wheat variety Fielder overexpressing the TaWLT14.2 gene after inoculation with the physiorace CYR 33. The resistance of Fielder over-expressing TaWLT14.2 gene to the rust stripe microspecies CYR33 is obviously improved, the reactivity is changed from high susceptibility to medium resistance to high resistance, and the reactivity of different strains is 0; 2 stages. OEWLT14.2-L4, OEWLT14.2-L6 and OEWLT14.2-L10 are 3 different overexpression transgenic lines, and the expression of the transgenic lines is used as a negative line for finding WLT14.2 transgene after Null is transgenic; WT stands for recipient wheat variety Fielder.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

BSMV-VIGS viral vectors (including alpha, beta and gamma plasmids) and recombinant gamma-GFP plasmids in the examples described below are disclosed in the literature "Holzberg S, Brosio P, Gross C, Pogue G.Bar strain viral-induced gene cloning in a monocot plant J.,2002,30: 315-. The public is available from institute of genetics and developmental biology, the institute of academy of sciences, and can only be used for repeated laboratories, and no other application is available.

The rust (Blumeragramis f.sp.tritici) physiological races CYR17, CYR33 in the literature "Hu X, Li J, Wang Y, Wang B, Li Q, Kang Z, Yang M, Peng Y, Liu T, Chen W, et al. race composition of Puccinia striiformis f.sp.tritici in Tibet, China. plant Dis,2012,96, 1615-.

Wheat variety water source 11(Triticum aestivum var. suwon 11) in the following examples is described in the literature "poplar grower. study of wheat for the inheritance of stripe rust resistance. crop press, 1981, 7 (2): 81-90. "is disclosed in the specification. The public can obtain from the institute of genetics and developmental biology of Chinese academy of sciences, and can only be used for repeated experiments, and no other purpose is available.

Wheat variety Fielder (Triticum aestivum var. Fielder) in the following examples "vain, zhangshu, fangchun, wangkun poplar, wang jing, wang trade, doulibu, phyllohu" document the effect of wheat haploid plants was doubled with different chemical reagents and treatment means. 811-820. "is disclosed in the specification. The public is available from institute of genetics and developmental biology, the institute of academy of sciences, and can only be used for repeated laboratories, and no other application is available.

The plasmids pTaU6, pJIT163-2NLSCas9 and pJIT163-GFP in the following examples are disclosed in the documents "Shann QW, Wang YP, Li J, Gao CX. genome injection in rice and while using the CRISPR/Cas system. Nature Protocols,2014,9(10): 2395-. The transformation of wheat immature embryos is completed by a transformation platform. The public is available from institute of genetics and developmental biology, the institute of academy of sciences, and can only be used for repeated laboratories, and no other application is available.

The plasmid pLGY-02 in the following examples was described in the literature "Liangfang, Liu-Fei, Zhendachi, Wang Haiyan, Liu Da Ji. Agrobacterium mediated method was used to rapidly identify the function of the protein gene TaPR1 associated with wheat disease course.Hebei university reports, 2019, 42 (2): 12-17. "is disclosed in the specification. The transformation of wheat immature embryos is completed by a transformation platform. The public is available from institute of genetics and developmental biology, the institute of academy of sciences, and can only be used for repeated laboratories, and no other application is available.

The criteria for response grading of resistance to wheat stripe rust described herein are described in detail in the literature "Li ZQ, Shang HS. wheat tests and the control.1989, Shanghai Science and Technology Press, Shanghai, China," which classifies stripe rust resistance as asymptomatic as immune grade (grade 0) according to a 0-4 response; producing withered spots or chlorosis, no summer sporangium, near immune grade (0; grade); the summer sporangium is small and few, and the periphery has withered and dead reactions, and is disease-resistant (level 1); the summer spore pile is small to medium, and the periphery has withered and green losing reactions, which is medium-grade (grade 2); the summer sporangium is medium in size, has no withered and dead reaction around the summer sporangium, but has slight green loss phenomenon, and is a medium grade (grade 3); summer sporangium is large and abundant, has no withered and dead reaction, and has no obvious phenomenon of green loss, and is of a disease grade (4 grade).

GKP Buffer in the following examples contains 50mM glycine (glycine), 30mM K ₂ HPO ₄ (pH9.2), 1% (mass percent) bentonite (bentonite) and 1% (mass percent) diatomaceous earth (celite).

Example 1 cloning of cDNA and DNA sequences of the TaWLT14.2 Gene

1. Obtaining of wheat cDNA and genome sequence

(1) Extraction of wheat genome DNA and total RNA

0.2g of fresh wheat leaves are taken and put into a 2mL centrifuge tube filled with steel balls, and after quick freezing by liquid nitrogen, the material is ground into powder in a tissue grinder. 0.8mL of lysis buffer (1% SLS (sodium lauryl sarcosinate), 417mM Tris/HCl pH8.0, 417mM NaCl, 83mM EDTA) was added, shaken vigorously, vortexed well and mixed well, and allowed to stand on ice for 10min to allow the cells to lyse well. Adding an extracting solution (Tris saturated phenol/chloroform/isoamyl alcohol is 25:24:1 in volume ratio) with the same volume as the lysate into a centrifuge tube, slowly shaking the mixture to be uniform to emulsion, standing the emulsion on ice (or at room temperature) for 10min, centrifuging the emulsion at 12000rpm for 10min, and transferring the supernatant into a new centrifuge tube. This operation was repeated once. The supernatant was aspirated into another 1.5mL centrifuge tube, 0.6 volume times the volume of the supernatant pre-cooled isopropanol was added, mixed well, and precipitated at-20 ℃ for 30 min. Centrifuging at 10000rpm for 10min at 4 ℃, removing the supernatant, adding 1mL of 75% ethanol to wash and precipitate twice, centrifuging at 10000rpm for 5min at 4 ℃ each time, and removing the supernatant. Then washing with absolute ethyl alcohol, centrifuging, removing the supernatant, inverting the centrifuge tube, and drying the DNA at room temperature. To the dried DNA, 100. mu.L of TE buffer was added, and after the DNA was sufficiently dissolved, RNaseA was added in a ratio of 1/1000 to digest the RNA. The concentration and purity of the DNA sample were determined by a NanoDrop micro nucleic acid protein analyzer, and the integrity of the DNA was verified by electrophoresis on a 1% agarose gel.

And extracting total RNA of the common wheat variety, namely the water source 11 and the wheat variety Fielder by adopting an RNA extraction kit. The RNA extraction kit is purchased from PEXBIO company (cargo number: A010400), and the specific extraction operation steps and the condition requirements are carried out according to the product instruction.

(2) Reverse transcription of RNA

The total RNA from wheat leaves obtained in the above step (1) was subjected to reverse transcription using a reverse transcription kit (cat # RR047A) of Takara to obtain cDNA. The specific operation steps and conditions are carried out according to the product specification.

2. PCR amplification and sequencing of TaWLT14.2 gene DNA and cDNA sequence

And (2) performing PCR amplification by taking the DNA obtained in the step (1) as a template, using a primer pair WLTCDS-F and WLTCDS-R and taking the genomic DNA of the Proaquaponia 11 (the anti-rust puccinia striiformis strain CYR17) as a template.

And (3) PCR reaction system: DNA template 0.5. mu.L (< 0.5. mu.g), dNTP (2.5mM) 4. mu.l, WLTCDS-F (10. mu.M) 1. mu.l, WLTCDS-R (10. mu.M) 1. mu.l, 5 XStartFastFU Buffer 10. mu.l, TransStartFastFU DNA Polymerase 1. mu.l (2.5U), and finally made up to 50. mu.l with water. PCR reaction procedure: pre-denaturation at 95 ℃ for 2 min; then denaturation at 95 ℃ for 20s, annealing at 58 ℃ for 20s, and extension at 72 ℃ for 20s for 35 cycles; finally, extension is carried out for 5min at 72 ℃.

WLTCDS-F：5’-ATGGCAAAGTATCTCGCCGTCG-3’；

WLTCDS-R：5’-TCATGGGTGAGCAGGGATCCC-3’。

Separating and purifying the PCR amplification product by 1% agarose gel, and connecting to

-Blunt Zero Cloning Vector (Transgen CB501) Vector to obtain recombinant plasmid pEASY-T-TaWLT14.2, transform E.coli competent cells DH 5. alpha. and sequence a bacterial single clone with the universal primer M13F for one generation.

A large number of sequencing results show that a DNA sequence with the length of 240bp obtained by PCR amplification is a full-length sequence of 3 TaWLT14.2 genomes of common wheat, and through sequence analysis, the DNA sequence of the TaWLT14.2 genome does not contain an intron, and the sequences are respectively shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6.

And (2) performing PCR amplification by taking the cDNA obtained in the step (1) as a template, utilizing a primer pair WLTCDS-F and WLTCDS-R and taking the cDNA of the water source 11 (the anti-rust fungus physiological race CYR17) as a template.

WLTCDS-F：5’-ATGGCAAAGTATCTCGCCGTCG-3’；

WLTCDS-R：5’-TCATGGGTGAGCAGGGATCCC-3’。

And (3) PCR reaction system: cDNA template 0.5. mu.L, dNTP (2.5mM) 4. mu.L, WLTCDS-F (10. mu.M) 1. mu.L, WLTCDS-R (10. mu.M) 1. mu.L, 5 × TransStartFastFastBuffer 10. mu.L, TransStartFast (10. mu.M) 1. mu.L, QCbHLHR2 (10. mu.M) 1. mu.L, 5 × TransStartFastFastBuffer 10. mu.L, TransStartFastFastFastFu DNA Polymerase 1. mu.L (2.5U), and finally made up to 50. mu.L with water. PCR reaction procedure: pre-denaturation at 95 ℃ for 2 min; then denaturation at 95 ℃ for 20s, annealing at 58 ℃ for 20s, and extension at 72 ℃ for 20s, and 35 cycles; finally, extension is carried out for 5min at 72 ℃.

Separating and purifying the PCR amplification product by 1% agarose gel, and connecting to

-Blunt Zero Cloning Vector (Transgen CB501) Vector to obtain the recombinant plasmid pEASY-T-TaWLT14.2, transform E.coli competent cells DH 5. alpha. and sequence bacterial clones one generation with the universal primer M13F.

A large number of sequencing results show that a cDNA sequence with the length of 240bp obtained by PCR amplification is the full-length sequence of the coding region of 3 TaWLT14.2 genes of common wheat, and the sequences are respectively shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6.

The three different gene copies are named as TaWLT14.2-1A, TaWLT14.2-1B and TaWLT14.2-1D, and the full-length sequences of the gene coding regions of the three copies of the TaWLT14.2 gene are SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6. Correspondingly, the proteins coded by the TaWLT14.2 gene are named TaWLT14.2-1A, TaWLT14.2-1B and TaWLT14.2-1D proteins respectively. The amino acid sequence of the TaWLT14.2-1A protein is shown in SEQ ID No. 1; the amino acid sequence of TaWLT14.2-1B protein is shown in SEQ ID No. 2; the amino acid sequence of TaWLT14.2-1D protein is shown in SEQ ID No. 3.

Example 2 Down-Regulation of TaWLT14.2 Gene expression Using the BSMV-VIGS experiment

Obtaining of wheat with silent TaWLT14.2 gene

1. Construction of BSMV-VIGS vector system for inducing TaWLT14.2 gene to down-regulate

(1) According to the TaWLT14.2 gene sequence (SEQ ID No.6) in the embodiment 1, two silent fragments with different lengths are selected, one fragment has specific positions of +9bp to +219bp and is 211bp long, the silent fragment is named as a silent sequence TaWLT14.2-1, and the silent sequence TaWLT14.2-F1 and V-TaWLT14.2-R1 are subjected to PCR amplification by using primers to obtain the silent sequence TaWLT14.2-F1. The other fragment starts from +10bp, ends from +113bp, has the length of 104bp, is named as a silent sequence TaWLT14.2-2, and is obtained by performing PCR amplification on V-TaWLT14.2-F2 and V-TaWLT14.2-R2 by using primers.

V-TaWLT14.2-F1：5’-ATTGCTAGCGTATCTCGCCGTCGCGCT-3’；

V-TaWLT14.2-R1：5’-TAAGCTAGCAGCGGGCCGGTAATGGT-3’。

V-TaWLT14.2-F2：5’-ATTGCTAGCGTATCTCGCCGTCGCGCT-3’；

V-TaWLT14.2-R2：5’-TAAGCTAGCGGCACCGGCACCGCGTG-3’。

The underlined sequence is the restriction recognition site of the restriction enzyme NheI.

(2) The vector was constructed according to the conventional molecular biology methods described in "Yanziying, Wang Hailin translation" Fine molecular biology laboratory Manual ", 2001, science publishers". The specific operation steps are as follows.

Reversely inserting the silencing sequences TaWLT14.2-1 and TaWLT14.2-2 obtained in the step (1) into NheI enzyme cutting sites of the BSMV-VIGS virus vector gamma respectively, and keeping other sequences unchanged to obtain recombinant vectors gamma-TaWLT14.2-1 and gamma-TaWLT14.2-2.

Carrying out PCR amplification and sequencing identification on the recombinant vector gamma-TaWLT14.2-1 by using a primer pair V-TaWLT14.2-F1 and gamma-strain-p, wherein positive cloning is to insert the sequence of 9 th to 219 th nucleotides from the 5' end of SEQ ID No.6 into the NheI enzyme cutting site of the gamma chain of the BSMV-VIGS virus vector in the opposite direction of gene expression, and keeping the gene silencing vector gamma-TaWLT14.2-1 of the TaWLT14.2 with other sequences unchanged.

Carrying out PCR amplification and sequencing identification on the recombinant vector gamma-TaWLT14.2-2 by using a primer pair V-TaWLT14.2-F2 and gamma-strain-p, wherein positive cloning is to insert the 10 th-113 th nucleotide sequence of SEQ ID No.6 from the 5' end into the NheI enzyme cutting site of the gamma chain of the BSMV-VIGS virus vector in the opposite direction of gene expression, and keeping the gene silencing vector gamma-TaWLT14.2-2 of the TaWLT14.2 with other sequences unchanged.

γ-strain-p：5’-CAACTGCCAATCGTGAGTAGG-3’。

The BSMV-VIGS viral vectors alpha, beta and gamma-GFP vectors jointly form a viral vector system BSMV-GFP.

The BSMV-VIGS virus vectors alpha and beta and the recombinant vector gamma-TaWLT14.2-1 jointly form a virus silencing vector system BSMV, TaWLT14.2-1 capable of silencing TaWLT14.2 genes.

The BSMV-VIGS system vectors alpha and beta and the recombinant vector gamma-TaWLT14.2-2 jointly form a virus silencing vector system BSMV, TaWLT14.2-2 which can silence the TaWLT14.2 gene.

2. BSMV in vitro transcription

(1) MluI is used for enzyme digestion of BSMV virus vector alpha, gamma-GFP vector, recombinant vector gamma-TaWLT14.2-1 and recombinant vector gamma-TaWLT14.2-2, SpeI is used for enzyme digestion of BSMV virus vector beta, and linearized plasmids are obtained respectively.

(2) And (2) carrying out in vitro transcription by taking the linearized plasmid obtained in the step (1) as a template to respectively obtain in vitro transcribed BSMV viral vectors alpha, beta, gamma-GFP, gamma-TaWLT14.2-1 and gamma-TaWLT14.2-2. In vitro transcription reaction according to RiboMAX ^TM The Large Scale RNA Production System-T7 (product of Promega corporation, cat. No.: P1300) was used as a manual. The transcription reaction system and conditions are respectively as follows: the reaction was carried out in a total volume of 20.0. mu.l, including 6.5. mu.l of linearized plasmid (1.0. mu.g) and RNase-free water, 4.0. mu.l of 5 × Transcription Buffer, 1.5. mu.l of m7G (5') ppp (5') G (Promega Co., Ltd.; cat. P1718), 6.0. mu.l of rNTPPreMix, 2.0. mu.l of Enzyme Mix, and at 37 ℃ for 4 hours, and the transcript was stored at-70 ℃ for further use. Wherein rNTPPreMix is prepared from 15. mu.l of four reagents in the kit, namely dATP (100mM), 15. mu.l of dCTP (100mM), 15. mu.l of dUTP (100mM), 1.2. mu.l of dGTP (100mM) and 13.8. mu.l of ddH ₂ And O is mixed.

3. Inoculation of BSMV

Sowing wheat variety Shuihuan 11 in nutrient soil, after growing to a two-leaf period, respectively taking 8 mul of BSMV: TaWLT14.2-1 recombinant virus vector solution and BSMV: TaWLT14.2-2 recombinant virus vector solution to respectively coat and inoculate on a second flat leaf of the Shuihuan 11, spraying leaf surfaces with sterilized ultrapure water after 10min, covering preservative films for preserving moisture for 24h, and then switching to normal condition culture at 22 ℃ to respectively obtain transformed BSMV: TaWLT14.2-1 plants and BSMV: TaWLT14.2-2 plants. Meanwhile, part of the plants are inoculated with a BSMV-GFP recombinant virus vector solution to obtain transformed BSMV-GFP plants, and part of the plants are coated with 1 XGKP Buffer to obtain simulated inoculated plants.

The BSMV-TaWLT14.2-1 recombinant virus vector solution is obtained by diluting in vitro transcribed BSMV-VIGS vectors alpha, beta and gamma-TaWLT14.2-1 by 3 times with RNase-free water, mixing the diluted vectors in equal amount, and adding equal volume of 2 XGKP Buffer.

The BSMV, TaWLT14.2-2 recombinant virus vector solution is obtained by diluting in vitro transcribed BSMV-VIGS vectors alpha, beta and gamma-TaWLT14.2-2 by 3 times with RNase-free water, mixing in equal volume, and adding 2 XGKP Buffer in equal volume.

The BSMV-GFP recombinant virus vector solution is obtained by diluting in vitro transcribed BSMV-VIGS vectors alpha, beta and gamma-GFP by 3 times with RNase-free water, mixing in equal volume, and adding 2 XGKP Buffer in equal volume.

The plant which is transformed with the BSMV, TaWLT14.2-1 and the BSMV, TaWLT14.2-2 is a wheat plant which silences the TaWLT14.2 gene, the plant which is transformed with the BSMV, GFP is a negative control plant, and the simulated inoculated plant which is smeared with 1 XGKP Buffer is a blank control plant.

4. RT-PCR verification of wheat for silencing TaWLT14.2 gene

The specific detection method for the gene silencing effect of TaWLT14.2 in the BSMV-VIGS system comprises the following steps: culturing the transformed BSMV (TaWLT14.2-1) plant and the BSMV (TaWLT14.2-2) plant, the transformed BSMV (GFP) plant and the simulated inoculation plant (MOCK) obtained in the step 3) for 10 days under normal conditions, taking the third leaf to extract total RNA, detecting the relative expression quantity of the TaWLT14.2 gene by quantitative PCR after reverse transcription, setting TaEF-1 alpha as an internal reference gene, calculating the relative expression quantity by a delta CT method, and calculating by an ABI7500 type fluorescence quantitative PCR instrument with a calculation formula.

qTaWLT14.2-F：5’-TGAACCAGAAGGACCAGGCACT-3’；

qTaWLT14.2-R：5’-CGAGATGATCATGGGTGAGCAGGGAT-3’。

qTaEF-F：5’-TGGTGTCATCAAGCCTGGTATGGT-3’；

qTaEF-R：5’-ACTCATGGTGCATCTCAACGGACT-3’。

The primer pair QTaWLT14.2-F and QTaWLT14.2-R can simultaneously detect the relative expression amounts of three genes of TaWLT14.2-1A, TaWLT14.2-1B and TaWLT14.2-1D.

The results of detecting the relative expression level of TaWLT14.2 gene are shown in FIG. 1. As can be seen from the figure, the relative expression amount of TaWLT14.2 gene in the BSMV-transformed and BSMV-transformed plants TaWLT14.2-1 and TaWLT14.2-2 is greatly reduced compared with that in the negative control plant (BSMV: GFP) and the blank control plant (MOCK) compared with that in the BSMV-transformed and MOCK plants, which indicates that the two silent fragments TaWLT14.2-1 and TaWLT14.2-2 selected in the experiment are effective.

II, analyzing the stripe rust resistance of TaWLT14.2 gene-silenced plants

Inoculating a plant (BSMV: TaWLT14.2-1 and BSMV: TaWLT14.2-2) effectively silencing TaWLT14.2-2 gene expression, a negative control plant (BSMV: GFP) and a blank control plant (MOCK) with a rust stripe fungus strain CYR17 (collecting 0.1g of fresh wheat stripe rust fungus summer spores) after a third leaf is unfolded (about 14 days old), adding 50ml of clear water into a culture dish, adding 25 mu l of Tween 20, slightly stirring by using a glass rod, then introducing into a spray pot, uniformly shaking to prepare a spore suspension, uniformly spraying and inoculating the prepared spore suspension on the treated wheat leaves by using the spray pot, placing in a 22 ℃ culture room for stripe rust culture after keeping out of light and moisture for 24 hours), and observing the incidence condition of the third leaf after inoculating for 14 days.

The results are shown in FIG. 2. Leaves of MOCK-inoculated blank control plants (MOCK) did not have any symptoms, were grade 0 (immunized) and had individual necrotic spots. The negative control plant leaves of the BSMV-GFP-transferred negative control plant can see obvious virus anaphylactic reaction (HR) spots, but no spores are generated, and the reaction type is 0-0; grade, indicated as immune or near immune. And the plants transformed with TaWLT14.2-1 and TaWLT14.2-2 with obvious gene silencing effect have no obvious disease resistance reaction, a large amount of rust streak rust spore piles appear, the reaction type is grade 3-4, and the strain presents a high-sensitivity phenotype.

The above results show that the reduction of TaWLT14.2 gene expression level completely loses the resistance of the water source 11 to the stripe rust germ strain CYR17, and the resistance is reduced to high sense. Thus proving that TaWLT14.2 is an important gene participating in the reaction process of wheat stripe rust resistance.

Example 3 knocking out endogenous TaWLT14.2 Gene of marine origin 11 by using CRISPR/Cas9 Gene editing method

Firstly, obtaining TaWLT14.2 gene editing plant

1. TaWLT14.2 gene editing guide sequence primer design

To ensure the effectiveness of gene function silencing, the sequence of NGG (or CCN) PAM was searched against the sequence of tawlt 14.2. Then, according to the Bbs I enzyme cutting site sequence of the pTaU6 vector, together with PAM and the upstream (or downstream) 19-20nt base sequences thereof, sgRNA (single guide RNA) primers WLT14.2-sg-F and WLT14.2-sg-R are designed.

WLT14.2-sg-F：5’-CTTGAATGGTGCTGGTACCGGTAA-3’；

WLT14.2-sg-R：5’-AAACTTACCACGACCATGGCCATT-3’。

The recognition sites for AgeI/BshTI are underlined.

In order to carry out subsequent enzyme digestion detection, PCR amplification primers gWLT14.2-F and gWLT14.2-R spanning the sgRNA part are designed for detecting the sgRNA guide activity and mutation effect.

gWLT14.2-F：5’-AGGGAGCTGAACCAGAAGG-3’；

gWLT14.2-R：5’-AGAACTGCAAAGAAATACAGATGAC-3’。

In order to amplify three members of the TaWLT14.2 gene simultaneously, a reverse primer gWLT14.2-R is designed from the untranslated region part (UTR) of the 3'.

2. Vector construction

Primers WLT14.2-sg-F and WLT14.2-sg-R were treated with ddH ₂ O is diluted to 10. mu.M, then 9. mu.L of the mixture is taken out respectively, and 2. mu.LddH is added ₂ O, preparing a 20 mu L reaction system, annealing the oligonucleotide sequence on a common PCR instrument according to the program of 94 ℃, 5min, 90-10 ℃ and 10 ℃ reduction every 1min, and placing the annealing product on ice for later use. The pTaU6 vector was digested, examined and constructed with Bbs I restriction enzyme according to the following method: 5 μ L

buffer, 2. mu.g pTaU6 vector plasmid, 1. mu.L Bbs I (20U/. mu.L), plus ddH ₂ O to 50 mu L, mixing evenly, reacting for 30min at 37 ℃, and adding 10 mu L of 6 Xgel loading dye to terminate the reaction; using 1% agarose gelElectrophoresis detection and gel cutting purification; the annealed sgRNA WLT14.2 was ligated with the digested pTaU6 vector using T4 DNA ligase to construct a gene editing vector pTaU6-WLT 14.2-sgRNA.

3. sgRNA-guided Activity detection

The pTaU6-WLT14.2-sgRNA vector plasmid and pJIT163-2NLSCas9 plasmid were co-transformed into a protoplast of Nongnong 199 belonging to the family Triticum aestivum variety by the PEG method, and the protoplast of wheat transformed with pJIT163-GFP plasmid alone was used as a positive control and cultured at 23 ℃ for 48 hours. The status of the protoplasts was observed under a fluorescence microscope and the transformation efficiency was calculated by counting the number of protoplasts with a fluorescent signal. The protoplast is full and round, the transformation efficiency is more than 70%, and the subsequent detection experiment can be carried out.

Protoplasts cultured for 48 hours were collected by centrifugation, and genomic DNA was extracted using a plant genomic DNA extraction kit (product of TIANGEN, cat # DP 305). The gene fragment containing the mutation target region was then amplified with the detection primers gWLT14.2-F and gWLT14.2-R. mu.L of the PCR reaction product was taken, and then 0.5. mu.L (2.5units) of AgeI/BshTI endonuclease was added to the reaction product, followed by digestion at 37 ℃ for 1 hour. The digested product was detected by electrophoresis on a 2% agarose gel. After the TaWLT14.2 gene fragment obtained by PCR amplification by taking protoplast DNA transformed by pTaU6-WLT14.2-sgRNA vector plasmid as a template is cut by AgeI/BshTI, a band with the size consistent with that of a PCR product fragment (control) which is not cut by enzyme appears in an electrophoresis map, and the mutation of the corresponding enzyme cutting site is shown (figure 3). The mutation of TaWLT14.2 gene in wheat protoplast transformed by pTaU6-WLT14.2-sgRNA vector plasmid is proved, namely, the pTaU6-WLT14.2-sgRNA has guide activity.

4. Obtaining of TaWLT14.2 Gene-edited plants

And (3) co-transforming the immature embryo of the aquatic source 11 by using the gene editing vector plasmid pTaU6-WLT14.2-sgRNA constructed in the step 2 and pJIT163-2NLSCas9, and regenerating a plant by using the aquatic source 11 through callus induction and differentiation culture. The transformation process is completed by a genetic transformation platform of the institute of genetics and developmental biology of Chinese academy of sciences, and the public can commercially contact the platform to complete the transformation. According to the detection method in step 3, the target gene mutation detection is carried out on all regeneration plants by using primer pairs gWLT14.2-F and gWLT14.2-R (example 3, step one, step 3), the obtained T0 generation mutant strain is subjected to cold chamber vernalization at 4 ℃ for 4 weeks, and then is transplanted into nutrient soil for conventional culture. T0 generation plants were selfed to obtain seeds of T0 generation TaWLT14.2 gene edited plants.

Second, the stripe rust resistance identification of TaWLT14.2 gene editing plants

1. First generation sequencing detection of the editing type of TaWLT14.2 gene in TaWLT14.2 gene editing plants

The seeds of T1 generation obtained in the above step 1 were sown in 10cm square pots, T1 generation gene editing lines and control lines grown for about 7 days were cut off and the leaves were extracted according to the method of example one, and PCR amplification was carried out using the extracted gene editing DNA as a template and primers gWLT14.2-F and gWLT14.2-R (example 3, step 3), and the PCR amplification product was separated and purified by 1% agarose gel and ligated to square pots

-Blunt Zero Cloning Vector (Transgen CB501) Vector to obtain recombinant plasmid pEASY-T-gTaWLT14.2, transform E.coli competent cell DH5 alpha, and perform primary sequencing on bacterial clones with the universal primer M13F. Three T1-generation TaWLT14.2 gene editions represent the sequencing results of a single plant as shown in FIG. 4, three copies of the ABD gene of TaWLT14.2 of the three representative plants are edited, and the same copy has different editing types.

2. Stripe rust resistance detection of TaWLT14.2 gene editing plant

The original 11TaWLT14.2 gene editing plant which grows for about 10 days and the blank control line thereof are inoculated with the rust streak strain CYR17 (same as the second step in example 2), and the leaf phenotype is observed after 14 days. The results of the mass inoculation identification are shown in FIG. 5. The leaf of the control plant has almost no rust streak spores (the reactivity is 0-0; grade), and the leaf of the TaWLT14.2 gene editing T1 generation strain has a large amount of rust streak spores (the reactivity is 3-4 grade).

The results show that the mutation of the TaWLT14.2 gene allels of the genome of the marine organism 11 can obviously reduce the expression of the TaWLT14.2 gene, so that the marine organism 11 is converted from an anti-rust bacterial line CYR17 to an influenza rust bacterial line CYR 17. Therefore, the gene TaWLT14.2 of the wheat is proved to participate in the resistance process of the wheat to stripe rust, and has a utilization value for wheat resistance breeding.

Example 4 overexpression of the TaWLT14.2 Gene in Fielder Using Agrobacterium-mediated transgenic approach

Firstly, obtaining TaWLT14.2 gene over-expression plant

1. Primer design and vector construction of TaWLT14.2 gene overexpression vector

In order to verify the function of the TaWLT14.2 gene in the stripe rust resistance of wheat, the TaWLT14.2-D1 is constructed into a pLGY-02 vector by driving a maize ubiquitin promoter (UBi1) (a promoter carried by the pLGY-02). The overexpression vector primers OEWLT14.2-F and OEWLT14.2-R were designed.

OEWLT14.2-F：5’-tctagaggatccccgggtaccATGGCAAAGTATCTCGCCGTC-3’；

OEWLT14.2-R：5’-ttcgagctctctagaactagtTCATGGGTGAGCAGGGATCC-3’。

And (3) PCR reaction system: the sequence product DNA of SEQ ID No.6 obtained in example 1 was 0.5. mu.L (< 0.5. mu.g), dNTP (2.5mM) 4. mu.l, OEWLT14.2-F (10. mu.M) 1. mu.l, OEWLT14.2-R (10. mu.M) 1. mu.l, 5 XStartFastFU Buffer 10. mu.l, TransStartFastFU DNA Polymerase 1. mu.l (2.5U), and finally made up to 50. mu.l with water. PCR reaction procedure: pre-denaturation at 95 ℃ for 2 min; then denaturation at 95 ℃ for 20s, annealing at 58 ℃ for 20s, extension at 72 ℃ for 40s, and 35 cycles; finally, extension is carried out for 5min at 72 ℃. The amplification product OEWLT14.2(SEQ ID No.6) is placed on ice until use.

The pLGY-02 plasmid was previously linearized by restriction with KpnI and SpeI: 5 μ L

buffer, 2. mu.g of pLGY-02 vector plasmid, 1. mu.L KpnI and 1. mu.L of SpeI (20U/. mu.L), plus ddH ₂ O to 50 mu L, mixing evenly, reacting for 30min at 37 ℃, and adding 10 mu L of 6 Xgel loading dye to terminate the reaction; detecting by using 1% agarose gel electrophoresis, cutting and purifying; one-step recombinant kit using Novozam (Vazyme, cat # C115)Carrying out recombination connection: mu.L of the linearized pLGY-02 after enzyme digestion recovery and 4 mu.L of the full-length fragment OEWLT14.2 react for 5min at 50 ℃ to construct a gene over-expression vector pLGY-02-WLT 14.2.

The recombinant expression vector pLGY-02-WLT14.2 is sequenced to show that the structure is described as follows: the DNA fragment shown in SEQ ID No.6 is inserted between the restriction sites KpnI and SpeI of the pLGY-02 vector to obtain the recombinant plasmid.

2. Obtaining of TaWLT14.2 overexpression plants

The gene over-expression vector plasmid pLGY-02-WLT14.2 constructed in the step 1 is transformed into an agrobacterium strain EHA105 to infect the immature embryo of a common wheat variety Fielder. Wheat immature embryos are subjected to callus induction and differentiation culture, meanwhile, hygromycin is used for screening to obtain regenerated plants, positive plants of T0 generation are obtained through real-time fluorescent quantitative PCR identification (example 2, step one and step 4), 3 strains of OEWLT-T0-4, OEWLT-T0-6 and OEWLT-T0-10 have higher expression of TaWLT14.2 in the positive plants, and a negative plant OEWLT-T0-15(Null) is determined at the same time. These four lines were therefore used for subsequent study analysis. The transformation process is completed by biological engineering limited of Jinan nation, and the public can be commercially connected with the platform to complete the transformation. And transplanting the obtained T0 generation mutant strain into nutrient soil for conventional culture. T0 generation plants are selfed for 2 times to obtain seeds of T1 generation TaWLT14.2 gene over-expression plants.

II, identifying stripe rust resistance of TaWLT14.2 gene overexpression plants

1. Fluorescent quantitative PCR detection of TaWLT14.2 gene expression in TaWLT14.2 gene over-expression plants

The seeds of T1 generation obtained in the first step above were sown in 10cm square pots, respectively, and the overexpression lines and control lines of T2 generation, which were grown for about 7 days, were excised and subjected to RNA extraction, RNA reverse transcription, and fluorescent quantitative PCR according to the methods of examples 1 and 2, to detect the relative expression level of the tawlt14.2 gene (example 2, step one, step 4). The quantitative PCR detection result is shown in FIG. 6, compared with the blank control, the expression level of the TaWLT14.2 gene is obviously improved in T2 generation over-expression plants.

2. Stripe rust resistance detection of TaWLT14.2 gene overexpression plant

The Fielder wild type, tawlt14.2 gene over-expressed plants and Null lines thereof which grew for about 10 days were inoculated with the rust streak fungus line CYR33 (same as the second step of example 2), and leaf phenotype was observed after 14 days. The results of the mass inoculation identification are shown in FIG. 7. A large number of rust-like fungus spores appear on leaves of a control plant, a large number of necroses appear in a strain with the TaWLT14.2 gene over-expressing T2 generation, and only a small number of rust-like fungus spores appear (the reaction type is changed from high sensitivity to high resistance, the reaction type of different strains is 0; -2 grade).

The results show that the TaWLT14.2 gene is over-expressed in wheat, so that the wheat variety Fielder is converted from a rust-sensitive strain CYR33 to an anti-rust strain CYR 33. Therefore, the gene TaWLT14.2 of the wheat is proved to participate in the resistance process of the wheat to stripe rust, and has a utilization value for wheat resistance breeding.

<110> institute of genetics and developmental biology of the Chinese academy of sciences

<120> wheat stripe rust resistance associated protein TaWLT14.2, coding gene and application thereof

<130> GNCLN200095

<160> 6

<170> PatentIn version 3.5

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<213> Artificial sequence

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Met Ala Lys Tyr Leu Ala Val Ala Leu Leu Leu Leu Val Ala Leu Ala

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Tyr Cys Asp Gly Arg Glu Leu Asn Gln Lys Asp Gln Ala Leu Ala Thr

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Pro Arg Gly Ala Gly Ala Gly Gly Ala Val Asp Glu Ser Lys Val Leu

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Gly Leu Pro Asp Leu Pro Val Val Gly Thr Val Thr Gly Thr Ser Thr

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Ile Thr Gly Pro Leu Val Val Leu Pro Gly Ile Pro Ala His Pro

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Met Ala Lys Tyr Leu Ala Val Ala Leu Leu Leu Leu Val Ala Leu Ala

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Tyr Cys Asp Gly Arg Glu Leu Asn Gln Lys Asp Gln Ala Leu Pro Thr

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Ile Thr Gly Pro Leu Val Val Leu Pro Gly Ile Pro Ala His Pro

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Met Ala Lys Tyr Leu Ala Val Ala Leu Leu Val Leu Val Ala Leu Ala

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Ala Arg Gly Ala Gly Ala Gly Gly Ala Val Asp Glu Ser Lys Val Leu

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Gly Leu Pro Asp Leu Pro Val Leu Gly Thr Phe Thr Gly Thr Ser Thr

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Ile Thr Gly Pro Leu Val Val Leu Pro Gly Ile Pro Ala His Pro

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atggcaaagt atctcgccgt cgcgcttctg ctactggtgg cgctcgcgta ctgtgacggc 60

agggagctga accagaagga tcaggcacta gcaacgccac gcggtgccgg tgccggtggt 120

gccgtcgacg agtcgaaggt gctggggctg ccagacctgc cggtcgtcgg aaccgttacc 180

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atggcaaagt atctcgccgt cgcgcttctg ctgctggtgg cgctcgcgta ctgcgacggg 60

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gccgtcgacg agtccaaggc gctagggctg ccagacctgc cgctcgtcgg aaccgttacc 180

ggtaccagca ccattaccgg cccgctggtg gtgcttcctg ggatccctgc tcacccatga 240

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atggcaaagt atctcgccgt cgcgctcctc gtactggtgg cgctcgcgta ctgcgacggg 60

agggagctga accagaagga ccaggcactg gcaacggcac gcggtgccgg tgccggtggt 120

gccgtcgacg agtccaaggt gctggggctg ccagacctgc cggtcctggg aacctttacc 180

ggtaccagca ccattaccgg cccgctggtg gtgcttcccg ggatccctgc tcacccatga 240

Claims (14)

1. A protein, which is P1 or P2:

p1: TaWLT14.2-A1 protein, TaWLT14.2-B1 protein and TaWLT14.2-D1 protein;

p2: TaWLT14.2-D1 protein;

the TaWLT14.2-A1 protein is the protein shown in the following (A1) or (A2):

(A1) protein with an amino acid sequence of SEQ ID No. 1;

(A2) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in (A1);

the TaWLT14.2-B1 protein is a protein shown as (B1) or (B2):

(B1) a protein having an amino acid sequence of SEQ ID No. 2;

(B2) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in (B1);

the TaWLT14.2-D1 protein is a protein shown as (D1) or (D2):

(D1) a protein having an amino acid sequence of SEQ ID No. 3;

(D2) and (D1) attaching a tag to the N-terminus and/or C-terminus of the protein defined in (D1).

2. A nucleic acid molecule, being Q1 or Q2:

q1: nucleic acid molecules encoding a tawlt14.2-a1 protein as defined in claim 1, nucleic acid molecules encoding a tawlt14.2-B1 protein as defined in claim 1, and nucleic acid molecules encoding a tawlt14.2-D1 protein as defined in claim 1;

q2: a nucleic acid molecule encoding a tawlt14.2-D1 protein as claimed in claim 1.

3. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule for coding the TaWLT14.2-A1 protein is a DNA molecule shown in SEQ ID No. 4.

4. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule for coding the TaWLT14.2-B1 protein is a DNA molecule shown in SEQ ID No. 5.

5. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule for coding the TaWLT14.2-D1 protein is a DNA molecule shown in SEQ ID No. 6.

6. An expression cassette, recombinant vector or recombinant bacterium comprising the nucleic acid molecule of any one of claims 2 to 5.

7. Use of the protein of claim 1 or the nucleic acid molecule of any one of claims 2-5 or the expression cassette, recombinant vector or recombinant bacterium of claim 6 for modulating the resistance of a plant to stripe rust;

the plant is wheat.

8. Use according to claim 7, characterized in that: the expression level of the protein or the coding gene thereof in the plant is reduced, and the resistance of the plant to the stripe rust is reduced; the expression level of the protein or the coding gene thereof in the plant is increased, and the resistance of the plant to the stripe rust is increased.

9. Use according to claim 8, characterized in that: the increased resistance to stripe rust is embodied as: increasing the expression level of the protein in the recipient plant, thereby reducing the number of yellow rust spores on the plant;

the reduced resistance to stripe rust is reflected in: after the expression level of the protein in the recipient plant is reduced, the number of yellow rust spores on the plant is increased.

10. Use according to claim 9, characterized in that: the stripe rust is stripe rust caused by stripe rust fungus physiological race CYR17 and/or CYR 33;

the stripe rust germs are stripe rust germ physiological race CYR17 and/or CYR 33.

11. A method of breeding a plant variety with altered resistance to stripe rust by method I or method II:

the method I comprises the following steps: a method of breeding a plant variety having increased resistance to stripe rust comprising the step of increasing the expression level of the protein of claim 1 in a recipient plant;

method II: a method of breeding a plant variety with reduced resistance to stripe rust comprising the step of reducing the expression of the protein of claim 1 in a recipient plant;

the plant is wheat.

12. A method of breeding a transgenic plant with altered resistance to stripe rust, method III or method IV:

method III: a method of breeding a transgenic plant with increased resistance to stripe rust comprising the steps of: introducing into a recipient plant a nucleic acid molecule capable of expressing the protein of claim 1 to produce a transgenic plant; the transgenic plant has increased resistance to stripe rust as compared to the recipient plant;

method IV: a method of breeding a transgenic plant with reduced resistance to stripe rust comprising the steps of: inhibiting the expression of a nucleic acid molecule capable of expressing the protein of claim 1 in a recipient plant to produce a transgenic plant; the transgenic plant has reduced resistance to stripe rust as compared to the recipient plant;

the plant is wheat.

13. The method of claim 12, wherein: the increased resistance to stripe rust is embodied as: increasing the expression level of the protein in the recipient plant, thereby reducing the number of yellow rust spores on the plant;

the reduced resistance to stripe rust is reflected in: after the expression level of the protein in the recipient plant is reduced, the number of yellow rust spores on the plant is increased.

14. The method of claim 13, wherein: the stripe rust is stripe rust caused by stripe rust fungus physiological race CYR17 and/or CYR 33;

the stripe rust germs are stripe rust germ physiological race CYR17 and/or CYR 33.

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