WO2024174954A1 - Wheat broad-spectrum powdery-mildew-resistant gene wtk7-tm cloning, functional marker, and use thereof - Google Patents

Abstract

Wheat broad-spectrum powdery-mildew-resistant gene WTK7-TM cloning, a functional marker and the use thereof. Provided is the use of a WTK7-TM protein, or a substance for regulating the expression of an encoding gene thereof, or a substance for regulating the activity or content of the protein in regulating the powdery mildew resistance of a plant, wherein the WTK7-TM protein may be a protein having an amino acid sequence as shown in SEQ ID No: 3, and the plant may be wheat. Provided are gene localization, map-based cloning and a powdery-mildew-resistant biological function identification method for the wheat broad-spectrum powdery-mildew-resistant gene WTK7-TM. The wheat broad-spectrum powdery-mildew-resistant gene WTK7-TM can be widely applied to the fields such as wheat disease-resistant genetic breeding, germplasm resource improvement, transgenesis, and genome editing breeding, and has an important effect on modifying and improving germplasm resources of crops such as wheat.

Description

Cloning, functional labeling and application of wheat broad-spectrum powdery mildew resistance gene WTK7-TM

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese patent application No. 202310138434.7, filed on February 20, 2023, and the entire contents of that patent application are incorporated herein by reference.

Technical Field

The present application belongs to the field of crop molecular biology and molecular breeding, and specifically relates to the cloning, functional labeling and application of the wheat broad-spectrum powdery mildew resistance gene WTK7-TM.

Background Art

Wheat powdery mildew is a worldwide fungal disease caused by the obligate parasitic Blumeria graminis f.sp.tritici, which seriously harms the yield and quality of wheat. Due to changes in wheat production and farming systems, especially the increase in close planting, irrigation and nitrogen fertilizer use, the occurrence of powdery mildew has become more serious year by year. Production practice shows that compared with chemical control, exploring disease-resistant genes and cultivating disease-resistant varieties are the most economical, effective and safe measures to prevent and control wheat powdery mildew. However, due to the continuous emergence of new pathogenic species of pathogens, the disease resistance of varieties is often at risk of reduction or loss.

Wild emmer wheat (Triticum dicoccoides, 2n=4x=28, AABB) is the direct ancestor of modern cultivated tetraploid and hexaploid wheat. It originated in the Fertile Crescent region of the Middle East and is widely distributed in Israel, Syria, Lebanon, and Turkey. Wild emmer wheat has experienced long-term and complex environmental evolution, accumulated rich genetic diversity, and has good resistance to wheat powdery mildew, making it an important genetic resource for the genetic improvement of modern wheat disease resistance. To date, eight officially named powdery mildew resistance genes have been discovered from wild emmer wheat, such as Pm16, Pm26, Pm30, Pm36, Pm41, Pm42, Pm64 and Pm69, as well as several temporarily named genes, such as MlZec1, MlIW72, PmG16, Ml3D232, MlIW170, PmAs846, PmG3M, MlIW172, MlIW30 and MlIW39. However, only Pm41 and Pm69 genes have been successfully cloned, and many powdery mildew resistance genes from wild emmer wheat are urgently needed to be cloned.

At present, several wheat powdery mildew resistance genes have been successfully cloned from wheat and its wild species, such as Pm1, Pm2, Pm3, Pm4, Pm5, Pm8, Pm17, Pm21, Pm24, Pm38/Lr34/Yr18/Sr57, Pm41, Pm46/Lr67/Yr46/Sr55, Pm55, Pm60 and Pm69. Except for Pm24 (tandem kinase protein), Pm38/Lr34/Yr18/Sr57 (ABC transporter) and Pm46/Lr67/Yr46/Sr55 (sugar transporter) with broad-spectrum disease resistance, the remaining cloned powdery mildew resistance genes all encode typical NLR type disease resistance proteins. NLR-type disease resistance proteins are only resistant to a single or a few specific species of pathogens. After a few years of large-scale use in production, they are very likely to lose their disease resistance, causing a powdery mildew epidemic. Therefore, further discovering and utilizing new wheat broad-spectrum powdery mildew resistance genes has important theoretical significance and production application value.

Invention Disclosure

The technical problem to be solved by this application is: how to regulate wheat resistance to powdery mildew and/or how to screen, Identification of wheat with resistance to powdery mildew.

To solve the technical problem, the present application provides the use of a protein or a substance that regulates gene expression or a substance that regulates the activity or content of the protein in any of the following items, wherein the gene encodes the protein, and the protein may be a WTK7-TM protein; A1), use in regulating plant stress resistance; A2), use in preparing a product that regulates plant resistance; A3), use in regulating plant powdery mildew resistance; A4), use in preparing a product that regulates plant powdery mildew resistance; A5), use in plant breeding or plant-assisted breeding;

The WTK7-TM protein can be any of the following proteins: a1), a protein whose amino acid sequence is shown in SEQ ID No. 3; a2), a protein that has more than 80% identity with the amino acid sequence shown in a1) and is related to plant stress resistance, obtained by replacing and/or deleting and/or adding amino acid residues in the amino acid sequence shown in a1); a3), a fusion protein obtained by connecting a tag to the N-terminus or/and C-terminus of a1) or a2).

Furthermore, in the above application, the evaluation index of plant breeding includes the powdery mildew resistance of the plant.

Furthermore, in the above application, the purpose of plant breeding includes cultivating plants resistant to powdery mildew.

Furthermore, in the above application, the protein is derived from wheat.

In this application, SEQ ID No. 3 consists of 667 amino acid residues.

The above proteins can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

The protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag can be a Flag protein tag, a His protein tag, an MBP protein tag, an HA protein tag, a myc protein tag, a GST protein tag and/or a SUMO protein tag, etc.

Furthermore, the connection described in a3) can be via a peptide bond.

Furthermore, in the above application, the substance regulating gene expression or the substance regulating the activity or content of the protein is a biological material, and the biological material can be any of the following: B1), a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the above protein or the activity of the above protein; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5), a transgenic plant containing the nucleic acid molecule described in B1). B6), transgenic plant tissue containing the nucleic acid molecule described in B1), transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3); B7), transgenic plant organ containing the nucleic acid molecule described in B1), transgenic plant organ containing the expression cassette described in B2), or transgenic plant organ containing the recombinant vector described in B3); B8), nucleic acid molecule encoding the above protein; B9), expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line containing the nucleic acid molecule described in B8).

In the above-mentioned related biological materials, the expression cassette described in B2) refers to a cassette capable of expressing interference in the host cell. The DNA of the interfering RNA may include not only a promoter for initiating transcription of the interfering RNA gene, but also a terminator for terminating transcription of the interfering RNA gene.

Among the above-mentioned related biological materials, the recombinant microorganisms described in B4) can specifically be yeast, bacteria, algae and fungi.

Among the above-mentioned related biological materials, the plant tissue described in B6) can be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

Among the above-mentioned related biological materials, the transgenic plant organs described in B7) can be roots, stems, leaves, flowers, fruits and seeds of transgenic plants.

Among the above-mentioned related biological materials, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs may or may not include propagation materials.

In the above-mentioned related biological materials, the expression cassette described in B9) refers to a DNA capable of expressing WTK7-TM protein in a host cell, which DNA may include not only a promoter for initiating transcription of the WTK7-TM protein encoding gene, but also a terminator for terminating transcription of the WTK7-TM protein encoding gene.

Furthermore, B9) the expression cassette may further include an enhancer sequence.

Among the above-mentioned related biological materials, the recombinant vector described in B9) may contain the DNA molecule for encoding WTK7-TM protein shown in SEQ ID No.2.

Furthermore, in the above application, the nucleic acid molecule in B1) may be an RNA or DNA molecule that expresses the gene targeted in the above application.

Furthermore, in the above application, the nucleotide sequence of the target sequence of the nucleic acid molecule described in B1) is SEQ ID No. 4 and/or SEQ ID No. 5.

Furthermore, in the above application, the nucleic acid molecule described in B8) can be a DNA molecule described in any one of the following g1)-g3): g1), a DNA molecule whose coding sequence of the coding chain is SEQ ID No.2; g2), a DNA molecule whose nucleotide sequence of the coding chain is SEQ ID No.1; g3), a DNA molecule that has more than 80% identity with the DNA molecule described in g1) or g2), and regulates plant stress resistance.

Furthermore, in the above application, the plant is selected from monocotyledonous plants.

Furthermore, in the above application, the monocotyledonous plant is selected from the Poaceae family.

Furthermore, in the above application, the grass plant is selected from the genus Triticum.

Furthermore, in the above application, the wheat plant is selected from wheat (Triticum aestivum L.).

Furthermore, the above-mentioned wheat may be wild emmer wheat (Triticum dicoccoides L.).

Further, in the application, the application may specifically be any one of the following: A1') A method for improving plant stress resistance, comprising introducing the nucleic acid molecule described in B1) into a recipient plant to obtain a target plant, wherein the stress resistance of the target plant is higher than that of the recipient plant. A2') A method for preparing a plant with improved stress resistance, comprising introducing the nucleic acid molecule described in B1) into a recipient plant to obtain a target plant with improved stress resistance. A3') A method for improving plant powdery mildew resistance, comprising introducing the nucleic acid molecule described in B1) into a recipient plant to obtain a target plant, wherein the powdery mildew resistance of the target plant is higher than that of the recipient plant. A4') A method for preparing a plant with improved powdery mildew resistance, comprising introducing the nucleic acid molecule described in B1) into a recipient plant to obtain a target plant with improved powdery mildew resistance. A5') A method for plant breeding, comprising introducing the nucleic acid molecule described in B1) into a recipient plant Recipient plant, obtain the target plant.

In this application, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence (or a nucleotide sequence) can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for a pair of amino acid sequences to calculate the identity, then the value of the identity (%) can be obtained.

The above-mentioned 80% or more identity may be 80%, 85%, 90% or 95% or more identity.

The 80% or more identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The 85% or more identity may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The 90% or more identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The greater than 95% identity may be at least 95%, 96%, 97%, 98% or 99% identity.

In the present application, the substance that regulates the activity or content of the protein may be a substance that downregulates or reduces or knocks out the gene encoding the protein and/or a substance that downregulates or reduces the expression of the gene encoding the protein. The regulation of plant stress resistance may be reducing plant stress resistance.

In the present application, the substance that regulates gene expression may be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcription level of the gene; 2) regulation after transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated by the gene).

In the present application, the regulating gene expression may be inhibiting or reducing the gene expression, and the inhibiting or reducing the gene expression may be achieved by gene knockout or gene silencing.

The gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout is the inactivation of a specific target gene by changing the DNA sequence.

Gene silencing refers to the phenomenon of making a gene non-expressed or low-expressed without damaging the original DNA. Gene silencing is based on the premise of not changing the DNA sequence, making the gene non-expressed or low-expressed. Gene silencing can occur at two levels. One is gene silencing at the transcriptional level caused by DNA methylation, heterochromatinization, and position effects. The other is post-transcriptional gene silencing, that is, at the level after gene transcription, the gene is inactivated by specifically inhibiting the target RNA, including antisense RNA, co-suppression, gene repression (quelling), RNA interference (RNAi) and micro RNA (miRNA). mediated translation inhibition, etc.

In the above application, the substance regulating gene expression may be an agent that inhibits or reduces the expression of the gene. The agent that inhibits or reduces the expression of the gene may be an agent that knocks out the gene, such as an agent that knocks out the gene by homologous recombination, or an agent that knocks out the gene by CRISPR-Cas9. The agent that inhibits or reduces the expression of the gene may include a polynucleotide that targets the gene, such as siRNA, shRNA, double-stranded RNA, miRNA or antisense RNA.

The present application also provides a method for cultivating plants with reduced powdery mildew resistance, the method comprising down-regulating or reducing the expression of the WTK7-TM protein in a recipient plant or down-regulating or reducing the activity or content of the WTK7-TM protein to obtain a target plant with reduced powdery mildew resistance.

Furthermore, the recipient plant contains the coding gene of the WTK7-TM protein or the WTK7-TM protein.

The present application also provides a method for regulating plant powdery mildew resistance, which comprises regulating plant powdery mildew resistance by regulating the expression of the encoding gene in a plant containing the above-mentioned WTK7-TM protein or by regulating the activity or content of the WTK7-TM protein in a plant containing the above-mentioned WTK7-TM protein.

In the method, the regulation may be down-regulation or reduction.

In the above-mentioned method for cultivating plants with reduced powdery mildew resistance and the method for regulating plant powdery mildew resistance, the regulation of the expression of the encoding gene in a plant containing the encoding gene of the WTK7-TM protein or the regulation of the activity or content of the WTK7-TM protein in a plant containing the WTK7-TM protein can be achieved by silencing the WTK7-TM gene in the receptor plant.

The WTK7-TM gene in the silencing recipient plant can be achieved by virus-induced gene silencing.

The virus-induced gene silencing includes introducing a recombinant virus targeting the WTK7-TM gene into the recipient plant, and the silencing target of the recombinant virus is SEQ ID No. 4 and/or SEQ ID No. 5.

In some embodiments of the present application, the recombinant virus is a recombinant barley streak mosaic virus (BSMV). The preparation method of the recombinant barley streak mosaic virus is:

The three plasmids were transfected into Agrobacterium tumefaciens EHA105 to prepare recombinant Agrobacterium tumefaciens bacterial solution, as follows:

Preparation of recombinant Agrobacterium tumefaciens EHA105/pCaBS-α: pCaBS-α was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain recombinant Agrobacterium tumefaciens EHA105/pCaBS-α.

Preparation of recombinant Agrobacterium tumefaciens EHA105/pCaBS-β: pCaBS-β was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain recombinant Agrobacterium tumefaciens EHA105/pCaBS-β.

Preparation of recombinant Agrobacterium tumefaciens EHA105/pCaBS-γbLIC: pCaBS-γbLIC was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain recombinant Agrobacterium tumefaciens EHA105/pCaBS-γbLIC.

Preparation of recombinant Agrobacterium tumefaciens EHA105/pCaBS-γbLIC-WTK7-TM ^KinⅠ : pCaBS-γbLIC-WTK7-TM ^KinⅠ was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain recombinant Agrobacterium tumefaciens EHA105/pCaBS-γbLIC-WTK7-TM ^KinⅠ .

Preparation of recombinant Agrobacterium tumefaciens EHA105/pCaBS-γbLIC-WTK7-TM ^KinⅡ : pCaBS-γbLIC-WTK7-TM ^KinⅡ was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain recombinant Agrobacterium tumefaciens Agrobacterium EHA105/pCaBS-γbLIC-WTK7-TM ^KinⅡ .

After activation treatment for 36-48h, recombinant Agrobacterium tumefaciens EHA105/pCaBS-α, recombinant Agrobacterium tumefaciens EHA105/pCaBS-β, recombinant Agrobacterium tumefaciens EHA105/pCaBS-γbLIC, recombinant Agrobacterium tumefaciens EHA105/pCaBS-γbLIC-WTK7-TM ^KinⅠ , and recombinant Agrobacterium tumefaciens EHA105/pCaBS-γbLIC-WTK7-TM ^KinⅡ were picked and inoculated into 1mL LB liquid medium, and cultured at 28℃, 220rpm shaking for 24h. Inoculate into 10mL LB liquid medium at a ratio of 1:100, and culture at 28℃, 200pm shaking for 12h to obtain bacterial liquid. The bacterial liquid was centrifuged at 6000 rpm for 5 min to collect the bacterial cells, and the bacterial cells were resuspended in an equal volume of tobacco infection liquid (10 mM MgCl ₂ , 10 mM MES, pH=5.2, 0.1 Mm AS) to obtain infection bacterial liquid.

Taking the recombinant virus BSMV:WTK7-TM ^KinⅡ as an example, the infection solution of EHA105/pCaBS-α, EHA105/pCaBS-β, EHA105/pCaBS-γbLIC-WTK7-TM ^KinⅡ was mixed in a ratio of 1:1:1, and then allowed to stand at 28°C for 3-5 hours before being injected into the unfolded leaves of Nicotiana benthamiana at the 6-8 leaf stage, marked as BSMV:WTK7-TM ^KinⅡ . 7-12 days after the injection, the injected Nicotiana benthamiana leaves and the first leaf on the upper part were collected, and the juice containing the recombinant virus BSMV:WTK7-TM ^KinⅡ was obtained by grinding them thoroughly in PBS (pH=7.2) buffer.

The preparation methods of recombinant virus BSMV:WTK7-TM ^KinⅠ and control group recombinant virus BSMV refer to the recombinant virus BSMV:WTK7-TM ^KinⅡ , with the only difference that the EHA105/pCaBS-γbLIC-WTK7-TM ^KinⅡ infection solution is replaced by EHA105/pCaBS-γbLIC-WTK7-TM ^KinⅠ infection solution and EHA105/pCaBS-γbLIC infection solution, respectively.

Furthermore, in the above-mentioned method for cultivating plants with reduced powdery mildew resistance and the method for regulating powdery mildew resistance of plants, the plants are selected from monocotyledons.

Furthermore, the monocotyledonous plant is selected from the Poaceae family.

Further, the grass plant is selected from the genus Triticum;

Furthermore, the Triticum plant is selected from wheat (Triticum aestivum L.).

In one embodiment of the present application, the expression of WTK7-TM gene in wheat is downregulated or reduced by virus-induced gene silencing, thereby reducing the powdery mildew resistance of wheat. This embodiment demonstrates the role of WTK7-TM gene or its encoded protein in regulating wheat powdery mildew resistance.

Specifically, in an embodiment of the present application, the wheat may be wheat line 3D232.

The present application also provides the protein in the above application and/or the biological material in the above application.

The present application also provides the use of WTK7-TM molecular markers and/or substances for detecting WTK7-TM molecular markers in any of the following:

D1), identification or auxiliary identification of wheat powdery mildew resistance;

D2), screening or assisting in screening wheat plants, strains, lines or varieties with resistance to powdery mildew;

D3), wheat assisted breeding;

The WTK7-TM molecular marker is a fragment in the wheat genome, and its nucleotide sequence is SEQ ID No.6;

The substance for detecting the WTK7-TM molecular marker may be any of the following:

E1), a PCR primer composition for amplifying a wheat genomic DNA fragment including the WTK7-TM molecular marker;

E2), a PCR reagent containing the PCR primer combination described in E1);

E3) A kit containing the PCR primer composition described in E1) or the PCR reagent described in E2).

Furthermore, the application may specifically be any of the following:

D1’), using the WTK7-TM molecular marker and substances that detect the WTK7-TM molecular marker to identify or assist in identifying wheat powdery mildew resistance.

D2'), using the WTK7-TM molecular marker and substances that detect the WTK7-TM molecular marker to screen or assist in screening wheat plants, lines, strains or varieties with resistance to powdery mildew.

D3’), using wheat containing the WTK7-TM molecular marker for wheat breeding or wheat assisted breeding.

Furthermore, in the application, the PCR primer composition includes a single-stranded DNA whose nucleotide sequence is SEQ ID No.7 and a single-stranded DNA whose nucleotide sequence is SEQ ID No.8.

The present application also provides a DNA molecule whose nucleotide sequence is SEQ ID No. 6 or/and the above-mentioned primer composition or/and the above-mentioned reagent or/and the above-mentioned kit.

The present application also provides a method for identifying or assisting in identifying the powdery mildew resistance trait of wheat, the method comprising detecting the WTK7-TM molecular marker in the wheat to be tested, and identifying or assisting in identifying the powdery mildew resistance of wheat based on whether the wheat to be tested contains the WTK7-TM molecular marker, the powdery mildew resistance of the wheat to be tested containing the WTK7-TM molecular marker being higher or having a potential to be higher than the powdery mildew resistance of the wheat to be tested that does not contain the WTK7-TM molecular marker.

Wherein, the nucleotide sequence of the WTK7-TM molecular marker is SEQ ID No. 6. SEQ ID No. 6 consists of 1198 nucleotides.

The wheat to be tested that contains the WTK7-TM molecular marker can use the above primer combination to amplify the DNA fragment of the WTK7-TM molecular marker, while the wheat to be tested that does not contain the WTK7-TM molecular marker cannot use the above primer combination to amplify the DNA fragment of the WTK7-TM molecular marker.

Furthermore, in the method described above, the method for detecting the WTK7-TM molecular marker in the wheat to be tested includes using the genomic DNA of the wheat to be tested as a template, amplifying using the above-mentioned PCR primer combination to obtain a PCR product, and performing agarose gel electrophoresis or sequencing on the PCR product.

The present application also provides a method for wheat breeding, which comprises selecting wheat containing the WTK7-TM molecular marker as a parent for breeding.

Furthermore, in the above method, the breeding target of the breeding includes wheat powdery mildew resistance. The purpose of the breeding includes cultivating wheat resistant to powdery mildew ((wheat powdery mildew resistance is higher than that of the parent)).

In the present application, the powdery mildew may be a fungal disease caused by the obligate parasitic Blumeria graminis f.sp.tritici (commonly referred to as powdery mildew). The disease resistance or powdery mildew resistance described in the present application may be resistance to diseases caused by Blumeria graminis f.sp.tritici.

In the embodiments of the present application, the powdery mildew described in the powdery mildew resistance experiment may specifically be powdery mildew physiological race E09.

The beneficial technical effects achieved by this application are as follows:

The present application provides a method for identifying the gene localization, map-based cloning and biological function of wheat broad-spectrum powdery mildew resistance gene WTK7-TM. The wheat broad-spectrum powdery mildew resistance gene WTK7-TM can be widely used in plant fields such as wheat disease resistance genetic breeding, germplasm resource improvement, transgenic and genome editing breeding, It plays an important role in improving and modifying the germplasm resources of crops such as wheat.

The functional marker WTK-TM-FM provided in the present application and the primer composition for amplification thereof can be used for auxiliary screening or identification of powdery mildew-resistant wheat and wheat breeding.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the identification of multiple races of powdery mildew fungus in common wheat line 3D232.

Figure 2 shows the positional cloning of the wheat powdery mildew resistance gene WTK7-TM.

Figure 3 shows the gene function verification of CYB561-Domon and TM9SF4 transgenes.

FIG. 4 shows the splicing pattern of WTK7-TM gene.

Figure 5 shows the verification of WTK7-TM's powdery mildew resistance function by BSMV-VIGS and EMS mutants.

FIG6 is a flow chart of the construction of a silencing vector.

FIG. 7 shows the development and application of WTK7-TM gene functional markers.

Best Mode for Carrying Out the Invention

The present application is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present application, not for limiting the scope of the present application. The examples provided below can be used as a guide for further improvements by ordinary technicians in the technical field, and do not constitute a limitation of the present application in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

The quantitative tests in the following examples were repeated three times unless otherwise specified, and the results were averaged.

The common wheat line 3D232 and common wheat Xue Zao described in the following examples are preserved in this laboratory. In the literature "Zhang, H. et al. Genetic and comparative genomics mapping reveals that a powdery mildew resistance gene Ml3D232 originating from wild emmer co-segregates with an NBS-LRR analog in common wheat (Triticum aestivum L.). Theor. Appl. Genet. 121, 1613-1621 (2010), the public can obtain the above biological materials from the applicant, and the obtained biological materials are only used for repeating the experiments of this application and cannot be used for other purposes.

The wheat line 5BIL-29 described in the following examples was kindly donated by Dr. Antonio Blanco of the University of Bari, Italy. According to the document "Blanco, A. et al. Molecular mapping of the novel powdery mildew resistance gene Pm36 introduced from Triticum turgidum var. dicoccoides in durum wheat. Theor. Appl. Genet. 117, 135-142 (2008)", the public can obtain the above biological materials from the applicant. The obtained biological materials are only used for repeating the experiments of this application and cannot be used for other purposes.

The powdery mildew strain E09 described in the following examples is preserved by this laboratory and is described in the document "Li, M. et al. A CNL protein in wild emmer wheat confers powdery mildew resistance. New Phytol. 228, 1027-1037 (2020). The public can obtain the above-mentioned biological materials from the applicant. The obtained biological materials are only used to repeat the experiments of this application and cannot be used for other purposes.

The gene silencing vectors pCaBS-α, pCaBS-β and pCaBS-γbLIC described in the following examples were kindly donated by Professor Li Dawei of China Agricultural University. Stripe mosaic virus vector for virus induced gene silencing in monocots and dicots. PLoS One 6, e26468 (2011). "The public can obtain the above biological materials from the applicant. The obtained biological materials are only used for repeating the experiments of this application and cannot be used for other purposes.

In the following examples, reagent AS represents acetosyringone.

In the following examples, MES represents 2-Morpholinoethanesulphonic acid, which is a zwitterionic buffer.

The following examples used SPSS11.5 statistical software to process the data, and the experimental results were expressed as mean ± standard deviation. One-way ANOVA test was used, * indicates a significant difference (P < 0.05), ** indicates an extremely significant difference (P < 0.01), and *** indicates an extremely significant difference (P < 0.001).

Table 1 Primer sequences of this study

Example 1: Powdery mildew resistance identification of common wheat 3D232

The common wheat line 3D232 is a hybrid of wild emmer wheat I222 (kindly donated by Dr. Gerechter-Amitai of the Volcani Center, Agricultural Research Organization, Israel) and common wheat line 87-1, and then backcrossed with common wheat Jing 411. The pedigree is 87-1/I222//Jing 411*7. 104 powdery mildew species collected from different ecological zones across the country were inoculated in vitro on leaves of 3D232 at the seedling stage. It was found that it was immune or highly resistant to all powdery mildew species, and is an excellent broad-spectrum powdery mildew resistant material (Figure 1).

Example 2: Fine localization of wheat broad-spectrum powdery mildew resistance gene M13D232

The seedling resistance of _F1 , _F2 segregating populations and _F3 families of common wheat lines 3D232, Xuezao and Xuezao×3D232 was identified using powdery mildew race E09, which is prevalent in Beijing. The identification results showed that 3D232 showed an immune response type (IT=0) and Xuezao showed a highly susceptible response type (IT=4). The _F1 generation of Xuezao/3D232 showed an immune response type (IT=0), which was consistent with the disease-resistant parent. The identification of 630 _F2 segregating populations of Xuezao/3D232 showed that 477 of them showed resistance and 153 showed susceptible. The chi-square test was consistent with the 3:1 segregation ratio controlled by a single gene. Among the 630 _F3 families of Xuezao/3D232, 157 families showed homozygous resistance, 320 families showed resistance-susceptibility segregation, and 153 families showed homozygous susceptibility. The chi-square test showed that the ratio was consistent with the 1:2:1 segregation ratio controlled by a single gene (Table 2). The above genetic analysis showed that the powdery mildew resistance of common wheat 3D232 was controlled by a dominant single gene, temporarily named Ml3D232.

Previously, the wheat powdery mildew resistance gene M13D232 was located in the 0.14 cM genetic interval between the near-terminal molecular markers XBD37670 and XBD37760 on chromosome 5BL by developing molecular markers (D Zhang, S H Ouyang, L L Wang, Y Cui, Q H Wu, Y Liang, Z Z Wang, J Z Xie, D Y Zhang, Y Wang, Y X Chen, Z Y Liu. Comparative genetic mapping revealed powdery mildew resistance gene MIWE4 derived from wild emmer is located in the same genomic region of Pm36 and MI3D232 on chromosome 5BL. Journal of Integrative Agriculture, 4 (2015), pp. 603-609). In order to further fine-tune the MI3D232 gene, we used Xue Zao and 3D232 hybrids to construct a genetic segregation population containing 15,893 _F2 individual plants. The genotypes of the _F2 genetic segregation population were identified using the molecular markers XBD37670 and XBD37760, which were closest to the MI3D232 gene localization interval, and the key recombinant exchange plants were screened. At the seedling stage, the powdery mildew resistance of the F2 _:3 family of the key recombinant exchange plants screened by the molecular markers XBD37670 and XBD37760 was identified using the powdery mildew physiological race E09. At the same time, based on the reference genome sequences of Chinese spring and wild emmer wheat Zavitan, new molecular markers that were closely linked and co-segregated with the MI3D232 gene were developed between molecular markers XBD37670 and XBD37760. A total of 9 molecular markers, WGGBM1-WGGBM9, were developed that were closely linked to MI3D232 (Table 1). These newly developed molecular markers WGGBM1-WGGBM9 were used to identify the genotypes of the F _2:3 families of key recombinant exchange plants selected by molecular markers XBD37670 and XBD37760, thereby finely positioning the MI3D232 gene. Finally, the MI3D232 gene was finely positioned in the genetic interval of 0.021 cM between molecular markers WGGBM2 and WGGBM7, among which molecular markers WGGBM3-WGGBM6 were co-segregated with the MI3D232 gene (Figure 2).

Previous studies have found that the MI3D232 gene is the same gene or allele as the officially named wheat powdery mildew resistance gene Pm36 (D Zhang, S H Ouyang, L L Wang, Y Cui, Q H Wu, Y Liang, Z Z Wang, J Z Xie, D Y Zhang, Y Wang, Y X Chen, Z Y Liu. Comparative genetic mapping revealed powdery mildew resistance gene MIWE4 derived from wild emmer is located in the same genomic region of Pm36 and MI3D232 on chromosome 5BL. Journal of Integrative Agriculture, 4 (2015), pp. 603-609). In order to clone the MI3D232 gene, we used the PacBio SMRT long-read genome sequencing strategy to resequence the genome of durum wheat material 5BIL-29 (kindly donated by Dr. Antonio Blanco, University of Bari, Italy) carrying the Pm36/MI3D232 gene. After quality control, a total of 172.53Gb HiFi reads were obtained, with a coverage of about 17× genome. Hifiasm 0.16.1-r375 software was used for data assembly, and a total of 15,347 contigs were obtained, of which N50 length was 3.2Mb, N90 length was 0.69Mb, the longest contig length was 25Mb, and the total genome length was about 10.7Gb. One of the contigs, utg0040681 (1.69Mb), can completely cover the Ml3D232 localization interval, of which the physical distance between the nearest molecular markers WGGBM2 and WGGBM7 on both sides of the Ml3D232 gene localization interval is about 1.17Mb, while the physical interval of the reference genome of the Chinese Spring is about 275Kb, indicating that there is a large genomic structural variation in this interval in different species. By annotating the 1.17Mb contig, a total of 11 genes were obtained, including CYB561-Domon, three F-boxes, five Serpins, TM9SF4, and WTK7-TM (Figure 2 and Table 3). RNA-seq analysis showed that CYB561-Domon, TM9SF4, and WTK7-TM were expressed in the leaves of 3D232 after inoculation with powdery mildew E09, while no expression of other genes was detected. We used Agrobacterium-mediated stable genetic transformation of CYB561-Domon and TM9SF4 genes in highly susceptible powdery mildew wheat Fielder to obtain transgenic plants. By transfecting CYB561-Domon and TM9SF4 at the seedling stage The powdery mildew resistance of the _T2 transgenic positive plants of the two genes was identified, and the results showed that the transgenic plants of the two genes were highly susceptible to powdery mildew (Figure 3), indicating that the CYB561-Domon and TM9SF4 genes do not have the function of powdery mildew resistance. Therefore, the WTK7-TM gene is the most likely candidate gene for the Ml3D232 gene.

By designing specific primers to amplify and sequence the WTK7-TM gene, it was found that its gene length was 3277bp. Further RACE experiments revealed that the WTK7-TM gene contains 9 different splicing forms (TV1-TV9). Among them, TV1 is the main splicing mode, with a gene coding region length of 2004bp, which can encode 667 amino acids (Figure 4). By analyzing the WTK7-TM protein, it was found that it contains two tandem kinases and a C-terminal transmembrane domain (Figure 2). Sequence analysis showed that the WTK7-TM gene is exactly the same in common wheat 3D232 and 5BIL-29, but is missing in the susceptible parents Xue Zao and Chinese Spring.

Specifically, the wheat broad-spectrum powdery mildew resistance gene WTK7-TM described in the present application is located on the wheat chromosome 5BL, the nucleotide sequence of its genomic sequence is SEQ ID No. 1, the nucleotide sequence of the cDNA sequence (coding sequence) is SEQ ID No. 2, and the amino acid sequence of the encoded WTK7-TM protein is SEQ ID No. 3.

Table 2 Genetic analysis of wheat powdery mildew resistance gene M13D232

Table 3 Candidate genes in the Ml3D232 localization interval

Example 3: Silencing of WTK7-TM gene by BSMV-VIGS technology

In order to verify whether WTK7-TM has the function of powdery mildew resistance, the BSMV-VIGS technology reported previously for silencing endogenous genes in wheat (Yuan C, Li C, Yan L, et al. A high throughput barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots. PLoS One. 2011; 6(10): e26468) was used to silence the WTK7-TM gene in 3D232.

The gene silencing vectors were pCaBS-α, pCaBS-β, and pCaBS-γbLIC, which were provided by Li Da from China Agricultural University. The plasmid containing the full-length WTK7-TM gene was amplified by VIGS-1F/VIGS-1R and VIGS-2F/VIGS-2R (Table 1) to obtain two target fragments for constructing the silencing vector. The target fragments amplified by VIGS-1F/VIGS-1R and VIGS-2F/VIGS-2R primers (containing silencing fragments with nucleotide sequences of SEQ ID No.4 and SEQ ID No.5, respectively) correspond to the kinase 1 (KinⅠ) and kinase 2 (KinⅡ) domains of the WTK7-TM gene, respectively. At the same time, the pCaBS-γbLIC vector was digested with restriction endonuclease ApaⅠ and the linearized product was recovered. Then, the two were connected by the LIC site connection principle to construct pCaBS-γbLIC-WTK7-TM ^KinⅠ and pCaBS-γbLIC-WTK7-TM ^KinⅡ , respectively. The specific operation is shown in Figure 6.

The sequencing results showed that the structure of pCaBS-γbLIC-WTK7-TM ^KinⅠ was a recombinant vector obtained by replacing the fragment between LIC1 and LIC2 on pCaBS-γbLIC with a DNA molecule having a nucleotide sequence of SEQ ID No.4, while keeping the other nucleotides of pCaBS-γbLIC unchanged. The structure of pCaBS-γbLIC-WTK7-TM ^KinⅡ was a recombinant vector obtained by replacing the fragment between LIC1 and LIC2 on pCaBS-γbLIC with a DNA molecule having a nucleotide sequence of SEQ ID No.5, while keeping the other nucleotides of pCaBS-γbLIC unchanged.

Preparation of recombinant Agrobacterium tumefaciens EHA105/pCaBS-α: pCaBS-α was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain recombinant Agrobacterium tumefaciens EHA105/pCaBS-α.

Preparation of recombinant Agrobacterium tumefaciens EHA105/pCaBS-β: pCaBS-β was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain recombinant Agrobacterium tumefaciens EHA105/pCaBS-β.

Preparation of recombinant Agrobacterium tumefaciens EHA105/pCaBS-γbLIC: pCaBS-γbLIC was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain recombinant Agrobacterium tumefaciens EHA105/pCaBS-γbLIC.

Preparation of recombinant Agrobacterium tumefaciens EHA105/pCaBS-γbLIC-WTK7-TM ^KinⅠ : pCaBS-γbLIC-WTK7-TM ^KinⅠ was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain recombinant Agrobacterium tumefaciens EHA105/pCaBS-γbLIC-WTK7-TM ^KinⅠ .

Preparation of recombinant Agrobacterium tumefaciens EHA105/pCaBS-γbLIC-WTK7-TM ^KinⅡ : pCaBS-γbLIC-WTK7-TM ^KinⅡ was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain recombinant Agrobacterium tumefaciens EHA105/pCaBS-γbLIC-WTK7-TM ^KinⅡ .

The procedure for BSMV-VIGS-induced WTK7-TM gene silencing is as follows:

1. After sowing, place the tobacco plants in a culture room at 20-22℃, with 16h light and 8h dark. When the tobacco plants grow to 6-8 leaves, they will be used for BSMV-VIGS experiments.

2. After activating the recombinant Agrobacterium tumefaciens EHA105/pCaBS-α, recombinant Agrobacterium tumefaciens EHA105/pCaBS-β, recombinant Agrobacterium tumefaciens EHA105/pCaBS-γbLIC, recombinant Agrobacterium tumefaciens EHA105/pCaBS-γbLIC-WTK7-TM ^KinⅠ and recombinant Agrobacterium tumefaciens EHA105/pCaBS-γbLIC-WTK7-TM ^KinⅡ for 36-48 hours, pick a single clone and inoculate it into 1 mL LB liquid medium (Kan+Rif), and culture it at 28°C and 220 rpm in a shaking incubator for 24 hours to obtain seed solution;

3. Inoculate into 10 mL LB liquid medium (Kan+Rif) at a ratio of 1:100, containing 20 μM AS and 100 μM MES, and culture at 28°C, 200 pm shaking for 12 h to obtain bacterial liquid;

4. Centrifuge the bacterial solution at 6000 rpm for 5 min to collect the bacterial cells, and resuspend the bacterial cells with an equal volume of tobacco infection solution (10 mM MgCl ₂ , 10 mM MES, pH=5.2, 0.1 Mm AS) to obtain the infection solution;

5. After adjusting the concentration of the infection solution to OD ₆₀₀ = 0.7, the cells were divided into three groups according to the different infection plasmids:

Group 1: EHA105/pCaBS-α, EHA105/pCaBS-β, and EHA105/pCaBS-γbLIC infection solutions were mixed at a ratio of 1:1:1, allowed to stand at 28°C for 3-5 hours, and then injected into the expanded leaves of Nicotiana benthamiana at the 6-8 leaf stage, marked as BSMV:00;

Group 2: EHA105/pCaBS-α, EHA105/pCaBS-β, and EHA105/pCaBS-γbLIC-WTK7-TM KinⅠ were mixed in a ratio of 1:1:1, and allowed to stand at 28°C for 3-5 hours before being injected into the unfolded leaves of Nicotiana benthamiana at the 6-8 leaf stage, and were labeled as BSMV:WTK7-TM ^{KinⅠ ;}

Group 3: EHA105/pCaBS-α, EHA105/pCaBS-β, and EHA105/pCaBS-γbLIC-WTK7-TM KinⅡ were mixed in a ratio of 1:1:1, and allowed to stand at 28°C for 3-5 hours before being injected into the unfolded leaves of Nicotiana benthamiana at the 6-8 leaf stage, and were labeled as BSMV:WTK7-TM ^{KinⅡ .}

6. 7-12 days after injection, collect the injected Nicotiana benthamiana leaves and the first leaf on top of them in groups, grind them thoroughly in PBS (pH=7.2) buffer to obtain juice, and inoculate the juice into the first leaf of 3D232 and 5BIL-29 wheat plants respectively by friction according to the grouping in step 5. The experiment was repeated 3 times, with 5 wheat plants/group in each repeat;

7. After 3 days of inoculation, observe the symptoms of virus expansion on wheat leaves, and inoculate powdery mildew E09 to identify the disease resistance of wheat seedlings to powdery mildew. The specific operation of identifying the resistance to powdery mildew in wheat seedlings is: first, use Xue Zao with high sensitivity to powdery mildew physiological race E09 as the breeding host of powdery mildew to breed powdery mildew. Place Xue Zao fully infected with powdery mildew around the test materials (control group BSMV:00, 3D232 and 5BIL-29 inoculated with BSMV:WTK7-TM ^KinⅠ and BSMV:WTK7-TM ^KinⅡ ), and use a chicken blanket to gently brush the powdery mildew spores in the breeding basin for inoculation, and brush and inoculate three times a day in the morning, noon and evening. According to the size, thickness and number of powdery mildew lesions on wheat leaves, as well as whether there is allergic necrosis and other reaction types, it is divided into 6 levels: 0 (immune), 0 (allergic necrosis), 1 (highly resistant), 2 (moderately resistant), 3 (moderately susceptible) and 4 (highly susceptible), among which 0-2 are disease-resistant reaction types, and 3-4 are disease-susceptible reaction types (Table 4).

Table 4 Grading standards for powdery mildew reaction types in wheat seedling stage

The results showed that on the 10th day after inoculation with powdery mildew E09, compared with the control group BSMV:00 (the wheat leaves in the control group were not infected with powdery mildew), the leaves of 3D232 and 5BIL-29 inoculated with BSMV:WTK7-TM ^KinⅠ or BSMV:WTK7-TM ^KinⅡ were susceptible to powdery mildew, and the wheat leaves were highly susceptible to powdery mildew (Figure 5b). At the same time, qRT-PCR detection found that in the 3D232 and 5BIL-29 plants susceptible to powdery mildew, compared with the control group BSMV:00 (the WTK7-TM gene in the wheat in the control group was not significantly downregulated), the leaves of 3D232 and 5BIL-29 inoculated with BSMV:WTK7-TM ^KinⅠ and BSMV:WTK7-TM ^KinⅡ could The above results show that silencing the expression of WTK7-TM gene in 3D232 and 5BIL-29 can make the plants susceptible to powdery mildew, further indicating that WTK7-TM is the Ml3D232 gene. qRT-PCR uses ACTIN gene as internal reference, and the nucleotide sequence of primers for qRT-PCR is shown in Table 1.

Example 4: EMS mutants verify the powdery mildew resistance function of WTK7-TM

In order to further confirm the powdery mildew resistance function of WTK7-TM, we used EMS mutagenesis to create a powdery mildew-susceptible mutant of 3D232. First, about 10,000 full seeds of the disease-resistant parent 3D232 were selected, treated with 0.6% ethyl methanesulfonate (EMS) mutagen, and sown in the experimental field, and finally 3,360 _M2 generation materials were harvested. In the greenhouse, the harvested _M2 generation materials were identified for powdery mildew resistance at the seedling stage according to the method of Example 3, and a total of 23 susceptible mutant families were obtained. Then, we amplified and analyzed the WTK7-TM gene in all susceptible mutants, and found that 17 susceptible mutants (M1-M17) (Figure 5 c) had mutations in the WTK7-TM gene, of which 4 were premature termination mutations, 1 variable splicing change and 12 missense mutations (Figure 5 d). The above mutant analysis results further confirmed that WTK7-TM is the Ml3D232 gene.

Example 5. Development and application of WTK7-TM gene functional marker

In order to use the WTK7-TM gene to breed wheat varieties resistant to powdery mildew, a primer combination for specific amplification of the functional marker WTK-TM-FM (WTK-TM-FMF and WTK-TM-FMR in Table 1) was developed based on the WTK7-TM gene sequence, and the genomic DNA of the 3D232 material was used as a template for amplification. The functional marker WTK-TM-FM is a DNA molecule with a nucleotide sequence of SEQ ID No.6.

The amplification procedure is:

PCR reaction system (10 μL): 2 μL of wheat leaf genomic DNA (25 ng/μL), 5 μL of 2×PCR Mix, 1 μL of primer WTK-TM-FMF aqueous solution (concentration 10 μmol/L), 1 μL of primer WTK-TM-FMR aqueous solution (concentration 10 μmol/L), and 1 μL of ddH ₂ O.

PCR reaction conditions: 94°C for 5 min; 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 7 min.

The amplified product was detected by 1% agarose gel electrophoresis and the band size was 1198bp. The amplified product was sequenced and found to be completely matched with the WTK7-TM gene sequence, indicating that WTK-TM-FMF and WTK-TM-FMR can specifically amplify the functional marker WTK-TM-FM from the wheat genome. If the functional marker WTK-TM-FM is present, a 1198bp band can be amplified, but if the functional marker WTK-TM-FM is not present, a band of the same size cannot be amplified.

The functional marker WTK7-TM-FM was used to construct an _F2 segregation population (the female parent was common wheat Xue Zao and the male parent was common wheat 3D232) by hybridization of the disease-resistant material common wheat 3D232 and the disease-susceptible material common wheat Xue Zao. 25 disease-resistant and 25 susceptible materials (the method for identifying powdery mildew resistance refers to Example 3) were tested and verified. It was found that the detection results of the functional marker WTK7-TM-FM were co-segregated with the phenotype, that is, the materials with amplified 1198bp bands all showed disease resistance, while the materials without amplified 1198bp bands all showed high sensitivity (Table 5).

A further selection of 442 wild emmer wheat accessions, 124 cultivated emmer wheat accessions, 230 durum wheat accessions, 262 Chinese wheat micro-core germplasms, and 394 foreign local varieties (Li, M. et al. A CNL protein in wild emmer wheat confers powdery mildew resistance.New Phytol.228,1027-1037(2020)), amplified with WTK7-TM-FM, and found that only 38 wild emmer wheat contained WTK7-TM, while there was no WTK7-TM gene in cultivated emmer wheat, durum wheat and common wheat (Figure 7).

Table 5 Functional markers of WTK7-TM-FM detection of Xue Zao × 3D232F ₂ population

In Table 5, R indicates that the phenotype of the tested wheat is resistant to powdery mildew, and S indicates that the phenotype of the tested wheat is susceptible to powdery mildew; amplification means that a target band of 1198 bp can be amplified; and no amplification means that no band is amplified.

During the research and development of this application, it took more than 20 years from the creation of the broad-spectrum powdery mildew resistant material 3D232 to the cloning of the Ml3D232 gene. During this period, several doctoral students participated in the public relations of the project and finely located the Ml3D232 gene by screening a large number of mapping populations. In the absence of a reference genome, a physical map covering the Ml3D232 gene was obtained by screening the genomic BAC library of wild emmer wheat IW2. Genetic analysis of the candidate interval revealed that only CYB561-Domon and TM9SF4 expression. However, after stable genetic transformation of these two genes into the highly susceptible powdery mildew wheat variety Fielder, it was found that neither gene had the function of powdery mildew resistance, indicating that the Ml3D232 gene does not exist in the currently known reference genome. In order to overcome this difficulty, the genome of durum wheat 5BIL-29 containing Ml3D232 was resequenced. Through assembly and gene annotation, it was found that the physical interval of the Ml3D232 interval in 5BIL-29 was 1.17Mb physical distance, and a total of 11 genes were annotated, including one encoding a tandem kinase protein WTK7-TM with a transmembrane domain. The BSMV-VIGS and EMS susceptible mutant experiments further verified that the WTK7-TM gene was Ml3D232.

Table 6 Partial sequence of this application

Industrial Applications

The present application provides a method for identifying the gene localization, map-based cloning and biological function of wheat broad-spectrum powdery mildew resistance gene WTK7-TM. The wheat broad-spectrum powdery mildew resistance gene WTK7-TM can be widely used in plant fields such as wheat disease resistance genetic breeding, germplasm resource improvement, transgenic and genome editing breeding, and plays an important role in improving and modifying the germplasm resources of crops such as wheat.

The functional marker WTK-TM-FM provided in the present application and the primer composition for amplification thereof can be used for auxiliary screening or identification of powdery mildew-resistant wheat.

The present application has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present application, and without the need to carry out unnecessary experimental conditions, the present application can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present application provides specific embodiments, it should be understood that further improvements can be made to the present application. In a word, according to the principles of the present application, the present application is intended to include any changes, uses or improvements to the present application, including departure from the disclosed scope in the present application and changes made with conventional techniques known in the art.

Claims (17)

1. Use of a protein or a substance regulating gene expression or a substance regulating the activity or content of the protein in any of the following items, wherein the gene encodes the protein, and the protein is WTK7-TM protein;

   A1) Application in regulating plant stress resistance; A2) Application in preparing products for regulating plant stress resistance; A3) Application in regulating plant powdery mildew resistance; A4) Application in preparing products for regulating plant powdery mildew resistance; A5) Application in plant breeding or plant-assisted breeding;

   The WTK7-TM protein is any of the following proteins: a1), a protein whose amino acid sequence is shown in SEQ ID No. 3; a2), a protein obtained by replacing and/or deleting and/or adding amino acid residues in the amino acid sequence shown in a1), which has more than 80% identity with the amino acid sequence shown in a1), and is related to plant stress resistance; a3), a fusion protein obtained by connecting a tag to the N-terminus or/and C-terminus of a1) or a2).
2. The use according to claim 1 is characterized in that: the substance regulating gene expression or the substance regulating the activity or content of the protein is a biological material, and the biological material is any one of the following:

   B1), a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the protein described in claim 1 or the activity of the protein described in claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5), a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2 or a transgenic plant cell line containing the recombinant vector described in B3); B6), a transgenic plant tissue containing the nucleic acid molecule described in B1), or a transgenic plant tissue containing the expression cassette described in B2), or a transgenic plant tissue containing the recombinant vector described in B3); B7), a transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3); B8), a nucleic acid molecule encoding the protein described in claim 1; B9), an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the nucleic acid molecule described in B8).
3. The use according to claim 2, characterized in that:

   B1) the nucleic acid molecule is an RNA or DNA molecule that expresses the gene targeted for use in claim 1;

   B8) The nucleic acid molecule is a DNA molecule as described in any one of the following g1)-g3): g1), a DNA molecule whose coding sequence of the coding chain is SEQ ID No.2; g2), a DNA molecule whose nucleotide sequence of the coding chain is SEQ ID No.1; g3), a DNA molecule that has more than 80% identity with the DNA molecule described in g1) or g2), and regulates plant stress resistance.
4. The use according to claim 3 is characterized in that: B1) the nucleotide sequence of the target sequence of the nucleic acid molecule is SEQ ID No. 4 and/or SEQ ID No. 5.
5. The use according to any one of claims 1 to 4, characterized in that the plant is selected from monocotyledonous plants.
6. The use according to claim 5, characterized in that: the monocotyledonous plant is selected from the family Poaceae plant.
7. The use according to claim 6 is characterized in that the grass plant is selected from the genus Triticum.
8. The use according to claim 7 is characterized in that the wheat plant is selected from wheat (Triticum aestivum L.).
9. A method for cultivating plants with reduced powdery mildew resistance, characterized in that: the method includes down-regulating or reducing the expression of the WTK7-TM protein in a recipient plant or down-regulating or reducing the activity or content of the WTK7-TM protein to obtain a target plant with reduced powdery mildew resistance.
10. The protein for use according to any one of claims 1 to 8.
11. The biomaterial for use according to any one of claims 2 to 8.
12. Application of WTK7-TM molecular marker and/or substance for detecting WTK7-TM molecular marker in any of the following: D1) identification or auxiliary identification of wheat powdery mildew resistance; D2) screening or auxiliary screening of wheat plants, lines, strains or varieties with powdery mildew resistance; D3) auxiliary wheat breeding;

    The WTK7-TM molecular marker is a DNA fragment of the WTK7-TM gene, and the nucleotide sequence of the DNA fragment is SEQ ID No.6;

    The substance for detecting the WTK7-TM molecular marker is any one of the following: E1), a PCR primer composition for amplifying a wheat genomic DNA fragment including the WTK7-TM molecular marker; E2), a PCR reagent containing the PCR primer composition described in E1); E3), a kit containing the PCR primer composition described in E1) or the PCR reagent described in E2).
13. The use according to claim 12 is characterized in that: the PCR primer composition includes a single-stranded DNA whose nucleotide sequence is SEQ ID No.7 and a single-stranded DNA whose nucleotide sequence is SEQ ID No.8.
14. The nucleotide sequence is a DNA molecule of SEQ ID No. 6 or/and the primer composition described in claim 12 or 13 or/and the reagent described in claim 12 or 13 or/and the kit described in claim 8 or 9.
15. A method for identifying or assisting in identifying wheat powdery mildew resistance, the method comprising detecting the WTK7-TM molecular marker described in claim 12 in the wheat to be tested, and identifying or assisting in identifying the wheat powdery mildew resistance based on whether the wheat to be tested contains the WTK7-TM molecular marker, wherein the powdery mildew resistance of the wheat to be tested containing the WTK7-TM molecular marker is higher or has a potential to be higher than that of the wheat to be tested that does not contain the WTK7-TM molecular marker.
16. A method for wheat breeding, characterized in that: the method comprises selecting wheat containing the WTK7-TM molecular marker described in claim 12 as a parent for breeding.
17. A method for regulating plant powdery mildew resistance, characterized in that: the method comprises regulating the plant powdery mildew resistance by regulating the expression of the coding gene in a plant containing the WTK7-TM protein as claimed in claim 1 or by regulating the activity or content of the WTK7-TM protein in a plant containing the WTK7-TM protein as claimed in claim 1.