Wheat powdery mildew resistance gene PmD479 and its application

Abstract

The present invention discloses a wheat powdery mildew resistance gene PmD479 and its application, and belongs to the technical field of molecular genetics. The present invention separates a new gene PmD479 from the wild two-grain wheat (Triticum turgidum ssp.dicoccoides), the ancestor of wheat, and confirms the gene function through CRISPR / Cas9 gene editing and wheat genetic transformation. The present invention finds that the gene is completely linked to the powdery mildew resistance phenotype of wild two-grain wheat DIC479, and the mutation of the gene will cause the wild two-grain wheat DIC479 to lose its resistance to powdery mildew. The present invention enriches the powdery mildew resistance gene resources, applies the PmD479 gene to the disease resistance breeding of wheat plants, and can promote the cultivation of powdery mildew resistance wheat varieties or germplasm materials.

Description

Technical Field

[0001] The invention relates to the technical field of molecular genetics, and in particular to a wheat powdery mildew resistance gene PmD479 and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Powdery Mildew is a fungal disease that seriously affects wheat yield and quality. It is caused by Blumeria graminis f.sp.tritici and is one of the most common diseases in wheat growth. The biggest harm of powdery mildew is the yield loss. As the severity of the disease increases, the yield loss is between 5% and 40% (He et al., 2021). If the disease is severe in the seedling stage, it may even cause plant death.

[0004] Wheat powdery mildew is mainly transmitted and transferred through air flow. It has a very strong parasitic ability, and physiological subspecies are prone to mutation (Hu Jinghuang, 2019). At the same time, since wheat powdery mildew resistance is mostly vertical resistance and the source of resistance is single, the mutation of the fungus easily leads to the loss of wheat's original resistance to the powdery mildew. At present, the cultivation and promotion of disease-resistant varieties and the aggregation breeding of disease-resistant genes are considered to be the most effective, economical and environmentally friendly methods to control losses caused by powdery mildew (Li et al., 2020a). The cloning of the Pm gene will help improve resistance to powdery mildew through transgenic means and help understand the resistance mechanism at the molecular level. So far, only a few genes such as Pm2, Pm3, Pm8, Pm21, Pm38, Pm39, Pm46, and Pm60 have been cloned (Sanchez-Martin et al. 2016; Herrera-Foessel et al. 2014; Hurni et al. 2013; Xing et al. 2018; Lagudah et al. 2009; Lillemo et al. 2008; Yahiaoui et al. 2004; Zou et al. 2018). Therefore, the isolation, identification, and cloning of new and effective Pm genes are crucial for the prevention and control of wheat powdery disease.

[0005] Wild emmer wheat material DIC479 carries a powdery mildew resistance gene throughout its growth period. Genetic analysis of the F2 population created by it and cultivated emmer wheat Langdon revealed that its powdery mildew resistance is controlled by a single dominant gene. The gene is located at the end of the long arm of wheat chromosome 2B (Yan Baiqiang, 2016). However, the sequence of the disease resistance gene and the information of the encoded protein have not yet been clarified, which limits its effective use in production. Summary of the invention

[0006] In view of the above-mentioned prior art, the purpose of the present invention is to provide a wheat powdery mildew resistance gene PmD479 and its application. The present invention isolates a new gene PmD479 from the wild two-grain wheat (Triticum turgidum ssp. dicoccoides), the ancestor of wheat, and confirms the gene function through CRISPR / Cas9 gene editing and wheat genetic transformation. The application of the PmD479 powdery mildew resistance gene in disease-resistant breeding of wheat plants can promote the cultivation of powdery mildew-resistant wheat varieties or germplasm materials.

[0007] The present invention is achieved through the following technical solutions:

[0008] In a first aspect of the present invention, a novel wheat powdery mildew resistance gene is provided, which is named as gene PmD479. The gene PmD479 is a nucleic acid molecule as shown in the following i) or ii) or iii) or iv):

[0009] i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1;

[0010] ii) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.3;

[0011] iii) a nucleic acid molecule having 90% or more identity with the nucleotide sequence of i) or ii) and expressing a protein with the same function;

[0012] iv) a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

[0013] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA, etc. Among them, the full-length cDNA sequence of the PmD479 gene is shown in SEQ ID NO.1; the full-length expression framework of the PmD479 genome, including the promoter, genomic coding region and terminator, has a nucleotide sequence shown in SEQ ID NO.3.

[0014] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. Identity can be evaluated using computer software, for example, the BLAST algorithm (Altschul et al. 1990. Journal of Molecular Biology 215: 403-410; Karlin and Altschul. 1993. Proceedings of the National Academy of Sciences 90: 5873-5877).

[0015] The second aspect of the present invention provides a protein encoded by the above-mentioned powdery mildew resistance gene PmD479, wherein the protein is a protein shown in any one of the following (A1)-(A3):

[0016] (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence listing;

[0017] (A2) the amino acid sequence shown in SEQ ID NO. 2 in the sequence list is substituted and / or deleted and / or added with one or more amino acid residues, and a protein derived from SEQ ID NO. 2 related to powdery mildew resistance;

[0018] (A3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein defined in (A1) or (A2).

[0019] The protein in (A2) can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0020] In order to facilitate the purification of the protein in (A1) or (A2), a tag may be attached to the amino terminus or carboxyl terminus of the protein in (A1) or (A2). The tag may be Poly-Arg (usually 6 RRRRR), Poly-His (usually 6 HHHHHH), FLAG (DYKDDDDK), Strep-tag II (WSHPQFEK) or c-myc (EQKLISEEDL).

[0021] The third aspect of the present invention provides a recombinant expression vector, a transgenic cell line or a genetically engineered bacterium containing the above-mentioned wheat powdery mildew resistance gene PmD479.

[0022] The recombinant expression vector can be constructed with existing plant expression vectors. The plant expression vector includes binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, such as pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other derived plant expression vectors. When the gene is used to construct the recombinant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before its transcription initiation nucleotide, such as cauliflower mosaic virus (CaMV) 35S promoter, ubiquitin gene Ubiquitin promoter (pUbi), stress-induced promoter rd29A, etc., which can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct the recombinant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers.

[0023] In a fourth aspect of the present invention, there is provided the use of the wheat powdery mildew resistance gene PmD479, the protein encoded by the powdery mildew resistance gene PmD479, the recombinant expression vector containing the wheat powdery mildew resistance gene PmD479, the transgenic cell line, the genetically engineered bacteria or the genetically engineered plant in any one of the following (1) to (3):

[0024] (1) Plant breeding and / or seed production;

[0025] (2) Regulate plant powdery mildew resistance;

[0026] (3) Prevention and control of plant powdery mildew.

[0027] In the above application, the plant can be either a monocot or a dicot, wherein the monocot can be a grass plant, such as barley or wheat.

[0028] A fifth aspect of the present invention provides a method for breeding powdery mildew resistant wheat or barley, the breeding method comprising: transferring the powdery mildew resistant gene shown in SEQ ID NO.1 or SEQ ID NO.3 into wheat or barley to obtain powdery mildew resistant wheat or barley;

[0029] Alternatively, the expression of the powdery mildew resistance gene shown in SEQ ID NO.1 or SEQ ID NO.3 in the wheat or barley genome is up-regulated to screen wheat or barley plants with improved powdery mildew resistance.

[0030] In the above breeding method, the method for transferring the powdery mildew resistance gene into wheat or barley includes: polyethylene glycol method, Agrobacterium-mediated method or gene gun bombardment method.

[0031] In the above-mentioned breeding method, the method for up-regulating the expression of the powdery mildew resistance gene in the wheat or barley genome includes: introducing a DNA fragment that can activate or increase the transcription level, translation level or protein activity of the wheat powdery mildew resistance gene; or controlling the synthesis of specific small RNA molecules to up-regulate the accumulation of the wheat powdery mildew resistance gene mRNA.

[0032] The specific small RNA molecules include: microRNA (miRNA), small interfering RNA (siRNA) or artificial miRNA (amiRNA).

[0033] In a sixth aspect of the present invention, a method for breeding wheat or barley with reduced powdery mildew resistance is provided, the method comprising: inhibiting the expression of the powdery mildew resistance gene shown in SEQ ID NO.1 or SEQ ID NO.3 in the wheat or barley genome, and screening wheat or barley plants with reduced powdery mildew resistance.

[0034] In the above method, the method for inhibiting the expression of the powdery mildew resistance gene in the wheat or barley genome includes: mutating or knocking out the entire or partial sequence of the gene shown in SEQ ID NO.1 or SEQ ID NO.3 in wheat or barley; or using interfering RNA to interfere with the expression of the gene shown in SEQ ID NO.1 or SEQ ID NO.3; or using a gene silencing system to silence the gene shown in SEQ ID NO.1 or SEQ ID NO.3.

[0035] In a seventh aspect of the present invention, a molecular marker for identifying the wheat powdery mildew resistance gene PmD479 is provided, which is named PmD479-GM. The molecular marker can distinguish different wheat varieties carrying or not carrying the PmD479 gene. PmD479-GM is a PCR marker designed based on the PmD479 coding region and 3' UTR, and its nucleotide sequence at the cDNA level is shown in SEQ ID NO.4, and its nucleotide sequence at the gDNA level is shown in SEQ ID NO.5.

[0036] The primers used to amplify PmD479-GM are PmD479-FP1 (SEQ ID NO.6) and PmD479-RP1 (SEQ ID NO.7). After cDNA and gDNA are amplified with primers, if bands of 520 bp and 898 bp appear respectively, it means that the wheat PmD479 gene is carried and the wheat powdery mildew resistance is obtained.

[0037] In an eighth aspect of the present invention, there is provided the use of the above-mentioned molecular marker or primer for amplifying a molecular marker in any of the following items:

[0038] 1) Identification of genes for resistance to powdery mildew in wheat or barley;

[0039] 2) Breeding of wheat plants;

[0040] 3) Prevention and control of powdery mildew of wheat or barley.

[0041] The ninth aspect of the present invention provides a method for obtaining plant cells carrying the above-mentioned powdery mildew resistance gene PmD479, which is obtained by transgenic or genome editing.

[0042] Furthermore, the present invention also provides a method for obtaining a plant carrying the powdery mildew resistance gene PmD479, and the plant cells obtained by the above method are regenerated into seedlings.

[0043] Beneficial effects of the present invention:

[0044] The present invention isolates the wheat powdery mildew resistance gene PmD479, which can effectively regulate the ability of wheat to resist powdery mildew and enrich the powdery mildew resistance gene resources. The present invention finds that the gene is completely linked to the powdery mildew resistance phenotype of wheat, and the mutation of the gene will cause the loss of wheat resistance to powdery mildew, indicating that the gene can provide the function of powdery mildew resistance. The present invention can be used to improve the powdery mildew resistance level of existing wheat, barley and other wheat plants through molecular marker-assisted breeding, genetic transformation and gene editing based on the PmD479 gene, and cultivate and create powdery mildew resistance intermediate materials and production varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 : Response of wild emmer wheat DIC479 and cultivated emmer wheat Langdon to Blumeria graminis.

[0046] Figure 2 : Molecular marker PmD479-GM at the cDNA level ( Figure 2 a) and gDNA levels ( Figure 2 b) Amplification status.

[0047] Figure 3 :Molecular marker sdau148B( Figure 3 a) and sdau149( Figure 3 b) Detection of single strain pop2-167-6.

[0048] Figure 4 :PmD479 gene-edited plants at target site 1 ( Figure 4 a) Target 2 ( Figure 4 b) Sequence variation; Figure 4 1-6 in the figure correspond to the transgenic plant numbers 1-6 in Table 3.

[0049] Figure 5 :Response type of PmD479 gene-edited plants inoculated with mixed spores of wheat powdery mildew.

[0050] Figure 6 :The reaction type of some T1-generation families of vector PC1218 inoculated with wheat powdery mildew physiological race E09 ( Figure 6 A) and target gene fusion at the gDNA level ( Figure 6 A\B).

[0051] Figure 7 :The reaction type of some T1-generation families of vector PC1219 inoculated with wheat powdery mildew physiological race E09 ( Figure 7 A) and target gene fusion at the gDNA level ( Figure 7 B).

[0052] Figure 8 :The reaction type of some T1-generation families of vector PC1220 inoculated with wheat powdery mildew physiological race E09 ( Figure 8 A) and target gene fusion at the gDNA level ( Figure 8 B) Target gene expression at cDNA level ( Figure 8 C) and the expression of wheat internal reference gene actin ( Figure 8 D). DETAILED DESCRIPTION

[0053] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0054] As mentioned above, due to the large and complex wheat genome, the cloned wheat powdery mildew resistance genes are very limited, and new disease resistance genes are urgently needed in production. In the previous study, the present invention found that wild two-grain wheat DIC479 has a high resistance to powdery mildew phenotype, from which the powdery mildew resistance gene locus was found and named PmD479 gene, which was located at the end of the long arm of wheat chromosome 2B, between the molecular markers sdau149 and sdau148B ("Location and Application of Wild Two-grain Wheat 'DIC479' Powdery Mildew Resistance Gene", Yan Baiqiang, Master's Thesis of Shandong Agricultural University, 2016). However, due to the large and complex wheat genome and the lack of the genome sequence of wild two-grain wheat DIC479, the further precise positioning of the PmD479 gene is hindered, resulting in the sequence of the disease resistance gene and the information of the encoded protein not yet clear, which limits its effective use in production.

[0055] In view of this, the present invention performed DNA resequencing on the disease-resistant parent DIC479, and used resequencing technology to accelerate the efficiency of molecular marker development. The F2 mapping population containing 3045 individual plants was analyzed, and PmD479 was finally located in the genetic interval of 0.1cM, completing the construction of the target gene fine genetic map. This genetic interval corresponds to an approximately 150kb physical segment of the wheat CS reference genome (IWGSC_Wheat_RefSeq_v2.0), and contains 4 high-confidence genes: TraesCS2B03G1572800 (NB-ARC), TraesCS2B03G1572900 (Calmodulin binding), TraesCS2B03G1573100 (NB-ARC&Rx_N), TraesCS2B03G1573300 (Pkinase); 3 low-confidence genes: TraesCS2B03G1572700LC, TraesCS2B03G1573000LC, TraesCS2B03G1573200LC. The resequencing data analysis and PCR amplification of the parent DIC479 confirmed that the TraesCS2B03G1573100 homologous gene only existed in the powdery mildew resistant parent DIC479, but was not detected in the susceptible parent Langdon. The DIC479 genome resequencing assembly sequence NODE_148_length_21600_cov_5.886633 (hereinafter referred to as NODE_148) carries a complete TraesCS2B03G1573100 homologous gene.

[0056] The gene editing vector was constructed using CRISPR / Cas9 technology, and gene editing was carried out on two targets of NODE_148. The vector name was PC1205. The powdery mildew-resistant single plant (F5 generation) in the hybrid offspring of DIC479 / Langdon was used as the material for genetic transformation, and 4 independent gene-edited strains were screened. Artificial inoculation of mixed subspecies of wheat powdery mildew in Tai'an City, Shandong Province, showed that the leaves of 3 gene-edited plants were highly susceptible to powdery mildew; while the 2 transgenic plants that were not edited showed high resistance to powdery mildew. The results showed that the TraesCS2B03G1573100 homologous gene contained in NODE_148 was the target gene PmD479.

[0057] Gene expression vectors were constructed by gene recombination technology, and transgenic complementation experiments were carried out on the full-length cDNA sequence and full-length gDNA sequence of NODE_148 gene. Three gene expression vectors were constructed, named PC1218 (Ubi::NODE_148-cDNA), PC1219 (Ubi::NODE_148-gDNA), and PC1220 (NP::NODE_148-gDNA::NT). Fielder, a common wheat variety highly susceptible to powdery mildew, was used as the material for genetic transformation, and 15 / 12 / 3 T0 generation transgenic positive lines were screened. The T1 generation transgenic families of the three vectors were phenotypic identified and molecularly detected using the powdery mildew physiological race E09. The results showed that the leaves of plants with excessive or natural expression of NODE_148 gene showed high resistance to powdery mildew, while the leaves of plants without NODE_148 gene expression showed high susceptibility to powdery mildew. It can be seen that the overexpression or natural expression of the cDNA / gDNA sequence of the NODE_148 gene confers resistance to the susceptible receptor Fielder powdery mildew. This further indicates that the NODE_148 gene is the target gene PmD479, which has the function of resisting wheat powdery mildew and has application value in the breeding of wheat powdery mildew-resistant varieties.

[0058] The full-length cDNA sequence of the PmD479 gene is shown in SEQ ID NO.1; the amino acid sequence of the protein encoded by the PmD479 gene (i.e., the PmD479 protein) is shown in SEQ ID NO.2; the nucleotide sequence of the full-length expression framework of the PmD479 genome, including the promoter, the genome coding region and the terminator, is shown in SEQ ID NO.3. The details are as follows:

[0059] The full-length cDNA sequence of PmD479 gene (SEQ ID NO.1):

[0060]

[0061] Protein encoded by PmD479 gene (SEQ ID NO.2):

[0062] MSLSAIGVVSAINECVTLWQWAKSAISSLHSRWSGSHDQILHDRVLQLESGLQLLRDTLPAMHDLINKAEWKSHERGVASLLPNLKDAVCDAEDLIDEFRWYELKLQVEGDANQSSLLDFFGTVIPGSFDKLNDVQLRLNHLSIQMENMGLRGVTQHFDKLVRPETTSLPNETKIFGRDKELKQVLGFLNVPMNSKRKRATCSVNASTSNLVNDTSRIYSLPILPIVGIGGVGKTTLSQHICNHQRVKSHFELIIWICVSDNFDVKRLTKEVIQSCNGKEATTDNLDSLQRALSNHVKNKRVLIVLDDMWDDSLKENGQCWKRFCAPFTSVQEGSMMLITTRCPKVAEGVRTMEHVKLEGLEDSIFWKFFKLCAFGSENSINDPELECIGRRILPKLKGSPLAAKTLGRMLSMDMQASHWNSILVSELWELRQEDTDILPALRLSYMYLPFYLKQCFAFCAVYPKDYKFQKACLAEIWVAEGFVDPQGRVPIEDIGCQYFEDLVARSFFQKASGGHIIHDLLHDMAQKVSEHDCFILRNKSEFDKVPQNVRHLYVLPSNDFDDSTLLSLSKYTKLRTLICKKNLGKMTGFVLDHWCTKLMRMRVISCASTNELPESIGNWKHLRYLEISRACPLKRIPSTFCWLYNLQILYAKKCKLDSLPSDFDKLTCLQKFESLGLAYCARNNTTLNRDHATEAEPENRKYLDELTLVMGSDMSQPPIGLKSLFLKNYGGSSLPRWFQPQNLPSLASLGFESCDGLKSINLNERPAVGGSITTFSFLTVLTVKYCDRLSTLDDLLTQEYVPAIEKIHIEDCPWLLSIPGQRFGSFCYLKHLAVIVCPSINWQRGLVLPSSLRSLSLLKCGDISPYVPSCLKNLTSLVSLNIDGLRITSIPGNIWHRNLASLEELEISDCPDLVSIGGAKAVAKIKRVLILRCPKLEEAEQINKRTHLKASMQESVAALFGRRKARGQGNLLVSDNE*

[0063] PmD479 genome full-length expression framework (SEQ ID NO.3):

[0064]

[0065] Those skilled in the art can easily adopt known methods, such as directed evolution and point mutation, to mutate the nucleotide sequence of non-critical positions of the PmD479 gene of the present invention. Those artificially modified nucleotides having 90% or higher identity with the nucleotide sequence of the PmD479 gene of the present invention, such as 90%, 92%, 94%, 96%, 97%, 98% or 99%, as long as the encoded protein is functionally equivalent to the protein shown in SEQ ID NO.2, are derived from the nucleotide sequence of the present invention and are equivalent to the PmD479 gene of the present invention, and should also fall within the scope of protection of the present invention.

[0066] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0067] The test materials used in the examples and comparative examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels. The experimental methods without detailed conditions are carried out in accordance with conventional test methods or the operating instructions recommended by the supplier. Among them:

[0068] The present invention uses wild two-grain wheat DIC479 and cultivated two-grain wheat Langdon as parents to create a hybrid F2 population. In order to test their powdery mildew resistance level, the present invention uses mixed spore inoculation and identification in Tai'an City, Shandong Province. The wheat is planted under greenhouse conditions, and the greenhouse is equipped with 16 hours of light during the day, a temperature of 22-25°C, 8 hours of darkness at night, and a temperature of 15-20°C. The inoculation adopts a natural propagation method and a sweeping method, and a sprayer is used to spray water and humidify every evening. After the control material Huixianhong is fully diseased (about 15 days), the traits are investigated, and the investigation is repeated once a week later.

[0069] The parental materials wild emmer wheat DIC479 and cultivated emmer wheat Langdon, as well as the control material Huixianhong, are publicly available from the applicant for use in replicating the present invention.

[0070] Example 1: Location and cloning of the PmD479 gene

[0071] 1. Construction of the fine genetic map of PmD479:

[0072] Wild emmer wheat DIC479 is highly resistant to wheat powdery mildew, while cultivated emmer wheat Langdon is highly susceptible to wheat powdery mildew ( Figure 1). Baiqiang Yan (Master's thesis of Shandong Agricultural University, 2016) used its F2 segregation population to locate PmD479 at the end of chromosome 2BL, with a genetic distance of 0.3cM from the proximal molecular marker sdau148B, and co-segregation with the distal molecular marker sdau149. In order to further develop distal markers and isolate the PmD479 gene, we performed DNA resequencing on the disease-resistant parent DIC479. Using the wheat CS genome sequence (IWGSC_Wheat_RefSeq_v2.0) and annotations (Chinese Spring v2.1) as references, DIC479 homologous sequences were obtained and molecular markers were further developed. Using encrypted molecular markers and an F2 fine-mapping population containing 3045 individuals, PmD479 was finally located in a genetic interval of 0.1cM, which corresponds to approximately 150kb on the wheat CS reference genome IWGSC_Wheat_RefSeq_v2.0.

[0073] 2. Preliminary analysis and screening of candidate genes:

[0074] Based on the reference physical map of wheat CS and combined with the annotation information of the reference genome of the target interval CS, a total of 4 candidate genes (high-confidence genes) were identified in the 150kb key region: TraesCS2B03G1572800 (NB-ARC), TraesCS2B03G1572900 (Calmodulin binding), TraesCS2B03G1573100 (NB-ARC&Rx_N), and TraesCS2B03G1573300 (Pkinase).

[0075] Comparing the four candidate genes with the DIC479 resequencing data, it was found that TraesCS2B03G1573100 had a homologous gene in DIC479, with a sequence similarity greater than 96% with contig NODE_148. The other three genes had lower sequence similarities with the DIC479 resequencing data, less than 92% or less than 85%.

[0076] In addition, we detected the expression of candidate gene NODE_148 in the two parents at the cDNA level and gDNA level using the molecular marker PmD479-GM (primer sequences are shown in SEQ ID NO.6 and SEQ ID NO.7), and found that this gene was only expressed in the disease-resistant parent DIC479, but not in the disease-susceptible parent Langdon ( Figure 2 ).

[0077] Example 2: Knockout verification of PmD479 candidate gene NODE_148

[0078] 1. Construction of CRISPR / Cas9 knockout vector:

[0079] The present invention uses NODE_148 as a target and constructs a dual-target knockout vector PC1205 using CRISPR / Cas9 technology for functional verification. The vector backbone uses pCBC-MT1T2 (https: / / www.addgene.org / 50593 / sequences / ) and pBUE413 (modified from pBUE411: https: / / www.addgene.org / 62200 / sequences / ). pBUE413 replaces the OsU3 promoter in pBUE411 with TaU3 promoter. The vector backbone is provided by the Ni Zhongfu Laboratory of China Agricultural University. The knockout vector carries two sgRNA target sites, the sequences of which are 5'-GCACCCCCTCCGCAACCTTG-3' (SEQ ID NO.12) and 5'-GTAATGCTGCCTCCAACTGC-3' (SEQ ID NO.13), respectively, located 1032 bp and 2302 bp downstream of the gene start codon ATG. The PCR reaction system and reaction conditions for knockout vector construction are shown in Table 1, and the system and reaction conditions for enzyme digestion and ligation reactions are shown in Table 2.

[0080] The primers used to construct the dual-target knockout vector are as follows:

[0081] MT1T2-F: 5'-AATAATGGTCTCAAGCGCACCCCCTCCGCAACCTTG-3'; (SEQ ID NO.14)

[0082] MT1T2-F0: 5'-GCACCCCCTCCGCAACCTTGGTTTTAGAGCTAGAAATAGC-3'; (SEQ IDNO.15)

[0083] MT1T2-R0: 5'-GCAGTTGGAGGCAGCATTACGCTTCTTGGTGCC-3'; (SEQ ID NO. 16)

[0084] MT1T2-R: 5'-ATTATTGGTCTCTAAACGCAGTTGGAGGCAGCATTA-3'; (SEQ ID NO. 17)

[0085] Table 1 Composition and conditions of PCR reaction system (knockout vector construction)

[0086]

[0087] Table 2 Enzyme digestion and ligation reaction system and conditions (knockout vector construction)

[0088]

[0089] The transformed recipient was pop2-167-6, a powdery mildew resistant plant in the hybrid offspring (F5) of the disease-resistant parent DIC479 and the disease-susceptible parent Langdon. Detection using molecular markers sdau148B and sdau149 showed that pop2-167-6 carried the homozygous PmD479 target segment ( Figure 3 ). The primer sequences for labeling sdau148B are shown in SEQ ID NO.8 and SEQ ID NO.9, and the primer sequences for labeling sdau149 are shown in SEQ ID NO.10 and SEQ ID NO.11.

[0090] Markers sdau148B and sdau149 are co-dominant markers, located on both sides of the target gene PmD479. If the results of the F5 generation genotype detection by the two markers are consistent with the parent DIC479, it means that the single plant is dominant homozygous in the entire sdau148B-sdau149 segment including the target gene PmD479, thereby ensuring that our recipient material carries the homozygous PmD479 target segment.

[0091] 2. Detection of gene editing:

[0092] The present invention obtained 6 transgenic seedlings through co-transformation. The sequences of the two target sites were amplified using the target detection primers LXM-168 / 169 and LXM-170 / 171, and the amplified products were sequenced using the first generation sequencing technology (Sangon Biotech). The results showed that 4 transgenic seedlings had different types of mutations at target sites 1 and 2 ( Figure 4 , Table 3).

[0093] The sequences of primers LXM-168 / 169 and LXM-170 / 171 used to detect the target are as follows:

[0094] LXM-168(F): 5'-GTGAATGCATCAACAAGCAACCTAGTT-3'; (SEQ ID NO.18)

[0095] LXM-169(R): 5'-TGTGTATTTGGATAGGCTCAACAAGG-3'; (SEQ ID NO.19)

[0096] LXM-170(F): 5'-GAAAAATTTAGGGAAGATGACAGGC-3'; (SEQ ID NO.20)

[0097] LXM-171(R): 5'-GTTATACGCAGACATCAATGTTTAGC-3'; (SEQ ID NO. 21)

[0098] Table 3 Gene editing test results of transgenic seedlings

[0099]

[0100] 1 This table describes the editing status of 6 transgenic seedlings at target sites 1 and 2.

[0101] 2 WT means that no editing has occurred, which is consistent with the wild-type gene sequence.

[0102] 3 & before and after indicate two editing effects detected on the same target site.

[0103] 3. Phenotypic identification of transgenic seedlings:

[0104] The present invention uses mixed spores of wheat powdery mildew naturally occurring in Tai'an City, Shandong Province to inoculate and identify 6 transgenic seedlings, disease-resistant parent DIC479 and control variety Huixianhong. The growth conditions are planting under plant growth room conditions, and the growth room is equipped with 16 hours of light during the day, a temperature of 22°C, 8 hours of darkness at night, and a temperature of 18°C. The inoculation adopts a sweeping method and a natural propagation method, and a sprayer is used to spray water and humidify every evening. After the control material Huixianhong is fully diseased (about 15 days), the characteristics are investigated, and the investigation is repeated once a week later.

[0105] The survey results show that:

[0106] (1) The control variety Huixianhong showed high susceptibility to powdery mildew, while the resistant parent DIC479 showed high resistance to powdery mildew ( Figure 5 );

[0107] (2) Transgenic plants #3 and #6 that were not edited showed high resistance to powdery mildew, and no powdery mildew spores appeared on the leaves;

[0108] (3) The edited transgenic plants #2, #4, and #5 showed high susceptibility to powdery mildew, with different levels of powdery mildew spores appearing on the leaves ( Figure 5 ).

[0109] In summary, combined with the results of fine positioning, gene editing results of CRISPR / Cas9 knockout vector transgenic seedlings and inoculation identification results, it can be determined that NODE_148 is the wheat powdery mildew resistance gene PmD479, and the mutation of this gene will lead to the loss of wheat powdery mildew resistance.

[0110] Example 3: Expression verification of PmD479 candidate gene NODE_148

[0111] 1. Construction of gene expression vector:

[0112] The present invention uses the gene sequence of NODE_148 to construct three expression vectors for transgenic complementation experiments. The vector backbones of PC1218 (Ubi::NODE_148-cDNA) and PC1219 (Ubi::NODE_148-gDNA) are PC186, and the overexpression is driven by the Ubi promoter; the vector backbone of PC1220 (NP::NODE_148-gDNA::NT) is PC1215, and the expression is driven by the gene's own promoter and terminator. The sequence of NODE_148-cDNA is 2,937bp (SEQ ID NO.1), which is the cDNA sequence between the gene start codon ATG and the stop codon TGA; the sequence of NODE_148-gDNA is 3,315bp (segment 2020-5334 of SEQ ID NO.3), which is the gDNA sequence between the gene start codon ATG and the stop codon TGA; the gene's own promoter used in the present invention is the region 2,019bp upstream of the gene's start codon (SEQ ID NO.22), and the gene's own terminator is the region 1,070bp downstream of the gene's stop codon (SEQ ID NO.23).

[0113] The vector was constructed using the seamless cloning kit of Novagen, and the primer sequences of PC1218 (PC1219) and PC1220 are shown in SEQ ID NO. 24 and SEQ ID NO. 25, SEQ ID NO. 26 and SEQ ID NO. 27, respectively. The PCR reaction system and reaction conditions for constructing the gene expression vector are shown in Table 4, and the system and reaction conditions for the ligation reaction are shown in Table 5.

[0114] Table 4 PCR reaction system composition and conditions (expression vector construction)

[0115]

[0116] Table 5 Ligation reaction system and conditions (expression vector construction)

[0117]

[0118] 1 20ng<=X<=200ng.

[0119] 2 Y = [0.02 × number of base pairs of fragment] ng, Y> = 10ng.

[0120] 3 The amount of each component added should be >= 1 μl.

[0121] 2. Obtaining T0 transgenic lines:

[0122] By using the Agrobacterium-mediated method, the present invention transforms and integrates plasmids PC1218 / PC1219 / PC1220 into the recipient Fielder, and obtains 20 / 12 / 3 regenerated plants, respectively. The Bar gene detection primers Bar-F / R (SEQ ID NO.28 and SEQ ID NO.29) were used to detect these regenerated plants at the gDNA level, and the results showed that the vectors PC1218, PC1219 and PC1220 respectively obtained 15, 12 and 3 T0 generation transgenic positive strains (Table 6).

[0123] Table 6 PC1218 / PC1219 / PC1220 transgenic wheat

[0124]

[0125]

[0126] 1 + indicates positive.

[0127] 2 - indicates negative.

[0128] 3. Expression and phenotypic identification of T1 transgenic lines:

[0129] The present invention selected 9 (3 each) T1 generation strains numbered PC1218-7 / 9 / 10, PC1219-3 / 4 / 6, and PC1220-1 / 2 / 3 for phenotypic identification and verification of the fusion expression of the target gene NODE_148.

[0130] On the one hand, the present invention uses the powdery mildew physiological race E09 to inoculate and identify the T1 generation families of 9 strains (20 plants in each family) as well as the disease-resistant parent DIC479 and the wild-type Fielder. The inoculation and identification process is carried out in an incubator, and the culture conditions are 16 hours of light and 22°C during the day; 8 hours of darkness and 18°C ​​at night. The inoculation adopts the sweeping method and the natural propagation method, and a sprayer is used to spray water and humidify every evening. After the wild-type Fielder is fully diseased (about 10 days), the characteristics are investigated, and the investigation is repeated once a week later.

[0131] The survey results show that:

[0132] (1) The susceptible control, Fielder, showed high susceptibility to powdery mildew, while the resistant parent, DIC479, showed high resistance to powdery mildew ( Figure 6A / 7A / 8A);

[0133] (2) The T1 generation of families 9 and 10 of vector PC1218 showed obvious resistance and susceptibleness segregation; family 7 had no susceptible plants and all showed high resistance to physiological race E09 ( Figure 6 A);

[0134] (3) All three T1 families of the vector PC1219 had obvious resistance and sensitivity segregation ( Figure 7 A);

[0135] (4) The T1 generation of family 1 and 2 of vector PC1220 had obvious separation of resistance and sensitivity; family 3 had no susceptible plants and all showed high resistance to physiological race E09 ( Figure 8 A).

[0136] On the other hand, the present invention uses specific primers LB046F / 8100R1 (SEQ ID NO.30 and SEQ ID NO.31) to perform target gene fusion detection on the T1 generation families of vectors PC1218 and PC1219 at the gDNA level. Specific primers 8100F4 / PC609R (SEQ ID NO.32 and SEQ ID NO.33) and 8100-F4 / R4 (SEQ ID NO.32 and SEQ IDNO.34) were used to perform target gene fusion detection and expression detection on the T1 generation families of vector PC1220 at the gDNA level and cDNA level, respectively. The expression detection at the cDNA level used the wheat internal reference gene actin as a control, and the amplification primers were NF482 / NF483 (SEQ ID NO.35 and SEQ ID NO.36). The results showed:

[0137] (1) The target gene NODE_148 was not expressed at the gDNA level or cDNA level in the wild-type Fielder, but was expressed at the gDNA level and cDNA level in the resistant parent DIC479 ( Figure 6 B / 7B / 8B / 8C);

[0138] (2) The T1 generation families 9 and 10 of the vector PC1218 showed target gene fusion segregation at the gDNA level and corresponded to the resistance and sensitivity segregation phenotypes one by one; all the individual plants of family 7 showed target gene fusion ( Figure 6 B);

[0139] (3) The three T1 generations of the vector PC1219 all showed target gene fusion segregation at the gDNA level and corresponded to the resistance and sensitivity segregation phenotypes one by one ( Figure 7 B);

[0140] (4) The T1 generation families 1 and 2 of the vector PC1220 showed target gene fusion segregation and expression segregation at the gDNA level and cDNA level, and the segregation phenotypes were one-to-one corresponding to the resistance and sensitivity segregation phenotypes; all the individual plants of family 3 showed target gene fusion and expression ( Figure 8 B / 8C).

[0141] It can be seen that the excessive or natural expression of the candidate gene NODE_148 cDNA sequence and gDNA sequence enables the disease receptor Fielder to acquire powdery mildew resistance.

[0142] In summary, combining the results of fine positioning, CRISPR / Cas9 knockout verification and transgenic complementation verification, it can be determined that NODE_148 is the wheat powdery mildew resistance gene PmD479, and the mutation or overexpression of this gene will lead to the loss or gain of wheat powdery mildew resistance.

[0143] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Wheat powdery mildew resistance gene PmD479, characterized in that: The gene PmD479 is a nucleic acid molecule as shown in the following i) or ii) or iii): i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.3; iii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

2. The protein encoded by the wheat powdery mildew resistance gene PmD479 according to claim 1, characterized in that: The protein is the protein shown in (A1) or (A2) below: (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence listing; (A2) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein defined in (A1).

3. A recombinant expression vector or genetically engineered bacteria containing the wheat powdery mildew resistance gene PmD479 according to claim 1.

4. Use of the wheat powdery mildew resistance gene PmD479 according to claim 1, the protein according to claim 2, or the recombinant expression vector or genetically engineered bacteria according to claim 3 in any one of the following (1) to (3): (1) Plant breeding and / or seed production; (2) Regulate plant powdery mildew resistance; (3) Control of powdery mildew of plants; The plant is barley or wheat.

5. A method for breeding powdery mildew resistant wheat or barley, characterized in that: The breeding method comprises: transferring the powdery mildew resistance gene shown in SEQ ID NO.1 or SEQ ID NO.3 into wheat or barley to obtain powdery mildew resistance wheat or barley; Alternatively, the expression of the powdery mildew resistance gene shown in SEQ ID NO.1 or SEQ ID NO.3 in the wheat or barley genome is up-regulated to screen wheat or barley plants with improved powdery mildew resistance.

6. A method for breeding wheat or barley with reduced resistance to powdery mildew, characterized in that: The method comprises: inhibiting the expression of the powdery mildew resistance gene shown in SEQ ID NO.1 or SEQ ID NO.3 in the wheat or barley genome, and screening wheat or barley plants with reduced powdery mildew resistance.

7. The method according to claim 6, characterized in that The method for inhibiting the expression of the powdery mildew resistance gene in the wheat or barley genome includes: mutating or knocking out the entire or partial sequence of the gene shown in SEQ ID NO.1 or SEQ ID NO.3 in wheat or barley; or using interfering RNA to interfere with the expression of the gene shown in SEQ ID NO.1 or SEQ ID NO.3; or using a gene silencing system to silence the gene shown in SEQ ID NO.1 or SEQ ID NO.

3.

8. A primer for amplifying a molecular marker, characterized in that: The molecular marker is a molecular marker used to identify the wheat powdery mildew resistance gene PmD479, and the nucleotide sequence of the molecular marker is shown in SEQ ID NO.4 or SEQ ID NO.5; The sequences of the primers are shown in SEQ ID NO.6 and SEQ ID NO.

7.

9. Use of the primers according to claim 8 in identifying a powdery mildew resistance gene in wheat or barley.

10. A method for obtaining a plant carrying the wheat powdery mildew resistance gene PmD479 according to claim 1, characterized in that: Plant cells carrying the wheat powdery mildew resistance gene PmD479 are obtained through transgenic or genome editing methods; and the obtained plant cells are then regenerated into seedlings.

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