Cloning and application of wheat broad-spectrum powdery mildew-resistant gene Pm26

Abstract

The invention discloses wheat broad-spectrum powdery mildew resistance gene Pm26 clone and application thereof. The invention provides a Pm26 protein which is a protein composed of an amino acid sequence as shown in SEQ ID No. 3. The invention also provides a gene Pm26, the coding region of which is a DNA molecule as shown in the 3001st-9489th sites of SEQ ID No.1, or the coding region of which is a DNA molecule as shown in SEQ ID No.2; the invention provides a gene localization, map-based cloning and powdery mildew resistance biological function identification method of a wheat broad-spectrum powdery mildew resistance gene Pm26. The wheat broad-spectrum powdery mildew-resistant gene Pm26 can be widely applied to the plant fields of wheat disease-resistant genetic breeding, germplasm resource improvement, transgenosis, genome editing breeding and the like, and plays an important role in improving germplasm resources of crops such as wheat and the like.

Description

Technical Field

[0001] The present invention belongs to the fields of crop molecular biology and molecular breeding, and particularly relates to the cloning and application of the wheat broad-spectrum powdery mildew resistance gene Pm26. Background Art

[0002] Wheat powdery mildew is a fungal disease caused by Blumeria graminis f. sp. tritici. Wheat powdery mildew is an important disease of wheat worldwide, posing an important challenge to wheat production safety. According to the statistics of the National Agricultural Technology Extension and Service Center, in the past decade, the occurrence area of wheat powdery mildew in China was about 100 million mu, accounting for about 1 / 5 of all wheat diseases in that year. Production practice has shown that compared with spraying chemical agents, breeding and promoting disease-resistant varieties is the most economical, safe and effective measure for controlling wheat powdery mildew, and the key to cultivating disease-resistant varieties lies in the discovery of disease-resistant genes. Up to now, 69 powdery mildew resistance genes have been officially named in common wheat and its related species. The discovery and utilization of these powdery mildew resistance genes have broadened the genetic resources for wheat disease-resistant breeding and played an important role in controlling wheat powdery mildew. However, new virulent physiological races of powdery mildew fungi are constantly evolving, resulting in the reduction or loss of resistance of some genes. For example, Pm8 was once the most widely used powdery mildew resistance gene in China, and the loss of its resistance led to a large-scale outbreak of powdery mildew at the end of the 20th century. At present, the powdery mildew resistance genes that are still effective or effective in some regions in the wheat varieties promoted in China are mainly a few genes such as Pm2, Pm4 and Pm21 and their allelic variations. Therefore, cloning and utilizing some powdery mildew resistance genes with better resistance and diverse sources are of great significance for improving wheat disease-resistant breeding in China.

[0003] Wild emmer wheat (Triticum dicoccoides, AABB) is a wild relative of wheat and the direct ancestor of tetraploid and hexaploid wheat. Wild emmer wheat grows in multiple ecological environments, has accumulated rich genetic diversity through long-term and complex environmental evolution, and contains rich powdery mildew resistance genes. Up to now, 8 officially named powdery mildew resistance genes have been discovered and mapped from wild emmer wheat, such as Pm16, Pm26, Pm30, Pm36, Pm41, Pm42, Pm64 and Pm69. Except for Pm41 and Pm69, other genes have not been cloned. Discovering and cloning the powdery mildew resistance genes in wild emmer wheat is of great significance for using powdery mildew resistance genes for modern wheat variety genetic improvement through modern genetic operations such as molecular marker-assisted selection and gene editing.

[0004] Currently, multiple wheat powdery mildew resistance genes have been successfully cloned from wheat and its wild relatives, such as Pm1, Pm2, Pm3, Pm4, Pm5, Pm8, Pm13, Pm17, Pm21, Pm24, Pm36, Pm38 / Lr34 / Yr18 / Sr57, Pm41, Pm46 / Lr67 / Yr46 / Sr55, Pm57, Pm60, Pm69, and WTK4. Among them, 61% of the powdery mildew resistance genes encode typical NLR (Nucleotide-binding leucine-rich repeat) type disease resistance proteins. NLR type disease resistance proteins can specifically recognize pathogen effectors, causing the formation of local programmed cell death at the pathogen infection site in plants, namely the hypersensitive response (HR), which restricts the growth and reproduction of pathogens, thereby protecting other parts of the plant from pathogen infection. Summary of the Invention

[0005] The object of the present invention is to provide the cloning and application of the wheat broad-spectrum powdery mildew resistance gene Pm26.

[0006] In the first aspect, the present invention provides a protein named Pm26 protein, which is as follows (1) or (2):

[0007] 1) A protein consisting of the amino acid sequence shown in SEQ ID No. 3;

[0008] 2) A protein derived from 1) with the same function, which is obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID No. 3.

[0009]

[0010] In the second aspect, the present invention provides a nucleic acid molecule encoding the protein described in the first aspect.

[0011] The above-mentioned nucleic acid molecule, named gene Pm26, is located on wheat chromosome 2BS and is a DNA molecule of any one of the following 1)-4):

[0012] 1) A DNA molecule with the coding region shown in positions 3001-9489 of SEQ ID No. 1, which is genomic DNA;

[0013] 2) A DNA molecule with the coding region shown in SEQ ID No. 2, which is cDNA;

[0014] 3) A DNA molecule that hybridizes with the DNA sequence defined in 1) or 2) under stringent conditions and encodes a protein with the same function;

[0015] 4) A DNA molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the DNA sequence defined in 1) or 2) and encodes a protein with the same function.

[0016] In a third aspect, the present invention provides a recombinant vector, expression cassette or recombinant bacterium containing the nucleic acid molecule described in the second aspect.

[0017] In a fourth aspect, the present invention provides the use of the protein described in the first aspect, the nucleic acid molecule described in the second aspect, or the recombinant vector, expression cassette or recombinant bacterium described in the third aspect in regulating plant resistance to powdery mildew; herein, regulating means enhancing.

[0018] Or, the present invention provides the use of the protein described in the first aspect, the nucleic acid molecule described in the second aspect, or the recombinant vector, expression cassette or recombinant bacterium described in the third aspect in cultivating powdery mildew-resistant plants.

[0019] In a fifth aspect, the present invention provides the use of a substance that inhibits the biological function (activity) of the protein described in the first aspect or a substance that inhibits the expression of the nucleic acid molecule described in the second aspect in reducing plant resistance to powdery mildew;

[0020] Or, the use of a substance that inhibits the biological function of the protein described in the first aspect or a substance that inhibits the expression of the nucleic acid molecule described in the second aspect in cultivating powdery mildew-susceptible plants.

[0021] Herein, the substance that inhibits the biological function (activity) of the protein described in the first aspect or the substance that inhibits the expression of the nucleic acid molecule described in the second aspect is specifically the BSMV-VIGS system, including pCaBS-α, pCaBS-β, pCaBS-γbLIC-Pm26. Among them, pCaBS-γbLIC-Pm26 is constructed by ligating the target fragment shown in SEQ ID No. 4 with the pCaBS-γbLIC vector to form a silencing vector pCaBS-γbLIC-Pm26.

[0022] In a sixth aspect, the present invention provides a method for cultivating transgenic plants resistant to powdery mildew, as follows: 1)

[0023] Or 2):

[0024] 1) The method includes the following steps: increasing the content, biological function and / or activity of the protein described in the first aspect in the starting plant to obtain a transgenic plant;

[0025] The powdery mildew resistance of the transgenic plant is higher than that of the starting plant;

[0026] 2) The method described above includes the following steps: increasing the expression of the nucleic acid molecule encoding the protein described in the first aspect in the starting plant to obtain a transgenic plant;

[0027] The powdery mildew resistance of the transgenic plant is higher than that of the starting plant.

[0028] In the seventh aspect, the present invention provides a method for cultivating a transgenic plant susceptible to powdery mildew, which is as follows 1) or 2):

[0029] 1) The method described above includes the following steps: reducing the content, biological function and / or activity of the protein described in the first aspect in the starting plant to obtain a transgenic plant;

[0030] The powdery mildew resistance of the transgenic plant is lower than that of the starting plant;

[0031] 2) The method described above includes the following steps: inhibiting the expression of the nucleic acid molecule encoding the protein described in the first aspect in the starting plant to obtain a transgenic plant;

[0032] The powdery mildew resistance of the transgenic plant is lower than that of the starting plant.

[0033] The plants include but are not limited to wheat.

[0034] In the eighth aspect, the present invention provides a primer pair for amplifying the full length or fragment of the nucleic acid molecule described in the second aspect.

[0035] In the examples of the present invention, the primer pairs are 666COM-1F / 1R and 666COM-2F / 2R, or 666OE-1F / 1R.

[0036] The present invention provides a BSMV-VIGS vector of the Pm26 gene, which is constructed by the method reported in the reference (Yuan C, Li C, Yan L, et al. A high throughput barley stripe mosaic virus vector for virusinduced gene silencing in monocots and dicots. PLoS One. 2011; 6(10):e26468). The nucleotide sequence of the Pm26 gene fragment used for constructing the BSMV-VIGS vector is shown in SEQ ID No. 4.

[0037] The present invention provides a complementary expression vector and an overexpression vector of the Pm26 gene, which are constructed by the method reported in the reference (Lue et al. A rare gain of function mutation in a wheat tandem kinase confers resistance to powdery mildew. Nat. Commun. 2020; 11, 680).

[0038] The present invention provides methods for gene mapping, map-based cloning, and identification of the biological function of wheat broad-spectrum powdery mildew resistance gene Pm26. The wheat broad-spectrum powdery mildew resistance gene Pm26 can be widely applied in plant fields such as wheat disease-resistant genetic breeding, germplasm resource improvement, transgenic and genome editing breeding, etc., and plays an important role in improving and modifying the germplasm resources of wheat and other crops. Brief Description of the Drawings

[0039] Figure 1 Identification of multiple races of powdery mildew fungus for wheat lines Pm26-40 and IW170.

[0040] Figure 2 Map-based cloning of the wheat powdery mildew resistance gene Pm26.

[0041] Figure 3 Verification of the powdery mildew resistance function of Pm26 by EMS mutant.

[0042] Figure 4 Verification of the powdery mildew resistance function of Pm26 by BSMV-VIGS.

[0043] Figure 5 Verification of the powdery mildew resistance function of Pm26 by transgenic method. Detailed Description of the Invention

[0044] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0045] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0046] The powdery mildew fungus strain E09 described in the following examples is preserved in this laboratory. In the literature "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 biological materials from the applicant. The obtained above biological materials are only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0047] In the following examples, the method for identifying powdery mildew resistance at the wheat seedling stage specifically refers to the following literature: liu ZY, Sun QX, Ni ZF, Yang TM (1999) Development of SCAR markers linked to the Pm21 gene conferring resistance to powdery mildew in common wheat. Plant Breeding 118: 215-219.

[0048] The following examples facilitate a better understanding of the present invention, but do not limit the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement made to the methods, steps or conditions of the present invention shall fall within the scope of the present invention.

[0049] Table 1 shows the primer sequences

[0050]

[0051]

[0052] Example 1. Discovery and cloning of the wheat broad-spectrum powdery mildew resistance gene Pm26

[0053] I. Identification of powdery mildew resistance

[0054] The common hexaploid wheat material Pm26-40 (described in the following literature: Zhang DY. Construction of physical maps of powdery mildew resistance genes MlIW170, Pm26, MlWE74 and PmWE35 in wild emmer wheat. (Doctoral dissertation), 2018.) is an introgression line material containing the Pm26 gene fragment obtained by crossing the wild emmer wheat material TTD140 with the common wheat variety Chinese Spring (CS) and then through multiple generations of backcrossing and self-crossing, and was kindly provided by Professor Rong Junkang of Zhejiang A&F University; the wild emmer wheat material IW170 with high resistance to powdery mildew was kindly provided by Professor Eviatar Nevo of the Institute of Evolution, University of Haifa, Israel. The common wheat variety Chinese Spring and the durum wheat 81086A were used as the corresponding powdery mildew-susceptible parents. The wheat lines Pm26-40 and IW170 were inoculated in vitro with multiple races at the seedling stage using 36 powdery mildew races collected from different locations across the country. Pm26-40 showed high resistance to immune responses against 28 powdery mildew races; IW170 showed high resistance to immune responses against all 36 powdery mildew races and was an excellent broad-spectrum powdery mildew-resistant material ( Figure 1 )

[0055] II. Fine mapping of the wheat broad-spectrum powdery mildew resistance gene Pm26

[0056] F was used to isolate the wheat lines Pm26-40, CS, IW170, 81086A, Pm26-40 / CS and IW170 / 81086A using the powdery mildew fungus E09 prevalent in Beijing. 1 Generation, F 2 Generation Segregating Populations and F 3 The disease resistance of the first generation families was identified at the seedling stage. The results showed that Pm26-40 and IW170 showed immune response (IT=0), and CS and 81086A showed highly susceptible response (IT=4). The F 1 Represents a high sensitivity response type (IT=3) ( Figure 2 a). For Pm26-40 / CS F 2 Single plant and F 2:3 The results of the seedling powdery mildew identification showed that among the 77 Pm26-40 / CS F 2 Among the segregating populations, 21 showed resistance and 56 showed susceptible. The chi-square test was consistent with the segregation ratio of 1:3 recessive single gene inheritance. Among the 205 Pm26-40 / CS F 2:3 45 families showed homozygous resistance, 110 families showed segregation, and 50 families showed homozygous susceptibility. The chi-square test was consistent with the segregation ratio of 1:2:1 recessive single gene inheritance. The above studies show that the powdery mildew resistance gene in Pm26-40 is controlled by the recessive single gene Pm26. F 2 Single plant and F 2:3 The family was tested for powdery mildew at seedling stage, and the results showed that in 130 F 2 Among the segregating populations, 30 showed resistance and 100 showed susceptible. The chi-square test was consistent with the segregation ratio of 1:3 recessive single gene inheritance. 2:3 Among the families, 43 showed homozygous resistance, 105 showed segregation, and 45 showed homozygous susceptibility. The chi-square test was consistent with the segregation ratio of 1:2:1 recessive single gene inheritance. This shows that the powdery mildew resistance gene in IW170 is controlled by the recessive single gene MlIW170 (Table 2).

[0057] Table 2 shows the genetic analysis of the wheat powdery mildew resistance gene Pm26

[0058]

[0059] ​​​​​​Dr. Rong Junkang identified and mapped a powdery mildew resistance gene from wild emmer wheat in 2000, named Pm26, which was located at the end of chromosome 2BS and co-segregated with the molecular marker Xwg516 (Rong JK, Millet E, Manisterski J, Feldman M. A new powdery mildew resistance gene: introgression from wild emmer into common wheat and RFLP-based mapping. Euphytica. 2000;115,121-126). Dr. Liu Ziji identified a powdery mildew resistance gene from wild emmer wheat in 2012, temporarily named MlIW170, which was also located at the end of chromosome 2BS, with a genetic distance of 2.69 cM between the molecular markers CJ945509 and BQ169830, and co-segregated with the molecular marker Xcau516 developed based on Xwg516 (Liu ZJ, Zhu J, Cui Y, et al. Identification and comparative mapping of a powdery mildew resistance gene derived from wild emmer (Triticum turgidum var. dicoccoides) on chromosome 2BS. Theor Appl Genet. 2012;124,1041-1049). Dr. Liang Yong screened the BAC library of wild emmer wheat TZ-2 in 2015, constructed the physical map of MlIW170, and found that MlIW170 was located in the R gene cluster (Liang Y, Zhang DY, Ouyang SH, et al. Dynamic evolution of resistance gene analogs in the orthologous genomic regions of powdery mildew resistance gene MlIW170 in Triticum dicoccoides and Aegilops tauschii. Theor Appl Genet. 2015,128(8),1617-1629). Previous research results showed that the mapped intervals of Pm26 were all located in the terminal region of chromosome 2BS ( Figure 2b), Molecular markers were developed based on the reference genome sequence of wild emmer wheat Zavitan corresponding to the gene mapping interval. Finally, it was found that polymorphic molecular markers WGGBH553, Xcau516, WGGBD412, WGGBH346, WGGD698, WGGD649, and WGGBD560 were tightly linked to the Pm26 gene. The common SSR molecular markers Xcau357 and Xwmc243 on both sides of the mapping interval were used to screen 4,664 F 2 single-plant genetic segregation populations of Pm26-40 / CS and 4,388 F 2 single-plant genetic segregation populations of IW170 / 81086A. In the F 2 genetic segregation population of Pm26-40 / CS, a total of 53 independent exchange single plants were screened, and 62 independent exchange single plants were screened in the IW170 / 81086 genetic segregation population. Molecular markers within the mapping interval were used to genotype the exchange single plants, and combined with phenotypic data, the Pm26 gene was finely mapped. Finally, Pm26 was mapped between molecular markers WGGBD412 and WGGBH346, corresponding to a genetic interval of 0.18 cM in the Pm26-40 / CS population( Figure 2 c), corresponding to a genetic interval of 0.07 cM in the IW170 / 81086A population( Figure 2 d), corresponding to a physical interval of 800 kb in the wild emmer wheat Zavitan V2.0 reference genome, in which a total of 8 genes were annotated, namely 2 phosphoglycerate mutase genes (TRIDC2BG005010, TRIDC2BG005040) and 6 CC-NBS-LRR genes related to disease resistance (TRIDC2BG005090-1, TRIDC2BG005100, TRIDC2BG005090-2, TRIDC2BG005110, TRIDC0UG006500, TRIDC2BG005230)( Figure 2 e). The above results indicate that the powdery mildew resistance gene Pm26 is located in the disease resistance R gene cluster.

[0060] To obtain the physical map of the Pm26 locus, the PacBio HiFi third-generation genome sequencing strategy was used to sequence and assemble the genome of wild emmer wheat IW170. The sequencing results are summarized as follows (Table 3).

[0061] Table 3 summarizes the PacBio sequencing results of IW170

[0062]

[0063] A total of 4 cells were used, with an average output of 35.35 Gb of data per cell, resulting in a total output of 141.4 Gb of data, covering a sequencing depth of approximately 14 times the wild emmer wheat genome. After sequence assembly, 3,208 contigs were obtained, with an N50 of 9.4 Mb. The molecular markers WGGBD412 and WGGBH346 flanking the MlIW170 gene were used to align these 3,208 contigs, and finally, the contig ptg000834l covering the mapped interval was obtained, with a full length of 4.1 Mb. The physical distance of the mapped interval where Pm26 is located is 909 kb( Figure 2 f).

[0064] III. Map-based cloning of the broad-spectrum powdery mildew resistance gene Pm26 in wheat

[0065] EMS mutagenesis was used to treat the seeds of the disease-resistant parents Pm26-40 and IW170 to create mutant libraries. Among them, 1,000 M 2 families were obtained from Pm26-40, and 3,300 M 2 families were obtained from IW170. At the seedling stage, the mutant families were identified for powdery mildew resistance using the powdery mildew physiological race E09 in the greenhouse. The obtained susceptible mutant plants were transplanted to obtain homozygous susceptible mutants. Finally, a total of 5 homozygous susceptible mutants with the Pm26-40 background and 9 homozygous susceptible mutants with the IW170 background were obtained( Figure 3 a). And 9 IW170 susceptible mutants and the IW170 wild type were subjected to RNA-Seq at the seedling stage.

[0066] The transcriptome data of the IW170 wild type and 9 mutants were anchored to the IW170 sequencing and assembled genomic sequence. The results showed that 6 R genes among the 9 candidate genes in the mapped interval were expressed, namely CNL1-CNL6. There were point mutations in the CNL1 gene in the RNA-Seq results of the IW170 mutants, resulting in amino acid changes. Among them, SNPs in the CNL1 gene of mutants M452, M3200, M1401, M2279, and M1241 compared with the IW170 wild type led to amino acid changes( Figure 3 b). Therefore, CNL1 was listed as a candidate gene for Pm26 for further analysis.

[0067] Specific primers were designed based on the sequence information of IW170 sequencing and genome assembly to amplify the Pm26 gene in 4 parental materials, Pm26-40, CS, IW170, and 81086A. Among them, the Pm26 gene could only be amplified in the two disease-resistant parents, Pm26-40 and IW170, and no amplification bands were observed in CS and 81086A. Sanger sequencing found that the Pm26 gene (denoted as CNL1 in the figure) had exactly the same sequence in Pm26-40 and IW170Figure 2 g). The NLR protein encoded by the Pm26 gene was annotated using NCBI, and it was found that Pm26 contains the CC, NB-ARC, and LRR domains typical of disease-resistant R proteins. In addition, an atypical NCKX domain was integrated between the CC domain and the NB-ARC domain ( Figure 3 b). A dominant marker WGGBM24 was designed based on the presence or absence of Pm26 in the parents. After verification in the exchange single plants of the Pm26-40 / CS segregation population and the IW170 / 81086A segregation population, WGGBM24 co-segregated with the phenotype in both cases. Figure 2 c, d).

[0068] The Pm26 sequences of all mutants in the backgrounds of Pm26-40 and IW170 were analyzed by Sanger sequencing. The results showed that among the 9 homozygous susceptible mutants in the IW170 background, mutations occurred in the Pm26 gene in 9 mutants. Among them, 1 mutant had a 6-bp nucleotide deletion resulting in the deletion of 2 amino acids, and 8 mutants had G / C-A / T type variations resulting in non-synonymous changes in amino acids. Among the 5 homozygous susceptible mutants in the Pm26-40 background, mutations occurred in Pm26 in 5 mutants. Among them, 1 G / C-A / T type variation led to premature termination, and 4 were G / C-A / T type variations resulting in non-synonymous changes in amino acids. Figure 3 b, c). It was thus speculated that Pm26 confers the function of powdery mildew resistance on Pm26-40 and IW170. And through sequence analysis and allelic tests, it was proven that Pm26 and MlIW170 are the same gene, the Pm26 gene.

[0069] After sequencing, the Pm26 gene is located on wheat chromosome 2BS. Its genomic sequence is as shown in positions 3001 - 9489 of SEQ ID No.1, the cDNA sequence is as shown in SEQ ID No.2, and the amino acid sequence is as shown in SEQ ID No.3.

[0070] Positions 1 - 3000 of SEQ ID No.1 are the upstream promoter region, positions 3001 - 9489 are the gene region, and positions 9490 - 12690 are the downstream regulatory region of the gene.

[0071] Example 2. Functional verification of the Pm26 gene

[0072] I. Silencing the Pm26 gene using the BSMV-VIGS technique

[0073] To verify whether Pm26 has the function of resisting powdery mildew, the endogenous gene system in wheat was silenced using the previously reported BSMV-VIGS technology (the pCaBS-α, pCaBS-β, and pCaBS-γbLIC used are all described in the following literature: 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), and the Pm26 genes in Pm26-40 and IW170 were silenced.

[0074] The gene silencing vectors were pCaBS-α, pCaBS-β, and pCaBS-γbLIC, which were kindly provided by Professor Li Dawei of China Agricultural University. The powdery mildew strain E09 was preserved in our laboratory.

[0075] 1. Construction of the silencing vector

[0076] Using Pm26-VIGSF / Pm26-VIGSR (Table 1) as primers and the Pm26 gene shown in SEQ ID No. 1 as the template, the target fragment (SEQ ID No. 4) was obtained.

[0077] Then, the pCaBS-γbLIC vector was digested with the restriction enzyme ApaⅠ and the linearized product was recovered. Then, based on the LIC site ligation principle, the above-mentioned target fragment shown in SEQ ID No. 4 was ligated with the pCaBS-γbLIC vector to construct the silencing vector pCaBS-γbLIC-Pm26.

[0078] After sequencing, the silencing vector pCaBS-γbLIC-Pm26 was the vector obtained by inserting the target fragment shown in SEQ ID No. 4 between the ApaⅠ digestion sites of the pCaBS-γbLIC vector.

[0079] The above-mentioned silencing vector pCaBS-γbLIC-Pm26 was transferred into Agrobacterium tumefaciens EHA105 to obtain the recombinant bacterium EHA105 / pCaBS-γbLIC-Pm26.

[0080] 2. BSMV-VIGS-induced silencing of the Pm26 gene leads to susceptibility of plants to powdery mildew

[0081] 1). Nicotiana benthamiana was sown and cultured in an incubator at 20 - 22 °C with a 16 h light and 8 h dark cycle. The tobacco plants were used for the BSMV-VIGS experiment when they grew to the 6 - 8 leaf stage.

[0082] 2), After activating and treating the recombinant bacterium EHA105 / pCaBS-γbLIC-Pm26 obtained in step 1) for 36 - 48 h, pick a single colony and inoculate it into 1 mL of LB liquid medium (Kan+Rif), and culture it in a shaker at 28 °C and 220 rmp for 24 h.

[0083] 3), Inoculate it into 10 mL of LB liquid medium (Kan+Rif) containing 20 μM AS and 100 μM MES at a ratio of 1:100, and culture it in a shaker at 28 °C and 200 rpm for 12 h to obtain a bacterial solution.

[0084] 4), Centrifuge the bacterial solution at 6000 rpm for 5 min to collect the bacteria, and resuspend the bacteria with an equal volume of tobacco infection solution (10 mM MgCl 2 , 10 mM MES, PH = 5.2, 0.1 mM AS).

[0085] 5), After adjusting the bacterial solution concentration to OD 600 = 0.7, mix the bacterial solutions containing pCaBS-α, pCaBS-β, and pCaBS-γbLIC vectors (recombinant bacteria obtained by transferring each vector into Agrobacterium EHA105) at a ratio of 1:1:1, or mix the bacterial solutions containing pCaBS-α, pCaBS-β, and pCaBS-γbLIC-Pm26 vectors (recombinant bacteria obtained by transferring each vector into Agrobacterium EHA105) at a ratio of 1:1:1. After standing at 28 °C for 3 - 5 h, inject the expanded leaves of Nicotiana benthamiana at the 6 - 8 leaf stage, and label them as BSMV:EV and BSMV:Pm26 respectively.

[0086] 6), 7 - 12 days after injection, collect the injected leaves and the first leaf above them, grind them thoroughly in PBS (PH = 7.2) buffer, and rub the juice onto the first leaf of Pm26 - 40 and IW170 wheat at the one - leaf - one - heart stage.

[0087] 7), 3 days after inoculation, observe the expansion symptoms of the virus on wheat leaves, and at the same time inoculate Blumeria graminis f. sp. tritici E09 for disease resistance identification.

[0088] It was found that on the 10th day after inoculating Blumeria graminis f. sp. tritici E09, the leaves of Pm26 - 40 and IW170 showed susceptibility to powdery mildew at the inoculated part of BSMV:Pm26 ( Figure 4 b, c).

[0089] B, qRT - PCR detection

[0090] For the Pm26 - 40 and IW170 wheat that showed susceptibility to powdery mildew on the 10th day after inoculating Blumeria graminis f. sp. tritici E09 in step 7) above, extract the genomic DNA of the leaves with white spots, and perform PCR amplification with the Pm26 - M4 primer.

[0091] The results are as Figure 4 shown in Figure a. It can be seen that in the Pm26-40 and IW170 plants showing powdery mildew susceptibility, compared with the control BSMV:EV, inoculation with BSMV:Pm26 can cause a significant down-regulation of the Pm26 gene expression.

[0092] The above results indicate that silencing the expression of the Pm26 gene in Pm26-40 and IW170 can render the plants susceptible to powdery mildew.

[0093] II. Verification of the powdery mildew resistance function of Pm26 by transgenic technology

[0094] To further confirm the powdery mildew resistance function of Pm26, the recombinant expression vectors pCAMBIA1300-Pm26 and pLGY-OE3-Pm26 were introduced into the recipient wheat cultivar Fielder by Agrobacterium-mediated genetic transformation method (Ishida Y, Tsunashima M, Hiei Y, Komari T. 2015. Wheat (Triticum aestivum L.) transformation using immature embryos. In: Wang K. (eds) Agrobacterium Protocols. Methods in Molecular Biology, vol. 1223, pp189-198. Springer, New York, NY.) to verify whether Pm26 can fully confer powdery mildew resistance to wheat materials. The pCAMBIA1300 plasmid is the basic vector for constructing the complementary transgenic vector, and the pLGY-OE3 plasmid is the basic vector for constructing the overexpression transgenic vector, both of which were kindly provided by Professor Li Genying of Shandong Academy of Agricultural Sciences.

[0095] 1. Construction of recombinant vectors

[0096] 1) Construction of the recombinant plasmid pCAMBIA1300-Pm26

[0097] (1) Take the pCAMBIA1300 plasmid (described in the following literature: Lu et al. A rare gain of function mutation in a wheat tandem kinase confers resistance to powdery mildew. Nat. Commun. 2020; 11, 680), and perform double digestion with the restriction endonucleases BamHⅠ and HindⅢ, and recover the vector backbone.

[0098] (2) Using the DNA fragment shown in SEQ ID No.1 as a template, PCR amplifications were performed respectively with 666COM-1F / 1R and 666COM-2F / 2R (see Table 1) to obtain 2 PCR amplification products.

[0099] (3) Using the pEASY-Uni Seamless Cloning and Assembly Kit (CU101-01) homologous recombination kit, the 2 PCR amplification products obtained in step (2) and the vector backbone recovered in step (1) were subjected to homologous recombination to obtain the recombinant plasmid pCAMBIA1300-Pm26.

[0100] The recombinant plasmid pCAMBIA1300-Pm26 is a plasmid obtained by homologous recombination of the DNA fragment shown in SEQ ID No.1 into the pCAMBIA1300 vector, and the Pm26 gene is driven by its own promoter (the 1st to 3000th positions from the 5'-end of SEQ ID No.1) for expression.

[0101] 2), Construction of the recombinant plasmid pLGY-OE3-Pm26

[0102] (1) Take the pLGY-OE3 plasmid (described in the following literature: Lu et al. A rare gain of function mutation in a wheat tandem kinase confers resistance to powdery mildew. Nat. Commun. 2020; 11, 680), perform double digestion with the restriction endonucleases BamHⅠ and Sac1, and recover the vector backbone.

[0103] (2) Using the DNA fragment shown in SEQ ID No.2 as a template, PCR amplification was performed with 666OE-1F / 1R (see Table 1) to obtain an amplification product.

[0104] (3) Using the pEASY-Uni Seamless Cloning and Assembly Kit (CU101-01) homologous recombination kit, the PCR amplification product obtained in step (2) and the vector backbone recovered in step (1) were subjected to homologous recombination to obtain the recombinant plasmid pLGY-OE3-Pm26.

[0105] The recombinant plasmid pLGY-OE3-Pm26 is a plasmid obtained by homologous recombination of the DNA fragment shown in SEQ ID No.2 into the pLGY-OE3 vector, and the Pm26 gene is driven by the Ubiquitin promoter of maize.

[0106] 2. Obtaining recombinant Agrobacterium

[0107] 1) Using the heat shock transformation method, the recombinant plasmid pCAMBIA1300 - Pm26 was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, named EHA105 / pCAMBIA1300 - Pm26.

[0108] 2) Using the heat shock transformation method, the recombinant plasmid pLGY - OE3 - Pm26 was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, named EHA105 / pLGY - OE3 - Pm26.

[0109] 3. Obtaining Pm26 - transgenic wheat

[0110] 1) Using the Agrobacterium - mediated genetic transformation method, EHA105 / pCAMBIA1300 - Pm26 was transformed into the common wheat material Fielder highly susceptible to powdery mildew (hereinafter referred to as wild wheat), and then complementary transgenic T 0 plants were obtained. A total of 2 positive plants were obtained through molecular identification, named COM - 1 to COM - 2.

[0111] The above - mentioned molecular identification: Genomic DNA was extracted from plant leaves, and PCR amplification was carried out using the WGGBM24 primer. Those with 870bp were positive.

[0112] 2) Using the Agrobacterium - mediated genetic transformation method, EHA105 / pTPCK303 - Pm26 was transformed into the common wheat material Fielder highly susceptible to powdery mildew (hereinafter referred to as wild wheat), and then over - expression transgenic T 0 plants were obtained. A total of 3 positive plants were obtained through molecular identification, named OE - 1 to OE - 3.

[0113] The above - mentioned molecular identification: RNA was extracted from plant leaves, reverse - transcribed to obtain cDNA, and PCR amplification was carried out using the WGGBM24 primer. Those with 870bp were positive.

[0114] 4. Powdery mildew resistance identification

[0115] The powdery mildew resistance of wheat was detected using the wheat seedling stage powdery mildew resistance identification method. The following phenotypic identification materials were Fielder, OE - 1, OE - 2, OE - 3, COM - 1, COM - 2. The specific method is as follows:

[0116] After sowing and cultivating the above wheat materials, the powdery mildew resistance was identified at the wheat seedling stage. The specific method is as follows. One week before planting the phenotypic identification materials, Xuezao was planted and inoculated with powdery mildew E09 to make it fully diseased to ensure an adequate amount of powdery mildew under laboratory conditions. Each phenotypic identification material was planted in a plug tray, with 15 seeds per hole. When it grew to the one-leaf and one-heart stage, the susceptible control material Xuezao was placed around the plug tray, and inoculation was carried out by natural transmission and artificial brushing. The disease resistance was recorded 15 days after inoculation. According to the distribution ratio of powdery mildew colonies on the leaves and the lesion size, the reaction type was divided into 6 grades, namely: 0 (immune), 0; (hypersensitive necrosis), 1 (highly resistant), 2 (moderately resistant), 3 (moderately susceptible), and 4 (highly susceptible). Among them, the 0-2 grades are the disease-resistant reaction types, and the 3-4 grades are the susceptible reaction types.

[0117] The results are as Figure 5 shown. It can be seen that compared with wild-type wheat, two Pm26 complementary transgenic T 0 families and three overexpressed T 0 families of CNL genes were all immune to powdery mildew.

[0118] The above results indicate that the Pm26 gene has good powdery mildew resistance function in common hexaploid wheat.

Claims

1. A protein, which is (1) or (2): 1) a protein consisting of the amino acid sequence shown in SEQ ID No. 3; 2) A protein derived from 1) with the same function as that of SEQ ID No. 3, wherein one or more amino acid residues are substituted and / or deleted and / or added.

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that: The nucleic acid molecule is a DNA molecule of any one of the following 1)-4): 1) The coding region is the DNA molecule shown in positions 3001-9489 of SEQ ID No.1; 2) The coding region is a DNA molecule shown in SEQ ID No. 2; 3) a DNA molecule that hybridizes with the DNA sequence defined in 1) or 2) under stringent conditions and encodes a protein with the same function; 4) A DNA molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA sequence defined in 1) or 2) and encodes a protein with the same function.

4. A recombinant vector, expression cassette or recombinant bacterium containing the nucleic acid molecule according to claim 2 or 3.

5. Use of the protein according to claim 1, the nucleic acid molecule according to claim 2 or 3, or the recombinant vector, expression cassette or recombinant bacterium according to claim 4 in regulating plant resistance to powdery mildew; Or, use of the protein according to claim 1, the nucleic acid molecule according to claim 2 or 3, or the recombinant vector, expression cassette or recombinant bacteria according to claim 4 in cultivating plants resistant to powdery mildew.

6. Use of a substance that inhibits the biological function of the protein according to claim 1 or a substance that inhibits the expression of the nucleic acid molecule according to claim 2 or 3 in reducing plant resistance to powdery mildew; Or, use of a substance that inhibits the biological function of the protein according to claim 1 or a substance that inhibits the expression of the nucleic acid molecule according to claim 2 or 3 in cultivating powdery mildew-sensitive plants.

7. A method for cultivating transgenic plants resistant to powdery mildew, comprising the following 1) or 2): 1) The method comprises the following steps: increasing the content, biological function and / or activity of the protein of claim 1 in the starting plant to obtain a transgenic plant; The transgenic plant has a higher resistance to powdery mildew than the starting plant; 2) The method comprises the following steps: increasing the expression of the nucleic acid molecule encoding the protein of claim 1 in the starting plant to obtain a transgenic plant; The transgenic plants have higher resistance to powdery mildew than the starting plants.

8. A method for cultivating transgenic plants susceptible to powdery mildew, comprising the following 1) or 2): 1) The method comprises the following steps: reducing the content, biological function and / or activity of the protein of claim 1 in the starting plant to obtain a transgenic plant; The transgenic plant has a lower resistance to powdery mildew than the starting plant; 2) The method comprises the following steps: inhibiting the expression of a nucleic acid molecule encoding the protein of claim 1 in a starting plant to obtain a transgenic plant; The transgenic plants have lower powdery mildew resistance than the starting plants.

9. A primer pair for amplifying the full length or fragment of the nucleic acid molecule according to claim 2 or 3.

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