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

Abstract

This invention discloses the cloning and application of the wheat broad-spectrum powdery mildew resistance gene Pm26. The invention provides the Pm26 protein, a protein composed of the amino acid sequence shown in SEQ ID No. 3; it also provides the gene Pm26, a DNA molecule with the coding region shown in positions 3001-9489 of SEQ ID No. 1, or a DNA molecule with the coding region shown in SEQ ID No. 2; this invention provides gene localization, map-based cloning, and methods for identifying the biological function of the wheat broad-spectrum powdery mildew resistance gene Pm26. The wheat broad-spectrum powdery mildew resistance gene Pm26 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 enhancing the germplasm resources of crops such as wheat.

Description

Cloning and Application of Pm26, a Broad-Spectrum Powdery Mildew Resistance Gene in Wheat Technical Field

[0001] This invention belongs to the fields of crop molecular biology and molecular breeding, specifically involving the cloning and application of the wheat broad-spectrum powdery mildew resistance gene Pm26. Background Technology

[0002] Wheat powdery mildew is a fungal disease caused by *Blumeria graminis* f. sp. *tritici*, a species of wheat. It is a significant global disease affecting wheat, posing a major challenge to wheat production safety. According to statistics from the National Agricultural Technology Extension Service Center, the affected area of ​​wheat powdery mildew in my country over the past decade has been approximately 100 million mu (about 6.67 million hectares), accounting for about one-fifth of all wheat diseases that year. Practical experience shows that, compared to spraying chemical agents, breeding and promoting resistant varieties is the most economical, safe, and effective measure for controlling wheat powdery mildew. The key to breeding resistant varieties lies in identifying resistance genes. To date, 69 powdery mildew resistance genes have been formally named in common wheat and its closely related species. The discovery and utilization of these resistance genes have broadened the genetic resources for wheat disease resistance breeding and played a crucial role in controlling wheat powdery mildew. However, the continuous mutation of new physiological races of virulent powdery mildew fungi has led to a decrease or loss of resistance in some genes. For example, Pm8 was once the most widely used powdery mildew resistance gene in my country, but its loss of resistance led to a major powdery mildew outbreak at the end of the 20th century. Currently, the powdery mildew resistance genes that are still effective or effective in some regions among wheat varieties promoted in my country 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 good resistance and diverse sources is of great significance for improving wheat disease resistance breeding in my country.

[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 undergone long-term and complex environmental evolution, accumulated rich genetic diversity, and contains abundant powdery mildew resistance genes. To date, eight formally named powdery mildew resistance genes have been identified and located from wild emmer wheat, namely Pm16, Pm26, Pm30, Pm36, Pm41, Pm42, Pm64, and Pm69. Except for Pm41 and Pm69, the other genes have not been cloned. The discovery and cloning of powdery mildew resistance genes in wild emmer wheat is of great significance for the genetic improvement of modern wheat varieties using modern genetic manipulation techniques such as molecular marker-assisted selection and gene editing.

[0004] Currently, several wheat powdery mildew resistance genes have been successfully cloned from wheat and its wild species, 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. 61% of these powdery mildew resistance genes encode typical NLR (Nucleotide-binding leucine-rich repeat) type resistance proteins. NLR-type resistance proteins can specifically recognize pathogen effector factors, causing localized programmed cell death at the pathogen infection site in the plant, i.e., a hypersensitive response (HR), which limits the growth and reproduction of the pathogen, thereby protecting other parts of the plant from infection. Summary of the Invention

[0005] The purpose of this invention is to provide a clone of the wheat broad-spectrum powdery mildew resistance gene Pm26 and its application.

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

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

[0008] 2) The amino acid sequence shown in SEQ ID No. 3 is modified by substitution of one or more amino acid residues and / or...

[0009] Proteins derived from 1) that are either missing or added and have the same function.

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

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

[0012] 1) The DNA molecule whose coding region is shown in positions 3001-9489 of SEQ ID No. 1 is genomic DNA;

[0013] 2) The coding region of the DNA molecule shown in SEQ ID No. 2 is cDNA;

[0014] 3) DNA molecules that hybridize with the DNA sequence defined in 1) or 2) under strict conditions and encode proteins 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 having the same function.

[0016] Thirdly, the present invention provides a recombinant vector, expression cassette, or recombinant bacteria containing the nucleic acid molecules described in the second aspect.

[0017] Fourthly, the present invention provides the application 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 bacteria described in the third aspect in regulating plant resistance to powdery mildew; in the above, regulation means enhancement.

[0018] Alternatively, 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 bacteria described in the third aspect in the cultivation of powdery mildew resistant plants.

[0019] Fifthly, the present invention provides the application of substances that inhibit the biological function (activity) of the proteins described in the first aspect or substances that inhibit the expression of nucleic acid molecules described in the second aspect in reducing plant resistance to powdery mildew;

[0020] Alternatively, the application of substances that inhibit the biological function of the proteins described in the first aspect or substances that inhibit the expression of nucleic acid molecules described in the second aspect in the cultivation of powdery mildew-sensitive plants.

[0021] In the preceding text, 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 specifically refers to the BSMV-VIGS system, including pCaBS-α, pCaBS-β, and pCaBS-γbLIC-Pm26. Specifically, pCaBS-γbLIC-Pm26 is constructed by linking the target fragment shown in SEQ ID No. 4 with the pCaBS-γbLIC vector to form the silencing vector pCaBS-γbLIC-Pm26.

[0022] Sixthly, the present invention provides a method for cultivating transgenic plants resistant to powdery mildew, comprising the following 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 transgenic plant exhibits higher resistance to powdery mildew than the original plant.

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

[0027] The transgenic plant exhibits higher resistance to powdery mildew than the original plant.

[0028] In a seventh aspect, the present invention provides a method for cultivating transgenic plants susceptible to powdery mildew, comprising the following 1) or 2):

[0029] 1) The method 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 transgenic plant exhibits lower powdery mildew resistance than the original plant.

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

[0032] The transgenic plant exhibits lower resistance to powdery mildew than the original plant.

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

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

[0035] In embodiments of the present invention, the primer pair is 666COM-1F / 1R and 666COM-2F / 2R, or 666OE-1F / 1R.

[0036] This invention provides a BSMV-VIGS vector for the Pm26 gene, which was constructed using the method reported in the reference (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). The nucleotide sequence of the Pm26 gene fragment used to construct the BSMV-VIGS vector is shown in SEQ ID No. 4.

[0037] This invention provides complementary expression vectors and overexpression vectors for the Pm26 gene, which are constructed using the method reported in the reference (Lu et al. A rare gain of function mutation in a wheat tandem kinase confersresistance to powdery mildew. Nat. Commun. 2020; 11, 680).

[0038] This invention provides a method for gene localization, map-based cloning, and identification of the biological function of the 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 resistance genetic breeding, germplasm resource improvement, transgenic and genome editing breeding, playing an important role in improving and enhancing germplasm resources of crops such as wheat. Attached Figure Description

[0039] Figure 1 shows the identification of multiple races of powdery mildew pathogens in wheat varieties Pm26-40 and IW170.

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

[0041] Figure 3 shows the EMS mutant verifying the Pm26 resistance to powdery mildew.

[0042] Figure 4 shows the BSMV-VIGS verification of Pm26's resistance to powdery mildew.

[0043] Figure 5 shows the transgenic verification of Pm26's resistance to powdery mildew. Detailed Implementation

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

[0045] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0046] The powdery mildew strain E09 described in the following examples was preserved in our laboratory. The above-mentioned biological material can be obtained from the applicant 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 obtained biological material is only used to repeat the experiments of the present invention and cannot be used for other purposes.

[0047] In the following examples, the method for identifying wheat seedling resistance to powdery mildew 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 embodiments are provided to better understand the present invention, but are not intended to limit the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0049] Table 1 shows the primer sequences.

[0050]

[0051]

[0052] Example 1: Discovery and Cloning of Pm26, a Gene for Broad-Spectrum Resistance to Powdery Mildew in Wheat

[0053] I. Identification of powdery mildew resistance

[0054] The common hexaploid wheat material Pm26-40 (described in the following literature: Zhang Deyun. 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 containing the powdery mildew resistance gene fragment Pm26, obtained by crossing wild emmer wheat material TTD140 with the common wheat variety Chinese Spring (CS), followed by multiple generations of backcrossing and self-pollination. It was kindly provided by Professor Rong Junkang of Zhejiang Agriculture and Forestry University. The highly powdery mildew-resistant wild emmer wheat material IW170 was kindly provided by Professor Eviatar Nevo of the Institute of Evolutionary Biology, University of Haifa, Israel. The common wheat varieties Chinese Spring and durum wheat 81086A were used as the corresponding susceptible parents. The wheat varieties Pm26-40 and IW170 were identified by in vitro seedling inoculation using 36 physiological races of powdery mildew collected from different locations across China. Pm26-40 showed high resistance to to immunity against 28 physiological races of powdery mildew; IW170 showed high resistance to to immunity against all 36 physiological races of powdery mildew, making it an excellent broad-spectrum anti-powdery mildew material (Figure 1).

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

[0056] Seedling resistance was assessed using the prevalent powdery mildew race E09 in the Beijing area against F1, F2 segregating populations and F3 families of wheat lines Pm26-40, CS, IW170, 81086A, Pm26-40 / CS, and IW170 / 81086A. The results showed that Pm26-40 and IW170 exhibited an immunoreactive phenotype (IT=0), while CS and 81086A showed a highly susceptible phenotype (IT=4). The F1 generation of CS × Pm26-40 and 81086A × IW170 showed a highly susceptible phenotype (IT=3) (Figure 2a). The resistance of F2 individual plants and F3 families of Pm26-40 / CS was also assessed. 2:3 Family pedigrees were tested for powdery mildew at the seedling stage. The results showed that in the F2 segregating population of 77 Pm26-40 / CS, 21 were resistant and 56 were susceptible, with a chi-square test consistent with a 1:3 recessive single-gene inheritance segregation ratio. In the F2 population of 205 Pm26-40 / CS... 2:3 Of the pedigrees, 45 families showed homozygous resistance, 110 families showed segregation, and 50 families showed homozygous susceptibility. The chi-square test showed a segregation ratio consistent with recessive single-gene inheritance (1:2:1). These findings indicate that the powdery mildew resistance gene in Pm26-40 is controlled by the recessive single gene Pm26. Further analysis was conducted on F2 and F3 plants of IW170 / 81086A. 2:3 Family pedigrees were tested for powdery mildew at the seedling stage. The results showed that in a segregating population of 130 F2 lines, 30 lines were resistant and 100 were susceptible. The chi-square test confirmed a 1:3 segregation ratio for recessive single-gene inheritance. In 193 F2 lines… 2:3 Of the pedigrees, 43 families showed homozygous resistance, 105 families showed segregation, and 45 families showed homozygous susceptibility. The chi-square test showed a segregation ratio consistent with recessive single-gene inheritance at 1:2:1. This indicates 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] In 2000, Dr. Rong Junkang discovered and located the powdery mildew resistance gene in wild emmer wheat and named it Pm26. It is located at the end of chromosome 2BS and co-segregates with the molecular marker Xwg516 (Rong JK, Millet E, Manisterski J, Feldman MA new powdery mildew resistance gene: introgression from wild emmer into common wheat and RFLP-based mapping. Euphatica. 2000; 115, 121-126). In 2012, Dr. Liu Ziji discovered a powdery mildew resistance gene from wild emmer wheat (Triticum turgidum var. dicoccoides) on chromosome 2BS. This gene was tentatively named MlIW170 and is also located at the end of chromosome 2BS. It is 2.69 cM apart from molecular markers CJ945509 and BQ169830 and co-segregates with 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 wildemmer (Triticum turgidum var. dicoccoides) on chromosome 2BS. Theor Appl Genet. 2012; 124, 1041-1049). In 2015, Dr. Liang Yong screened the BAC library of wild emmer wheat TZ-2 and constructed the physical map of MlIW170, finding that MlIW170 is 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 studies have shown that the Pm26 gene locus is located at 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 locus. Ultimately, the polymorphic markers WGGBH553, Xcau516, ​​WGGBD412, WGGBH346, WGGD698, WGGD649, and WGGBD560 were found to be closely linked to the Pm26 gene. Using the common SSR markers Xcau357 and Xwmc243 flanking the locus, 4664 F2 individual plants from the Pm26-40 / CS genetic segregating population and 4388 F2 individual plants from the IW170 / 81086A genetic segregating population were screened. A total of 53 independently exchanged individuals were selected from the Pm26-40 / CS F2 genetic segregating population, and 62 independently exchanged individuals were selected from the IW170 / 81086 genetic segregating population. Genotyping of exchanged plants was performed using molecular markers within the localization interval, and the Pm26 gene was finely localized by combining phenotypic data. Finally, Pm26 was located between molecular markers WGGBD412 and WGGBH346, corresponding to a genetic interval of 0.18 cM for the Pm26-40 / CS population (Fig. 2c), a genetic interval of 0.07 cM for the IW170 / 81086A population (Fig. 2d), and a physical interval of 800 kb for the wild emmer wheat Zavitan V2.0 reference genome. A total of 8 genes were annotated, including 2 phosphoglycerate mutase genes (TRIDC2BG005010, TRIDC2BG005040) and 6 CC-NBS-LRR genes related to disease resistance (TRIDC2BG005090-1, TRIDC2BG005100, TRIDC2BG005090-2, TRIDC2BG005110, TRIDC0UG006500, TRIDC2BG005230) (Fig. 2e). The above results indicate that the powdery mildew resistance gene Pm26 is located in the resistance R gene cluster.

[0060] To obtain the physical map of the Pm26 site, the genome of wild wheat IW170 was sequenced and assembled using the PacBio HiFi third-generation genome sequencing strategy. The sequencing results are summarized in Table 3.

[0061] Table 3 shows the statistics of IW170 PacBio sequencing results.

[0062]

[0063] Four cells were used, with an average of 35.35 Gb of data per cell, for a total of 141.4 Gb of data, covering approximately 14 times the sequencing depth of the wild emmer wheat genome. After sequence assembly, 3208 contigs were obtained, with an N50 of 9.4 Mb. These 3208 contigs were aligned using the molecular markers WGGBD412 and WGGBH346 flanking the MlIW170 gene. Finally, the contig ptg000834l, covering the localization region, was obtained, with a total length of 4.1 Mb. The physical distance of the localization region containing Pm26 was 909 kb (Figure 2f).

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

[0065] A mutant library was created by treating seeds of the resistant parents Pm26-40 and IW170 with EMS mutagenesis. 1000 M2 families were obtained from Pm26-40, and 3300 M2 families were obtained from IW170. Seedling resistance to powdery mildew was identified in a greenhouse using the physiological race E09 of powdery mildew. The obtained susceptible mutant plants were transplanted to obtain homozygous susceptible mutants. Ultimately, a total of 5 homozygous susceptible mutants with a background of Pm26-40 and 9 homozygous susceptible mutants with a background of IW170 were obtained (Figure 3a). Seedling RNA-Seq was performed on the 9 susceptible IW170 mutants and the wild-type IW170.

[0066] Transcriptome data from the wild-type IW170 and nine mutants were anchored to the IW170 sequencing-assembled genome sequence. The results showed that six of the nine candidate genes in the localization region were expressed as R genes, namely CNL1-CNL6. Point mutations in the CNL1 gene in the IW170 mutant RNA-Seq results led to amino acid changes. Specifically, the CNL1 genes in mutants M452, M3200, M1401, M2279, and M1241 exhibited SNPs with the wild-type IW170, resulting in amino acid changes (Figure 3b). Therefore, CNL1 was listed as a candidate gene for Pm26 for further analysis.

[0067] Based on the sequence information from IW170 sequencing and genome assembly, specific primers were designed to amplify the Pm26 gene in four parental materials: Pm26-40, CS, IW170, and 81086A. The Pm26 gene was amplified only in the two resistant parents, Pm26-40 and IW170, with no amplification bands observed in CS and 81086A. Sanger sequencing revealed that the Pm26 gene (denoted as CNL1 in the figure) was completely identical in sequence in Pm26-40 and IW170 (Figure 2g). Annotation of the NLR protein encoded by the Pm26 gene using NCBI showed that Pm26 contains the typical CC, NB-ARC, and LRR domains found in resistant R proteins. Furthermore, an atypical NCKX domain was integrated between the CC and NB-ARC domains (Figure 3b). Based on whether the dominant marker WGGBM24 was designed in the parental line of Pm26, and after verification in exchange plants in the Pm26-40 / CS segregating population and the IW170 / 81086A segregating population, WGGBM24 was found to co-segregate with the phenotype (Fig. 2c, d).

[0068] Sanger sequencing was used to analyze the Pm26 sequence of all mutants in the Pm26-40 and IW170 backgrounds. The results showed that in nine homozygous susceptible mutants in the IW170 background, all nine mutants had mutations in the Pm26 gene. One mutation resulted in a 6 bp nucleotide deletion leading to a two-amino acid deletion, and eight mutations were G / CA / T type variations leading to nonsynonymous amino acid changes. In five homozygous susceptible mutants in the Pm26-40 background, all five mutants had mutations in Pm26. One G / CA / T type variation led to premature termination, and four mutations were G / CA / T type variations leading to nonsynonymous amino acid changes (Figures 3b and 3c). Therefore, it is inferred that Pm26 confers resistance to powdery mildew to both Pm26-40 and IW170. Furthermore, sequence analysis and allelic tests confirmed that Pm26 and MlIW170 are the same Pm26 gene.

[0069] Sequencing revealed that the Pm26 gene is located on the wheat 2BS chromosome, and its genome sequence is shown in SEQ ID No. 1 (3001-9489), cDNA sequence is shown in SEQ ID No. 2, and amino acid sequence is shown in SEQ ID No. 3.

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

[0071] Example 2: Functional Verification of the Pm26 Gene

[0072] I. BSMV-VIGS technology for silencing the Pm26 gene

[0073] To verify whether Pm26 has the function of resisting powdery mildew, the Pm26 gene in Pm26-40 and IW170 was silenced using the previously reported BSMV-VIGS technology system for silencing endogenous genes in wheat (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).

[0074] The gene silencing vectors, pCaBS-α, pCaBS-β, and pCaBS-γbLIC, 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 silent carrier

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

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

[0078] Sequencing revealed that the silencing vector pCaBS-γbLIC-Pm26 was obtained by inserting the target fragment shown in SEQ ID No. 4 between the ApaⅠ restriction sites of the pCaBS-γbLIC vector.

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

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

[0081] 1) After sowing, place the tobacco seedlings in a culture room at 20-22℃ with 16 hours of light and 8 hours of darkness. Once the tobacco has grown to 6-8 leaves, use them for the BSMV-VIGS experiment.

[0082] 2) After activating the recombinant bacteria EHA105 / pCaBS-γbLIC-Pm26 obtained in step 1 above for 36-48 h, single clones were picked and inoculated into 1 mL LB liquid medium (Kan+Rif) and cultured in a shaker at 28 °C and 220 rpm for 24 h.

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

[0084] 4) Centrifuge the bacterial solution at 6000 rpm for 5 min to collect the bacterial cells, and resuspend the bacterial cells in an equal volume of tobacco infection solution (10 mM MgCl2, 10 mM MES, pH = 5.2, 0.1 Mm AS).

[0085] 5) Adjust the bacterial concentration to OD 600 After reaching 0.7, bacterial suspensions containing pCaBS-α, pCaBS-β, and pCaBS-γbLIC vectors (recombinant bacteria obtained by transforming each vector into Agrobacterium EHA105) were mixed in a 1:1:1 ratio, or bacterial suspensions containing pCaBS-α, pCaBS-β, and pCaBS-γbLIC-Pm26 vectors (recombinant bacteria obtained by transforming each vector into Agrobacterium EHA105) were mixed in a 1:1:1 ratio. After standing at 28℃ for 3-5 hours, the mixture was injected into the unfolded leaves of 6-8 leaf stage Nicotiana spp., and labeled as BSMV:EV and BSMV:Pm26, respectively.

[0086] 6) 7-12 days after injection, collect the injected leaf and its upper first leaf, grind it thoroughly in PBS (pH=7.2) buffer, and rub the juice onto the first leaf of Pm26-40 and IW170 wheat.

[0087] 7) Three days after inoculation, observe the spread of the virus on wheat leaves and simultaneously inoculate with powdery mildew E09 to identify disease resistance.

[0088] The results showed that on the 10th day after inoculation with powdery mildew E09, the leaves of Pm26-40 and IW170 showed susceptibility to powdery mildew in the BSMV:Pm26 inoculation area (Fig. 4b, c).

[0089] B. qRT-PCR detection

[0090] On the 10th day after inoculation with powdery mildew E09 in step 7) above, genomic DNA was extracted from leaves of wheat infected with powdery mildew, including Pm26-40 and IW170 wheat, and PCR amplification was performed using Pm26-M4 primers.

[0091] As shown in Figure 4a, it can be seen that in Pm26-40 and IW170 plants susceptible to powdery mildew, inoculation with BSMV:Pm26 significantly downregulated the expression of the Pm26 gene compared with the control BSMV:EV.

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

[0093] II. Verification of Pm26's resistance to powdery mildew via 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 variety Fielder using Agrobacterium-mediated transformation (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.). This was to verify whether Pm26 would adequately confer powdery mildew resistance to the wheat material. The pCAMBIA1300 plasmid was used as the base vector for constructing the complementary transgenic vector, and the pLGY-OE3 plasmid was used as the base vector for constructing the overexpression transgenic vector; both were kindly provided by Professor Genying Li of the Shandong Academy of Agricultural Sciences.

[0095] 1. Construction of recombinant vectors

[0096] 1) Construction of 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 powderymildew. Nat. Commun. 2020; 11, 680), and double digest it with restriction endonucleases BamHI and HindIII to recover the vector backbone.

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

[0099] (3) Homologous recombination was performed using the pEASY-Uni Seamless Cloning and Assembly Kit (CU101-01) to obtain the recombinant plasmid pCAMBIA1300-Pm26.

[0100] The recombinant plasmid pCAMBIA1300-Pm26 is obtained by homologously recombining the DNA fragment shown in SEQ ID No. 1 into the pCAMBIA1300 vector, and the Pm26 gene is expressed by its own promoter (positions 1-3000 from the 5' end of SEQ ID No. 1).

[0101] 2) Construction of 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 powderymildew. Nat. Commun. 2020; 11, 680), and double digest it with restriction endonucleases BamHI and Sac1 to recover the vector backbone.

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

[0104] (3) Homologous recombination was performed using the pEASY-Uni Seamless Cloning and Assembly Kit (CU101-01) to obtain the recombinant plasmid pLGY-OE3-Pm26.

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

[0106] 2. Obtaining recombinant Agrobacterium

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

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

[0109] 3. Obtaining Pm26 transgenic wheat

[0110] 1) Using Agrobacterium-mediated genetic transformation, EHA105 / pCAMBIA1300-Pm26 was transformed into Fielder, a common wheat material highly susceptible to powdery mildew (hereinafter referred to as wild wheat), and then complementary transgenic T0 plants were obtained. After molecular identification, two positive plants were obtained and named COM-1 to COM-2.

[0111] The above molecular identification: Genomic DNA was extracted from plant leaves and amplified by PCR using WGGBM24 primers. A result of 870 bp was considered positive.

[0112] 2) Using Agrobacterium-mediated genetic transformation, EHA105 / pTPCK303-Pm26 was transformed into Fielder, a common wheat material highly susceptible to powdery mildew (hereinafter referred to as wild wheat), and then overexpressing transgenic T0 plants were obtained. After molecular identification, three positive plants were obtained and named OE-1 to OE-3.

[0113] The above molecular identification was performed by extracting RNA from plant leaves, reverse transcribing it to obtain cDNA, and then performing PCR amplification using WGGBM24 primers. A result of 870 bp was considered positive.

[0114] 4. Identification of powdery mildew resistance

[0115] The powdery mildew resistance of wheat was detected using a method for identifying powdery mildew resistance at the seedling stage. The following phenotypic identification materials are Fielder, OE-1, OE-2, OE-3, COM-1, and COM-2. The specific methods are as follows:

[0116] After sowing and cultivating the above-mentioned wheat materials, powdery mildew resistance was identified during the seedling stage. The specific method was as follows: One week before planting the phenotypic identification materials, *Xuezao* seedlings were planted and inoculated with powdery mildew fungus E09 to ensure sufficient powdery mildew fungus under laboratory conditions. Each phenotypic identification material was planted in a seedling tray with 15 seeds per cell. When the seedlings reached the one-leaf-one-heart stage, the susceptible control material *Xuezao* was placed around the seedling trays and inoculated through natural dispersal and manual dusting. Resistance was recorded 15 days after inoculation. Based on the distribution ratio of powdery mildew colonies on the leaves and the size of lesions, the reaction type was divided into 6 levels: 0 (immune), 0 (hypoallergic necrosis), 1 (highly resistant), 2 (moderately resistant), 3 (moderately susceptible), and 4 (highly susceptible). Levels 0-2 represent resistant reactions, and levels 3-4 represent susceptible reactions.

[0117] As shown in Figure 5, compared with wild-type wheat, the two Pm26 complementary transgenic T0 families and the three CNL gene overexpression T0 families all showed immunity to powdery mildew.

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

Claims

1. A protein consisting of the amino acid sequence shown in SEQ ID No.

3.

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 any one of the following 1)-2): 1) DNA molecule whose coding region is shown in positions 3001-9489 of SEQ ID No. 1; 2) DNA molecule whose coding region is shown in SEQ ID No.

2.

4. A recombinant vector, expression cassette, or recombinant bacteria containing the nucleic acid molecule described in claim 2 or 3.

5. The application of the protein of claim 1, the nucleic acid molecule of claim 2 or 3, or the recombinant vector, expression cassette, or recombinant bacteria of claim 4 in improving wheat resistance to powdery mildew.

6. A method for breeding transgenic wheat resistant to powdery mildew, comprising the following steps: 1) or 2): 1) The method comprises the following steps: increasing the content of the protein of claim 1 in the starting wheat to obtain transgenic wheat; the transgenic wheat has higher powdery mildew resistance than the starting wheat; 2) The method comprises the following steps: increasing the expression of the nucleic acid molecule encoding the protein of claim 1 in the starting wheat to obtain transgenic wheat; the transgenic wheat has higher powdery mildew resistance than the starting wheat.

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