Wheat broad-spectrum multi-effect disease-resistant gene WAI-D1 clone and application thereof

Abstract

The invention discloses cloning of a wheat broad-spectrum multiple-effect disease-resistant gene WAI-D1 and application of the wheat broad-spectrum multiple-effect disease-resistant gene The invention provides a protein which is as shown in (1) or (2): (1) a protein consisting of an amino acid sequence as shown in a sequence 3 in a sequence table; and (2) a protein which is derived from (1), has the same function and is obtained by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in the sequence 3 in the sequence table. The wheat broad-spectrum multiple-effect disease-resistant gene WAI-D1 can be widely applied to the fields of wheat genetic breeding, germplasm resource improvement, genome editing breeding and cultivation of WAI-D1 transgenic plants, and plays an important role in improving germplasm resources of crops such as wheat.

Description

Technical Field

[0001] The invention belongs to the field of crop molecular biology and molecular breeding, and specifically relates to cloning of a wheat broad-spectrum pleiotropic disease resistance gene WAI-D1 (Wheat AutoImmunity-D1) and application thereof. Background Art

[0002] Wheat is the most widely distributed food crop in the world, and more than 40% of the world's population relies on it as their staple food. Currently, wheat in almost all regions is affected by diseases, among which stripe rust, leaf rust, stem rust and powdery mildew seriously threaten production safety. A large number of chemical pesticides are used to control the occurrence and spread of diseases, which not only pollutes the environment, but also seriously affects people's food safety and health. Production practice shows that breeding disease-resistant varieties is the most economical, environmentally friendly and effective measure to control diseases, and it is also the only way for my country's green agricultural development in the future.

[0003] The key to breeding disease-resistant varieties lies in the discovery of disease-resistant genes. Most of the disease-resistant genes identified so far are only resistant to a single or a few specific species of pathogens. However, due to the toxicity variation of pathogens, new pathogenic types and dominant physiological species continue to emerge, causing wheat disease-resistant varieties to "lose" disease resistance after a few years of use, resulting in serious economic losses. Therefore, the discovery and use of durable, broad-spectrum, and multi-effect disease-resistant genes that are resistant to multiple diseases at the same time has important theoretical significance and production application value.

[0004] At present, there are four multi-effect disease resistance genes reported in wheat, including Lr34 / Yr18 / Sr57 / Pm38 / Ltn1 / Sb1, Lr27 / Yr30 / Sr2, Lr46 / Yr29 / Sr58 / Pm39 / Ltn2 and Lr67 / Yr46 / Pm46 / Sr55, of which only Lr34 / Yr18 / Sr57 / Pm38 / Ltn1 / Sb1 and Lr67 / Yr46 / Pm46 / Sr55 genes have been cloned. Lr34 (Lr34 / Yr18 / Sr57 / Pm38 / Ltn1 / Sb1) is the earliest discovered multi-effect disease resistance gene, which has been used in breeding for more than 100 years and still has a long-lasting and broad-spectrum resistance to wheat stripe rust, leaf rust, stem rust and powdery mildew. Krattinger et al. (2009) successfully cloned the Lr34 gene using a positional cloning strategy, which encodes an ATP-binding cassette (ABC) transporter protein (Krattinger et al., 2009). By transferring the Lr34 gene into crops such as wheat, barley, rice, corn and sorghum (Risk et al., 2012; Risk et al., 2013; Krattinger et al., 2016; Sucher et al., 2017; Schnippenkoetter et al., 2017; Boni et al., 2018), it was found that the gene can improve the resistance of these crops to a variety of (semi-) living parasitic fungal diseases, such as stripe rust, leaf rust, stem rust and powdery mildew in wheat, leaf rust and powdery mildew in barley, rice blast in rice, rust and large spot in corn, rust and anthracnose in sorghum, and other diseases. Lr67 (Lr67 / Yr46 / Sr55 / Pm46 / Ltn3) is another multi-effect disease resistance gene cloned from wheat, which encodes a hexose transporter. By transferring Lr67 into wheat and barley, the resistance to wheat stripe rust, leaf rust, stem rust and powdery mildew, as well as barley leaf rust and powdery mildew can be significantly improved (Moore et al. 2015; Ricky J. Milne et al., 2019). These studies show that the excavation and cloning of multi-effect disease resistance genes can not only improve wheat's resistance to a variety of fungal diseases, but will also help other gramineous crops to resist a variety of fungal diseases, which has extremely important theoretical and application value.

[0005] Therefore, there is an urgent need to discover and clone new broad-spectrum and multi-effect disease-resistant genes in this field to provide new gene resources for the breeding of disease-resistant wheat varieties. Summary of the invention

[0006] The purpose of the present invention is to provide a wheat broad-spectrum multi-effect disease resistance gene WAI-D1 and its application.

[0007] In a first aspect, the present invention provides a protein, which is as follows (1) or (2) or (3):

[0008] (1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing;

[0009] (2) a protein derived from (1) having the same function as the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing, wherein one or more amino acid residues are substituted and / or deleted and / or added;

[0010] (3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein described in (1).

[0011] The specific labels are shown in Table 1.

[0012] Table 1 shows the sequence of tags

[0013] Label Residue sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHQ c-myc 10 EQKLISEEDL HA 9 YPYDVPDYA

[0014] In a second aspect, the present invention provides a nucleic acid molecule encoding the protein described in the first aspect.

[0015] The nucleic acid molecule described above is a DNA molecule of any one of the following 1)-3):

[0016] 1) The coding region is the DNA molecule shown in Sequence 1 or Sequence 2 in the sequence list;

[0017] 2) A DNA molecule that hybridizes with the DNA sequence defined in 1) under stringent conditions and encodes a protein with the same function;

[0018] 3) 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) and encodes a protein with the same function.

[0019] In a third aspect, the present invention provides a recombinant vector, an expression cassette or a recombinant bacterium containing the nucleic acid molecule described in the second aspect.

[0020] 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 bacteria described in the third aspect in regulating plant disease resistance.

[0021] In a fifth 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 bacteria described in the third aspect in cultivating highly disease-resistant substances.

[0022] In the above applications, the disease resistance is resistance to powdery mildew, resistance to stripe rust and / or resistance to leaf rust.

[0023] In a sixth aspect, the present invention provides a method for cultivating highly disease-resistant transgenic plants, which is as follows 1) or 2):

[0024] 1) The method comprises the following steps: increasing the content and / or activity of the protein of the first aspect in the target plant to obtain a transgenic plant;

[0025] 2) The method comprises the following steps: increasing the expression of the nucleic acid molecule encoding the protein of the first aspect in the target plant to obtain a transgenic plant;

[0026] The disease resistance of the transgenic plant is higher than that of the target plant.

[0027] In the method described above, increasing the content and / or activity of the protein described in the first aspect in the target plant, or increasing the expression of the nucleic acid molecule encoding the protein described in the first aspect in the target plant, are achieved by introducing the nucleic acid molecule described in the second aspect into the target plant.

[0028] In the above method, the disease resistance is resistance to multiple diseases, specifically resistance to powdery mildew, resistance to stripe rust and / or resistance to leaf rust.

[0029] The plants mentioned above include but are not limited to wheat.

[0030] The present invention provides gene localization and map-based cloning of a wheat broad-spectrum multi-effect disease resistance gene WAI-D1, as well as biological functions in improving wheat resistance to powdery mildew, stripe rust and leaf rust. The wheat broad-spectrum multi-effect disease resistance gene WAI-D1 of the present invention can be widely used in wheat genetic breeding, germplasm resource improvement, genome editing breeding and cultivation of plants with WAI-D1 gene, and plays an important role in improving and modifying germplasm resources of crops such as wheat. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The plant and leaf phenotypes of mutant M1885 and wild-type XY54.

[0032] Figure 2 The distribution of SNPs loci associated with the WAI-D1 gene on wheat chromosomes.

[0033] Figure 3 This is the positional cloning of WAI-D1 gene.

[0034] Figure 4 Functional verification of WAI-D1 transgene.

[0035] Figure 5 To identify the disease resistance of WAI-D1 transgenic plants. DETAILED DESCRIPTION

[0036] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0037] The experimental methods in the following examples, unless otherwise specified, are all conventional methods well known to those skilled in the art. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

[0038] The wheat overexpression vector pTCK303 is disclosed in the document "Li et al., A CNL protein in wild emmerwheat confers powdery mildew resistance. New Phytologist, 2020, 228: 1027-1037" and is named pTCK303 in the document; the public can obtain the above-mentioned biological materials from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The above-mentioned biological materials are only used to repeat the experiments of the present invention and cannot be used for other purposes.

[0039] The transgenic receptor material Fielder is highly susceptible to powdery mildew disease and is preserved by this laboratory and disclosed in the document "Li et al., ACNL protein in wild emmer wheat confers powdery mildew resistance. New Phytologist, 2020, 228: 1027-1037". The public can obtain the above-mentioned biological materials from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The above-mentioned biological materials are only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0040] Powdery mildew physiological races E09, E21, HB-24, 3-53 and 5-83 are all disclosed in the document "Li et al., A CNL protein in wild emmer wheat confers powdery mildew resistance. New Phytologist, 2020, 228: 1027-1037".

[0041] The physiological races of stripe rust CYR32, CYR33, CYR34, ZS and Gui22-1 are published in the document "Bai Bin et al., Current status and strategies of utilization of stripe rust resistance genes in winter wheat breeding in the Northwest stripe rust source area. Chinese Agricultural Science, 2023, DOI:10.3864 / j.issn.0578-1752.220611".

[0042] Example 1. Creation of wheat spontaneous immune mutant M1885

[0043] The common wheat variety Xiaoyan 54 (XY54) was provided and preserved by the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. 10,000 fully grown Xiaoyan 54 seeds were selected and soaked in a large beaker filled with tap water for 12 hours. The seeds were then treated with 0.8% EMS phosphate buffer for 8 hours, rinsed with tap water for 24 hours, and placed in a culture dish to whiten. The whitened M0 generation seeds were planted in the field and managed normally. When mature, the individual plants were harvested and numbered. 20 fully grown M2 generation seeds were randomly taken out of each package and sown in the field in rows with a row length of 1.5 meters and a row spacing of 0.25 meters. Normal field management was carried out, and phenotypic investigations were carried out throughout the growth period to screen mutants. A mutant segregation family M1885 with spontaneous immune necrosis of leaves was identified. The leaves of the mutant plants showed spontaneous immune necrosis at heading, showing a cell death phenotype similar to that in disease resistance (see Figure 1 ). The M1885 mutant was harvested and sown in the field to obtain a stable homozygous mutant line M1885.

[0044] Example 2: Genetic analysis of wheat pleiotropic disease resistance gene WAI-D1

[0045] The common wheat variety Jimai 20 (JM20) was provided by the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The leaves did not show the leaf necrosis phenotype of the mutant M1885 at the heading stage. In order to conduct genetic analysis on the leaf autoimmune necrosis trait of M1885, F1 was obtained by hybridization of M1885 and JM20. The leaves of F1 plants showed a slight leaf necrosis phenotype at the heading stage, and the leaf necrosis in the F2 generation plants was 3:1 (see Table 2). The above genetic analysis showed that the gene controlling the leaf necrosis phenotype in the autoimmune mutant M1885 was controlled by a semi-dominant gene, named WheatAutoImmunity-D1 (WAI-D1).

[0046] Table 2 shows the genetic analysis of WAI-D1 gene

[0047]

[0048] Example 3: Positional cloning of wheat pleiotropic disease resistance gene WAI-D1

[0049] In order to locate the WAI-D1 gene, 35 families with homozygous normal leaves at heading stage and 35 families with homozygous normal leaves at heading stage were selected from the F3 generation segregating population constructed by hybridization of M1885 and JM20. Equal amount of leaves were taken from each family to construct mixed pools of normal leaves and necrotic leaves to extract RNA. RNA-Seq was performed using Illumina Hiseq 4000. After that, the transcriptome data were quality controlled, aligned and filtered. Finally, SNPs were mined for variation to find candidate SNP sites significantly associated with normal leaves and necrotic leaves for molecular marker development and prediction of the genomic segment where the target gene is located. The results of BSR-Seq analysis showed that 323 SNPs / InDels sites were associated with the WAI-D1 gene, of which the most candidate SNP sites (173 / 323) were on chromosome 5D (see Figure 2 ), indicating that the WAI-D1 gene is very likely located on chromosome 5DL.

[0050] In order to precisely locate the WAI-D1 gene, molecular markers were developed using candidate SNPs / InDels sites on wheat chromosome 5DL based on the results of BSR-Seq analysis, and a total of 14 molecular markers XM1-XM14 were developed that were polymorphic between JM20 and M1885 (Table 3).

[0051] Table 3 shows the primer sequences of this study

[0052]

[0053]

[0054] Using the F from the hybrid combination of JM20×M1885 2:3 Ten homozygous normal and ten homozygous necrotic families were used for verification, and it was determined that molecular markers XM1-XM14 were all linked to the WAI-D1 gene. The mapping population was further expanded, and genotyping was performed on 5,628 F2 plants of the JM20×M1885 hybrid combination to construct a genetic linkage map of the WAI-D1 gene (see Figure 3The positioning results showed that the WAI-D1 gene was located at a genetic distance of 0.1 cM between molecular markers XM9-XM12 on chromosome 5DL, and the physical distance corresponding to Chinese Spring was 175.7 kb, including 4 predicted protein-coding genes, TraesCS5D01G342300 (Agmatine coumaroyltransferase-2), TraesCS5D01G342400 (Agmatine coumaroyltransferase-2), TraesCS5D01G342500 (Always early 2) and TraesCS5D01G342600 (Ankyrin repeat family protein) (see Table 4). By amplifying and comparing the four genes in the localized interval between the wild-type Xiaoyan54 and the mutant M1885, it was found that only in TraesCS5D01G342600, there was a missense mutation c.G1637A in the third exon of both the wild-type and mutant, resulting in the amino acid substitution G546D. Therefore, this gene was used as a candidate gene for WAI-D1 gene in the M1885 spontaneous immune mutant for functional analysis (see Figure 3 ).

[0055] Table 4 shows candidate genes for the WAI-D1 localization interval

[0056]

[0057] The genomic nucleotide sequence of the WAI-D1 gene is sequence 1, the cDNA nucleotide sequence is sequence 2, the protein encoded by it is named WAI-D1 protein, and the amino acid sequence of the protein is sequence 3.

[0058] Example 4: Functional verification of WAI-D1 gene

[0059] 1. Obtaining WAI-D1 transgenic plants

[0060] 1) Construction of WAI-D1 transgenic vector

[0061] The WAI-D1 transgenic vector is a recombinant plasmid pUBI:WAI-D1, specifically a plasmid obtained by replacing the fragment between the BamH I and SacI restriction sites of the pTCK303 plasmid with the WAI-D1 gene shown in sequence 2.

[0062] (1) Using the WAI-D1 gene shown in SEQ ID NO: 2 as a template, the WAI-D1-1 specific primers in Table 3 were used for amplification to obtain the WAI-D1 gene product.

[0063] (2) The wheat overexpression vector pTCK303 plasmid was taken and double-digested with restriction endonucleases BamH I and SacI to recover the linearized vector backbone.

[0064] (3) The WAI-D1 gene product and the pTCK303 linearized vector backbone were homologously recombined using the pEASY-Uni Seamless Cloning and Assembly Kit (Beijing Quanshijin Biotechnology Co., Ltd., CU101-01) to obtain the recombinant plasmid pUBI:WAI-D1.

[0065] Sequencing further verified that the pUBI:WAI-D1 vector was complete and correct and could be used in the next experiment.

[0066] 2) Obtaining WAI-D1 transgenic plants

[0067] The above-mentioned overexpression vector pUBI:WAI-D1 was introduced into Agrobacterium EHA105, and Agrobacterium-mediated genetic transformation of common wheat variety Fielder (hereinafter referred to as wild-type wheat) was carried out to obtain T0 generation transgenic strains.

[0068] The above transgenic method is referenced in: Ishida Y, Tsunashima M, Hiei Y, Komari T. Wheat (Triticum aestivum L.) transformation using immature embryos. In: Wang K, ed. Agrobacterium protocols. methods in molecular biology, vol. 1223. New York, NY, USA: Springer, 2015, 189.

[0069] (1) Molecular identification of T0 transgenic lines

[0070] The genomic DNA of the leaves of the T0 transgenic wheat line was extracted, and the genomic DNA was used as a template, and the primer WAI-D1-2 was used for PCR amplification. The recombinant expression plasmid pUBI:WAI-D1 was used as a positive control, and the genome of wheat Fielder was used as a negative control. PCR amplification was performed, and the expected amplification product fragment size of about 600bp was obtained as a positive plant. The PCR reaction program was: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 58℃ renaturation for 30s, 72℃ extension for 30s, 35 cycles; 72℃ extension for 10min. The PCR amplification product was detected by 1% agarose gel electrophoresis, UV photography was used, and the results were recorded.

[0071] Three T0 generation transgenic positive plants were obtained by PCR detection and named OE#1, OE#2 and OE#3 respectively.

[0072] (2) Molecular identification of WAI-D1 transgenic T1 generation

[0073] The T0 generation transgenic positive plants were self-pollinated to obtain T1 generation transgenic plants, and the T1 generation transgenic plants were further subjected to molecular identification using the same method as described in (1). If the amplification product was 600 bp, it was a T1 generation transgenic positive plant; if the amplification product was not 600 bp, it was a T1 generation transgenic negative plant.

[0074] 2. Molecular and phenotypic identification of WAI-D1 transgenic plants

[0075] Fifteen individual plants were selected from each line of the T0 generation transgenic positive plants and T1 generation transgenic wheat plants obtained in 1 above for phenotypic identification.

[0076] The details are as follows: The transgenic lines were sown in the plant culture greenhouse of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, with 16 hours of light and 8 hours of darkness, and a growth temperature of 22 degrees. The leaf necrosis phenotype of the plants was identified during the flowering period of wheat (about 60 days after sowing).

[0077] Wild-type wheat was used as the control.

[0078] The results are shown in Table 5 and Figure 4 As shown, it can be seen that the leaves of T0 or T1 transgenic positive plants all showed immune necrotic spots in the adult stage, while the leaves of T1 transgenic negative plants all appeared normal.

[0079] The above results indicate that WAI-D1 gene is the target gene controlling spontaneous immune necrosis of mutant M1885.

[0080] Table 5 shows the genotype and phenotype identification results of WAI-D1 transgenic

[0081]

[0082] In the above table, leaf necrosis corresponds to transgenic positive plants, and normal leaves correspond to transgenic negative plants.

[0083] Example 5: Analysis of the disease resistance function of the WAI-D1 gene

[0084] In order to study whether the wheat WAI-D1 gene has disease resistance, the WAI-D1 transgenic T2 homozygous transgenic line was used for disease resistance phenotype identification in the adult stage. The powdery mildew, stripe rust and leaf rust used in the identification were all mixed strains composed of multiple strains. The specific identification method is as follows:

[0085] The T1 transgenic plants were self-pollinated to obtain T2 homozygous transgenic plants, which were named WAI-D1 homozygous transgenic lines OE#1, OE#2, and OE#3.

[0086] 1. Powdery mildew inoculation and identification methods

[0087] The wheat variety Fielder was used as the material for the propagation of powdery mildew. The wheat variety Fielder was inoculated with five powdery mildew physiological races (E09, E21, HB-24, 3-53 and 5-83) donated by Yu Dazhao's research group at the Institute of Plant Protection and Soil and Fertilizer of Hubei Academy of Agricultural Sciences to make it fully diseased. During this period, continuous planting and inoculation were carried out to ensure the supply of powdery mildew.

[0088] The WAI-D1 homozygous transgenic line and the wild-type wheat susceptible control Fielder were sown in growth pots and placed in the plant culture greenhouse of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, with 16 hours of light and 8 hours of darkness, and a growth temperature of 22 degrees. At the wheat heading stage (about 50 days after sowing), the WAI-D1 transgenic line and the wild-type wheat susceptible control Fielder were inoculated with powdery mildew using Fielder materials: specifically, the inoculation was carried out by artificial dusting and natural propagation, with one inoculation every other day, for a total of 7 inoculations.

[0089] When the wild-type wheat susceptible control Fielder was fully diseased, the WAI-D1 transgenic line was graded and identified for disease resistance. For specific resistance grading, see the reference: Liu ZY, et al. Development of SCAr markers linked to the Pm21 gene conferring resistance to powdery mildew in common wheat. Plant Breeding 1999, 118: 215-219.

[0090] 2. Stripe rust inoculation and identification methods

[0091] The wheat variety Fielder was used as the material for breeding stripe rust. The stripe rust physiological species CYR32, CYR33, CYR34, ZS and Gui22-1 donated by Du Jiuyuan's research group at the Wheat Research Institute of Gansu Academy of Agricultural Sciences were mixed and dissolved in water, and 0.001% Tween solution (Tween8) was dropped in. After mixing, it was evenly sprayed on the leaves of the breeding material Fielder, and kept in the dark for 24 hours. After that, it was placed in a culture room with 16h of light, 8h of darkness, and a growth temperature of 18°C ​​to make the wheat variety Fielder fully diseased. During this period, continuous planting and inoculation were carried out to ensure the supply of stripe rust.

[0092] The WAI-D1 homozygous transgenic line and the wild-type wheat susceptible control Fielder were sown in growth pots and placed in the plant culture greenhouse of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, with a light period of 16h, a dark period of 8h, and a growth temperature of 22 degrees. At the wheat heading stage (about 50 days after sowing), the WAI-D1 transgenic line and the wild-type wheat susceptible control Fielder were inoculated with Fielder materials infected with stripe rust by artificial dusting and natural propagation. When the wild-type wheat susceptible control Fielder was fully diseased, the disease resistance was graded and identified in the adult stage. For specific resistance grading, please refer to the literature: Wang Y et al. Mapping stripe rust resistance gene YrZH22 in Chinese wheat cultivar Zhoumai 22 by bulked segregant RNA-Seq (BSR-Seq) and comparative genomics analyses. Theoretical and Applied Genetics 2017, 130: 2191–2201.

[0093] 3. Leaf rust inoculation and identification methods

[0094] The wheat variety Fielder was used as the material for propagating leaf rust. The physiological subspecies THJS, PGTS, THND and PHTT of leaf rust donated by Li Zaifeng's research group at Hebei Agricultural University were mixed and dissolved in water to prepare a spore suspension with a concentration of about 2-3 mg / ml. 0.001% Tween solution (Tween8) was added and evenly sprayed on the leaves of the propagation material Fielder after mixing. The leaves were kept in the dark and moisturized for 18 hours. After that, they were placed in a culture room with a light of 16 hours, a dark of 8 hours, and a growth temperature of 22°C to make the wheat variety Fielder fully diseased. During this period, continuous planting and inoculation were carried out to ensure the supply of leaf rust.

[0095] The WAI-D1 homozygous transgenic line and the wild-type wheat susceptible control Fielder were sown in growth pots and placed in the plant culture greenhouse of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, with 16 hours of light, 8 hours of darkness, and a growth temperature of 25 degrees. At the wheat heading stage (about 50 days after sowing), the WAI-D1 transgenic line and the wild-type wheat susceptible control Fielder were inoculated by artificial dusting and natural propagation using Fielder materials infected with leaf rust. When the wild-type wheat susceptible control Fielder was fully diseased, the disease resistance was graded and identified in the adult stage. For specific resistance grading and physiological races of leaf rust, please refer to the literature: "Zhang, et al., QTL mapping of adult-plant resistance to leaf and stripe rust in wheat cross SW 8588 / Thatcher using the wheat 55K SNP array. Plant Disease, 2019, 103: 3041-3049"

[0096] The identification results are as follows Figure 5 As shown, the transgenic receptor Fielder was highly susceptible to all three diseases (level 4), the WAI-D1 transgenic line was highly resistant to powdery mildew (level 1), highly resistant to stripe rust (level 1+), and highly resistant to leaf rust (level 1), indicating that the WAI-D1 gene can simultaneously improve wheat resistance to powdery mildew, stripe rust and leaf rust, and is a broad-spectrum and multi-effect disease resistance gene.

Claims

1. A protein, which is (1) or (2) or (3): (1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing; (2) a protein derived from (1) having the same function as the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing, wherein one or more amino acid residues are substituted and / or deleted and / or added; (3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein described in (1).

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)-3): 1) The coding region is the DNA molecule shown in Sequence 1 or Sequence 2 in the sequence list; 2) A DNA molecule that hybridizes with the DNA sequence defined in 1) under stringent conditions and encodes a protein with the same function; 3) 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) 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 disease resistance.

6. 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 cultivating highly disease-resistant organisms.

7. The use according to claim 5 or 6, characterized in that: The disease resistance is powdery mildew resistance, stripe rust resistance and / or leaf rust resistance.

8. A method for cultivating highly disease-resistant transgenic plants, comprising the following 1) or 2): 1) The method comprises the following steps: increasing the content and / or activity of the protein of claim 1 in the target plant to obtain a transgenic plant; 2) The method comprises the following steps: increasing the expression of the nucleic acid molecule encoding the protein of claim 1 in the target plant to obtain a transgenic plant; The disease resistance of the transgenic plant is higher than that of the target plant.

9. The method according to claim 8, characterized in that: The increasing of the content and / or activity of the protein according to claim 1 in the target plant, or the increasing of the expression of the nucleic acid molecule encoding the protein according to claim 1 in the target plant, is achieved by introducing the nucleic acid molecule according to claim 2 or 3 into the target plant.

10. The method according to claim 8 or 9, characterized in that: The disease resistance is powdery mildew resistance, stripe rust resistance and / or leaf rust resistance.

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