Application of ERF100-6A protein or coding gene thereof in improving powdery mildew resistance of plants

Abstract

The invention provides application of ERF100-6A protein or a coding gene thereof in improving powdery mildew resistance of plants, and belongs to the technical field of molecular biology. The invention provides an application of an ERF100-6A protein or a coding gene of the ERF100-6A protein or a derivative of the ERF100-6A protein in at least one of improvement of powdery mildew resistance of plants, cultivation of powdery mildew resistant transgenic plants and breeding of powdery mildew resistant plant varieties. According to the invention, mutant wheat of ERF100-6A protein and overexpressed transgenic wheat are respectively used as research objects, and phenotypes after powdery mildew inoculation are detected. The result shows that the mutant wheat of the ERF100-6A protein is more sensitive to pathogenic bacteria of powdery mildew, and the over-expressed transgenic wheat shows higher resistance. Therefore, the ERF100-6A protein can be used for improving the powdery mildew resistance of plants.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology, and particularly relates to application of ERF100-6A protein or a coding gene thereof in improving plant resistance to powdery mildew. Background Art

[0002] Powdery mildew is an obligate parasitic fungal disease caused by Blumeria graminis f.sp.tritici (Bgt). It is a disease with a wide range of prevalence, high frequency of occurrence and strong outbreak in global crop production, which seriously threatens crop production. At present, the area of ​​powdery mildew is gradually expanding. Vigorously cultivating and applying disease-resistant varieties is the most economical, effective and lasting method to control powdery mildew, and it is also an important goal of plant breeding today and in the future. Traditional breeding has a long cycle, is difficult, and requires a lot of manpower and material resources. In recent years, molecular biology technology has been widely used in biological breeding. Through transgenic breeding, plants can be improved in a targeted manner and the breeding cycle can be shortened. However, there are few studies on genes related to powdery mildew resistance, which seriously hinders the development of molecular breeding. Summary of the invention

[0003] The object of the present invention is to provide an application of an ERF100-6A protein or a gene encoding the ERF100-6A protein or a derivative thereof in at least one of improving plant resistance to powdery mildew, cultivating powdery mildew-resistant transgenic plants and breeding powdery mildew-resistant plant varieties, so as to provide resources for transgenic breeding of powdery mildew-resistant plant varieties.

[0004] The present invention provides an application of an ERF100-6A protein, a gene encoding the ERF100-6A protein or a derivative of the gene encoding the ERF100-6A protein in at least one of improving plant resistance to powdery mildew, cultivating powdery mildew-resistant transgenic plants and breeding powdery mildew-resistant plant varieties;

[0005] 1) a protein having an amino acid sequence as shown in SEQ ID NO: 1;

[0006] 2) A fusion protein formed by the protein described in item 1) and a tag sequence.

[0007] Preferably, the improving the resistance of plants to powdery mildew comprises reducing the number of spores and / or spore biomass of powdery mildew microcolonies in plants infected with powdery mildew.

[0008] Preferably, the nucleotide sequence of the gene encoding the ERF100-6A protein is shown in SEQ ID NO:2.

[0009] Preferably, the derivative of the coding gene includes at least one of the following: a recombinant expression vector containing the coding gene of the ERF100-6A protein or a recombinant bacterium containing the coding gene of the ERF100-6A protein and a gene expression cassette containing the coding gene of the ERF100-6A protein.

[0010] Preferably, the plant comprises wheat.

[0011] The invention provides a method for improving the resistance of plants to powdery mildew, which increases the expression amount of ERF100-6A protein or a gene encoding the ERF100-6A protein in the plants.

[0012] Preferably, the method for increasing the expression level of ERF100-6A protein or a gene encoding ERF100-6A protein in a plant is to transfer the gene encoding ERF100-6A protein or a derivative of the gene encoding the gene into the plant for overexpression.

[0013] The present invention provides a method for breeding powdery mildew-resistant plant varieties, which comprises detecting the expression level of the ERF100-6A protein or the gene encoding the ERF100-6A protein in the genome of the plant:

[0014] Plants with upregulated expression of the ERF100-6A protein or the gene encoding the ERF100-6A protein are selected as breeding materials for breeding resistant plant varieties.

[0015] Preferably, the detection reagent includes a reagent for detecting ERF100-6A protein and / or a reagent for detecting a gene encoding ERF100-6A protein;

[0016] The reagent for detecting ERF100-6A protein includes MYC antibody;

[0017] The reagent for detecting the gene encoding the ERF100-6A protein comprises a forward primer qF having a nucleotide sequence as shown in SEQ ID NO:3 and a reverse primer qR having a nucleotide sequence as shown in SEQ ID NO:4.

[0018] Preferably, the plant comprises wheat.

[0019] Beneficial effects:

[0020] The present invention provides an application of an ERF100-6A protein, a gene encoding the ERF100-6A protein, or a derivative of the gene encoding the ERF100-6A protein in at least one of improving plant resistance to powdery mildew, cultivating transgenic plants resistant to powdery mildew, and breeding powdery mildew resistant plant varieties. The ERF100-6A protein, the gene encoding the ERF100-6A protein, or a derivative of the gene encoding the ERF100-6A protein can effectively improve wheat powdery mildew resistance. The embodiment of the present invention analyzes the powdery mildew resistance of erf100-6A mutant materials and wild-type Fielder materials. The results show that compared with the wild-type Fielder control, the number of microcolonies of the erf100-6A mutant material increased significantly after 3 days of inoculation, the colony area of ​​wheat leaves increased significantly after 7 days of inoculation, and the leaf disease became more severe, which indicates that the mutation of the ERF100 gene causes the mutant material to be more sensitive to powdery mildew pathogens and more susceptible to disease. The present invention further analyzes the powdery mildew resistance of the ERF100-6A overexpression transgenic strain and the wild-type Fielder. The results show that compared with the wild-type Fielder control, the number of microcolonies of the ERF100-6A overexpression transgenic strain 3 days after inoculation is extremely significantly reduced. Analysis of the colony area of ​​wheat leaves 7 days after inoculation found that the leaf disease of the ERF100-6A overexpression transgenic strain was significantly reduced. This shows that the antibodies of the overexpression transgenic strain to powdery mildew are significantly improved, showing resistance to powdery mildew. It can be seen that the ERF100-6A can improve the powdery mildew resistance of plants, and the ERF100-6A protein and its encoding gene or derivatives of the encoding gene will play an important role in breeding disease-resistant wheat varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Figure 1 is a graph showing the results of the test of the powdery mildew resistance of wheat erf100-6A mutant material and wild-type Fielder material; wherein A is a graph showing the gene sequence changes of erf100-6A mutant material, B is a graph showing the microcolony phenotype of leaves of erf100-6A mutant material and wild-type Fielder material 3 days after inoculation with powdery mildew, C is a graph showing the powdery mildew microcolony index of erf100-6A mutant material and wild-type Fielder material 3 days after inoculation with powdery mildew, D is a graph showing the powdery mildew spore phenotype of leaves of erf100-6A mutant material and wild-type Fielder material 7 days after inoculation with powdery mildew, and E is a graph showing the powdery mildew spore area index of erf100-6A mutant material and wild-type Fielder material 7 days after inoculation with powdery mildew;

[0022] Figure 2The figures are the test results of the powdery mildew resistance of wheat ERF100-6A overexpressing transgenic materials compared with the control wild-type Fielder; among them, A is the protein content in the ERF100-6A overexpressing transgenic lines, B is the leaf microcolony phenotype of the ERF100-6A overexpressing transgenic lines and the wild-type Fielder materials 3 days after inoculation with powdery mildew, C is the powdery mildew microcolony index statistical graph of the ERF100-6A overexpressing transgenic lines and the wild-type Fielder materials 3 days after inoculation with powdery mildew, D is the powdery mildew spore phenotype of the ERF100-6A overexpressing transgenic lines and the wild-type Fielder materials 7 days after inoculation with powdery mildew, and E is the powdery mildew spore area index statistical graph of the ERF100-6A overexpressing transgenic lines and the wild-type Fielder 7 days after inoculation with powdery mildew. DETAILED DESCRIPTION

[0023] The present invention provides an application of an ERF100-6A protein, a gene encoding the ERF100-6A protein or a derivative of the gene encoding the ERF100-6A protein in at least one of improving plant resistance to powdery mildew, cultivating powdery mildew-resistant transgenic plants and breeding powdery mildew-resistant plant varieties, wherein the ERF100-6A protein is at least one of the following proteins:

[0024] 1) a protein whose amino acid sequence is shown in SEQ ID NO: 1 (MLLLNPASEAAALDSIRQQLLEEPARPA YCRSASFGSLVADQWSESLPFRPNDADDMVVYGALRDAFSCGWLPDGSFAAVKPEPLPSPDGSYDGSCLGSFLAPPAPGPDAPWAEEEAEVAAAASRGKHFRGVRQRPWGKFAAEIRDPAKNGARVWLGTFDSAEDAAVAYDRAAYRMRGSRALLNFPLRIGSEIAAAAAAAGQKRPSPQPASPDSSSPSCSAPGSSKRRKRGEAAAASMAMALVPPPPAQAPVQLTLPAQPWLAAGAVQQLVS);

[0025] 2) A fusion protein formed by the protein described in item 1) and a tag sequence.

[0026] In the present invention, the tag sequence described in item 2) refers to a polypeptide or protein that is fused and expressed together with the target protein in order to facilitate the expression, detection, tracing and / or purification of the target protein in the DNA in vitro recombination technology. The tag sequence preferably includes at least one of the following: Flag tag, polyhistidine tag, MBP tag, hemagglutinin tag, Myc tag, GST tag and SUMO tag, more preferably Myc tag. The tag sequence is preferably connected to the 5' or 3' end of the ERF100-6A protein encoding gene.

[0027] In the present invention, the nucleotide sequence of the coding gene can be codon-optimized according to the codon preferences of different hosts. The nucleotide sequence of the coding gene is preferably as shown in SEQ ID NO: 2 (ATGCTGCTGCTTAATCCGGCGTCCGAGGCGGCGGCGCTGGACAGCATCC).The nucleotide sequence of the gene encoding the ERF100-6A protein is preferably amplified using wild-type hexaploid wheat (Fielder) as a template, using a forward primer such as SEQ ID NO:7 (CCACAAAATCCACTGACAAACG) and a reverse primer such as SEQ ID NO:8 (TTCCTCACGTCGCTCAACACAT). The nucleotide sequence of the ERF100-6A protein encoding gene connected to a Myc tag is preferably SEQ ID NO:6.

[0028] (ATGCTGCTGCTTAATCCGGCGTCCGAGGCGGCGGCGCTGGACAGCATCC GGCAGCAGCTCCTGGAGGAGCCGGCGCGGCCGGCGTACTGCCGGAGCGCGAGCTTCGGCAGCCTGGTGGCGGACCAGTGGAGCGAGTCGCTCCCGTTCCGTCCCAACGACGCCGACGACATGGTCGTCTACGGCGCCCTCCGCGACGCCAs shown in TTCTCCTGCGGCTGGCTCCCCGACGGCTCCTTCGCGGCCGTCAAGCCCGAGCCCCTGCCCTCCCCCGACGGCTCCTACGACGGCTCCTGCCTCGGCAGCTTCCTCGCGCCGCCGGCGCCCGGGCCGGACGCGCCGTGGGCGGAGGAGGAGGCCGAGGTCGCGGCGGCGGCGTCGAGGGGGAAGCACTTCAGAGGCGTGAGGCAGCGGCCGTGGGGCAAGTTCGCGGCGGAGATCCGGGACCCGGCCAAGAACGGCGCGCGCGTGTGGCTCGGCACCTTCGACAGCGCCGAGGACGCCGCCGTGGCCTACGACCGCGCCGCCTACCGCATGCGCGGCTCCCGCGCGCTCCTCAACTTCCCGCTCCGCATCGGCTCCGAGATCGCCGCCGCCGCCGCAGCCGCGGGCCAGAAGCGTCCGTCTCCCCAGCCGGCGAGCCCCGACTCTTCATCTCCCTCCTGCAGCGCGCCCGGGTCGTCGAAGAGGAGAAAGAGAGGCGAGGCCGCGGCAGCGTCCATGGCCATGGCTCTGGTGCCGCCCCCGCCGGCGCAGGCTCCGGTCCAGCTGACCCTCCCAGCCCAGCCGTGGCTGGCCGCCGGCGCCGTCCAGCAGCTGGTGAGCGGTACCAAGCTTGAGCAAAAACTGATAAGTGAAGAAGACCTCAATGAGATGGAACAGAAACTTATTTCGGAGGAGGACTTGAATGAGATGGAACAGAAACTTATTTCAGAAGAGGACCTGAATGAGATGGAACAAAAACTCATTTCCGAAGAGGACCTGAATGAAATGGAGCAGAAACTGATCTCTGAAGAAGACCTCAATGAAATGGAGTCTCTCGGAGACCTTACAATGGAACAGAAACTGATCTCAGAAGAAGATTTGAATGAGTGA).

[0029] In the present invention, the derivative of the coding gene includes at least one of the following: a recombinant expression vector containing the coding gene of the ERF100-6A protein or a recombinant bacterium containing the coding gene of the ERF100-6A protein and a gene expression cassette containing the coding gene of the ERF100-6A protein.

[0030] In the present invention, the backbone vector type of the recombinant expression vector preferably includes at least one of the following: plasmid, cosmid and bacteriophage. The host bacteria type of the recombinant bacteria preferably includes bacteria and / or fungi. The bacteria preferably include Agrobacterium or Escherichia coli. The strain of Agrobacterium is preferably Agrobacterium EHA105 and / or Agrobacterium C58C1. The gene expression cassette preferably includes DNA capable of expressing ERF100-6A protein in host cells. The DNA may include not only a promoter for initiating transcription of ERF100-6A, but also a terminator for terminating transcription of ERF100-6A. The expression cassette preferably also includes an enhancer sequence.

[0031] In an embodiment of the present invention, the construction method of the recombinant expression vector is preferably: the coding gene of the ERF100-6A protein is seamlessly cloned into the backbone vector, verified, and the recombinant expression vector is obtained. The backbone vector of the recombinant expression vector is preferably a pUbi vector, the cloning site is SacI, and the pUbi vector is preserved by Wang Daowen's research group of Henan Agricultural University (reference to the prior art: New Phytologist (2020) 225: 2526-2541 doi: 10.1111 / nph.16305). The verification method is preferably verified by sequencing, and the forward primer qF shown by the nucleotide sequence such as SEQ ID NO: 9 (ATCCCCGGGTACCGAGCTC) and the reverse primer shown in SEQ ID NO: 10 (GTCAGGGTGCAGAGCAGGCA) are preferably used for sequencing. The construction method of the recombinant bacteria is preferably to transfect the recombinant vector into the host bacteria, and the recombinant bacteria are obtained by screening and verification. The transfection method is preferably any one of the following: electric shock method, calcium phosphate method and freeze-thaw method, more preferably freeze-thaw method. The host bacteria is preferably Agrobacterium C58C1, purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd., with a product number of ZC1504. The screening method is preferably screened by antibiotics, and the antibiotics are preferably rifampicin and kanamycin sulfate, and the concentration is preferably 50 mg / mL. The verification method is preferably sequencing verification, and the sequencing is preferably performed using a forward primer shown by a nucleotide sequence such as SEQ ID NO: 11 (ATGCTGCTGCTTAATCCGGC) and a reverse primer shown by a nucleotide sequence such as SEQ ID NO: 10 to amplify the sequence.

[0032] In the present invention, the improvement of plant resistance to powdery mildew preferably includes reducing the number of powdery mildew microcolony spores and / or spore biomass of powdery mildew plants infected with powdery mildew. The plant preferably includes wheat. The present invention does not specifically limit the variety of wheat and the physiological race of the powdery mildew pathogen. In an embodiment of the present invention, the wheat variety is wild-type hexaploid wheat (Fielder), and the powdery mildew pathogen is the E09 race.

[0033] In one embodiment of the present invention, the powdery mildew resistance of erf100-6A mutant material and wild-type Fielder material was analyzed, and the results showed that compared with the wild-type Fielder control, the number of microcolonies of erf100-6A mutant material increased significantly after 3 days of inoculation, the colony area of ​​wheat leaves increased significantly after 7 days of inoculation, and the leaf disease became more severe. It was preliminarily determined that the ERF100-6A gene can regulate wheat resistance to powdery mildew. The present invention further analyzed the powdery mildew resistance of ERF100-6A overexpression transgenic strain and wild-type Fielder, and the results showed that compared with the wild-type Fielder control, the number of microcolonies of ERF100-6A overexpression transgenic strain 3 days after inoculation was extremely significantly reduced, and the analysis of the colony area of ​​wheat leaves after 7 days of inoculation found that the leaf disease of ERF100-6A overexpression transgenic strain was significantly reduced, showing good powdery mildew resistance. Therefore, ERF100-6A can improve the powdery mildew resistance of plants.

[0034] The invention provides a method for improving the resistance of plants to powdery mildew, which increases the expression amount of ERF100-6A protein or a gene encoding the ERF100-6A protein in the plants.

[0035] In the present invention, the method for increasing the expression level of ERF100-6A protein or the gene encoding ERF100-6A protein in plants is to transfer the gene encoding ERF100-6A protein or the derivative of the gene encoding into the plant for overexpression.

[0036] In the present invention, the overexpression method is preferably to introduce the coding gene of the ERF100-6A protein into the recipient plant; more preferably, the above-mentioned recombinant bacteria are infected with the host plant, and the plants with high expression of the ERF100-6A protein or the coding gene of the ERF100-6A protein are screened and detected. The overexpression method is preferably referred to the prior art New Phytologist (2020) 225: 2526-2541 doi: 10.1111 / nph.16305. The detection preferably detects the expression level of the ERF100-6A protein or the coding gene of the ERF100-6A protein. The method for detecting the ERF100-6A protein is preferably detected by immunoblotting, and the reagent for detecting the ERF100-6A protein is preferably a MYC antibody, purchased from ABmart, item number M20002. The reagent for detecting the expression amount of the gene encoding the ERF100-6A protein preferably includes a forward primer qF whose nucleotide sequence is shown in SEQ ID NO:3 (TTCATCTCCCTCCTGCAGCGCG) and a reverse primer qR whose nucleotide sequence is shown in SEQ ID NO:4 (TGCTGGACGGCGCCGGCGGCC). The reaction system for the detection is preferably:

[0037] 2×ChamQ Universal SYBR qPCRMasterMix 10.0μl

[0038] Primer 1 (10 μM) 0.4 μl

[0039] Primer 2 (10 μM) 0.4 μl

[0040] Template cDNA 1μl

[0041] ddH2O 8.2μl

[0042] The reaction procedure preferably comprises:

[0043] Rep:1 95℃ 5min

[0044] Reps:40 95℃ 10s 60℃ 30s*

[0045] 2×ChamQ Universal SYBR qPCRMasterMix was purchased from Novozymes Biotech Co., Ltd. with the trade name Q711 ChamQ Universal SYBR qPCRMasterMix and the catalog number Q711-02.

[0046] When the ERF100-6A protein content in the plant is increased or the expression level of the ERF100-6A gene is increased, the plant's resistance to powdery mildew is improved, which is specifically manifested in a decrease in the number of powdery mildew spores and / or the biomass of powdery mildew spores after inoculation with powdery mildew fungi.

[0047] The present invention provides a method for breeding powdery mildew-resistant plant varieties, which comprises detecting the expression level of the ERF100-6A protein or the gene encoding the ERF100-6A protein in the genome of the plant:

[0048] Plants with upregulated expression of the ERF100-6A protein or the gene encoding the ERF100-6A protein are selected as breeding materials for breeding resistant plant varieties.

[0049] In the present invention, the detection reagent and detection method are preferably the same as the reagent for detecting the expression level of ERF100-6A protein or the gene encoding the ERF100-6A protein used in the above-mentioned method for improving plant resistance to powdery mildew, and are not described in detail here.

[0050] In the present invention, the types of the plant and powdery mildew pathogens are preferably the same as those in the above-mentioned application technical solution, and will not be described in detail here.

[0051] In any of the above-mentioned uses or methods, the plants overexpressing ERF100-6A protein include not only the first generation transgenic plants obtained by transforming the coding gene of ERF100-6A protein into the target plant, but also its progeny. For transgenic plants, the gene can be propagated in the species, or the gene can be transferred into other varieties of the same species, especially including commercial varieties, by conventional breeding techniques. The transgenic plants include seeds, callus tissues, whole plants and cells.

[0052] In order to further illustrate the present invention, the embodiments provided by the present invention are described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be understood as limiting the protection scope of the present invention.

[0053] Example 1

[0054] Acquisition and testing of erf100-6A mutant materials

[0055] 1. Obtaining erf100-6A mutant material

[0056] The erf100-6A mutant material was obtained by gene editing of the common wheat variety Fielder. For specific steps, please refer to the prior art (New Phytologist (2020) 225: 2526-2541 doi: 10.1111 / nph.16305).

[0057] 2. Sequence Analysis of erf100-6A Mutant Materials

[0058] The genomic DNA of the erf100-6A mutant material was extracted, and the sequence containing the ERF100 mutant gene was amplified using a forward primer represented by a nucleotide sequence such as SEQ ID NO:7 (CCACAAAATCCACTGACAAACG) and a reverse primer represented by a nucleotide sequence such as SEQ ID NO:8 (TTCCTCACGTCGCTCAACACAT), such as SEQ ID NO:5 (CCACAAAATCCACTGACAAACGCTCGGCGAATCCCAAGAGCGAACTCA GATCATCCTACGACCAGACGCGACCACACAGGATAAGATGCTGCTGCTTAATCCGGCGTCCGAGGCGGCGGCGCTGGACAGCATCCGGCAGCAGCTCCTGGAGGAGCCGGCGCGGCCGGCGTACTGCCGGAGCGAGCTTCGGCAGCCTGGTGGCGGACCAGTGGAGCGAGTCGCTCCCGTCCGTCCCCAACGACGCCGACGACATGGT CGTCTACGGCGCCCTCCGCGACGCCTTCTCCTGCGGCTGGCTCCCCGACGGCTCCTTCGCGGCCGTCAAGCCCGAGCCCCTGCCCTCCCCCGACGGCTCCTACGACGGCTCCTGCCTCGGCAGCTTCCTCGCGCCGCCGGCGCCCGGGCCGGACGCGCCGTGGGCGGAGGAGGAGGCCGAGGTCGCGGCGGCGGCGTCGAGGGGGAAGCAC TTTCAGAGGCGTGAGGCAGCGGCCGTGGGGCAAGTTCGCGGCGGAGATCCGGGACCCGGCCAAGAACGGCGCGCGCGTGTGGCTCGGCACCTTCGACAGCGCCGAGGACGCCGCCGTGGCCTACGACCGCGCCGCCTACCGCATGCGCGGCTCCCGCGCGCTCCTCAACTTCCCGCTCCGCATCGGCTCCGAGATCGCCGCCGCCGCCGCAGCCGCGGGCCAGAAGCG TCCGTCTCCCCAGCCGGCGAGCCCCGACTCTTCATCTCCCTCCTGCAGCGCGCCCGGGTCGTCGAAGAGGAGAAAGAGAGGCGAGGCCGCGGCAGCGTCCATGGCCATGGCTCTGGTGCCGCCCCCGCCGGCGCAGGCTCCGGTCCAGCTGACCCTCCCAGCCCAGCCGTGGCTGGCCGCCGGCGCCGTCCAGCAGCTGGTGAGCTGAAGCGGCGAAGCGACCAGTGATCGTTCTCACTTCTCACGAGCGATTAGTTGCTTGATGTGTTGAGCGACGTGAGGAA, where the underlined bases are bases with insertion mutations). After analysis, compared with the wild-type wheat Fielder genome sequence, the erf100-6A mutant material has a base T inserted between the 384th and 385th positions of the gene sequence encoding ERF100 on wheat chromosome 6A, resulting in a protein translation frameshift mutation ( Figure 1 (A).

[0059] Example 2

[0060] Acquisition of ERF100 overexpression materials and detection of protein expression

[0061] 1) Construction of the recombinant vector pUbi::ERF100-6A-MYC

[0062] The sequence shown in SEQ ID NO:2 was added with a MYC tag to obtain the ERF100-6A-MYC gene (nucleotide sequence shown in SEQ ID NO:6). The ERF100-6A-MYC gene shown in SEQ ID NO:6 was connected to the SacI site of the pUbi vector using the Infusion method to obtain the recombinant vector pUbi::ERF100-6A-MYC, which was sequenced and verified.

[0063] The primers used for sequencing were pUbi-Seq-f: ATCCCCGGGTACCGAGCTC (SEQ ID NO: 9) and pUbi-Seq-r: GTCAGGGTGCAGAGCAGGCA (SEQ ID NO: 10). Sequencing showed that there was no mutation in the ERF100-6A gene in the recombinant vector.

[0064] 2) Obtaining recombinant bacteria

[0065] The recombinant vector pUbi::ERF100-6A-MYC was transformed into Agrobacterium C58C1 to obtain the recombinant bacteria pUbi::ERF100-6A-MYC / C58C1.

[0066] The specific conversion method is as follows:

[0067] Take the competent Agrobacterium C58C1 stored at -70℃ and place it in room temperature or ice water bath for a while until it partially melts. When it is in the ice-water mixing state, insert it into the ice bath; add 1μg plasmid DNA for every 100μl competent state, stir the bottom of the tube by hand to mix, and place it on ice for 5min, liquid nitrogen for 5min, 37℃ water bath for 5min, and ice bath for 5min in sequence;

[0068] After an ice bath, add 800 μl of TY liquid culture medium and culture at 28°C with shaking for 2 h; collect the bacteria by centrifugation at 5000 rpm for 1 min, retain about 100 μl of supernatant, gently blow to resuspend the bacteria, spread on a TY plate containing the corresponding antibiotics, and invert and culture in a 28°C incubator for 3 days.

[0069] 3) Obtaining pUbi::ERF100-6A-MYC transgenic wheat

[0070] The recombinant bacteria pUbi::ERF100-6A-MYC / C58C1 was used to infect the wild-type hexaploid spring wheat Fielder in good growth condition, and genetic transformation was carried out by embryo stripping. After callus differentiation, the callus was screened to obtain the ERF100-6A transgenic wheat line.

[0071] The method for wheat genetic transformation is as follows:

[0072] Take the seeds with embryos as small as 2 mm and place them at 4°C. Wash the seeds with 75% alcohol for 1 min, place them in 15% NaClO solution for shaking culture for about 10 min, wash them with sterile water 4 times, peel the embryos, place the peeled embryos in WLS culture medium, centrifuge them at 4°C and 7500 rpm for 10 min, remove the supernatant, and add WLS to clean them.

[0073] Infection: The recombinant bacteria were inoculated into 4 mL of TY culture medium containing rifampicin and kanamycin and shaken at 28°C overnight. The bacteria were collected and resuspended with WLS. The resuspended bacteria were added to the embryos and allowed to stand for 5 minutes. The bacteria were removed and the embryos were placed on sterilized filter paper. After absorbing the bacteria, the embryos were placed on AS medium with the scutellum facing down and grown in the dark at 23°C for 48 hours. After cutting the tip, the incision was placed on the recovery medium with the incision facing down for 5 days and transferred to a sieve plate. After 14 days, the callus was cut in half and transferred to a second sieve medium. After 20 days, the callus was observed for differentiation. After budding, the plants were transferred to differentiation medium. After 14 days, the plants were transferred to 1 / 2MS medium to allow them to root. After rooting, the ERF100-6A overexpressing transgenic lines ERF100-OE12# and ERF100-OE19# were obtained.

[0074] For the specific steps of the wheat genetic transformation method, please refer to the prior art (New Phytologist (2020) 225: 2526-2541 doi: 10.1111 / nph.16305).

[0075] 4) Gene level identification

[0076] The expression levels of the gene encoding ERF100-6A protein in the wild-type hexaploid spring wheat Fielder and the above-mentioned ERF100-6A overexpressing transgenic line were detected using the forward primer qF with a nucleotide sequence as shown in SEQ ID NO:3 and the reverse primer qR with a nucleotide sequence as shown in SEQ ID NO:4. The results showed that the expression level of the gene encoding ERF100-6A protein in the ERF100-6A overexpressing transgenic line was higher than that in the wild-type hexaploid spring wheat Fielder.

[0077] 5) Protein level identification

[0078] The crude proteins of wild-type hexaploid spring wheat Fielder and the above-mentioned ERF100-6A overexpressing transgenic lines were extracted respectively, and the expression level of ERF100-6A protein was detected by Western-blot.

[0079] The specific method of plant crude protein extraction is as follows: weigh 2g wheat leaves and grind them thoroughly with liquid ammonia; add 2mL Lysis Buffer and shake thoroughly to mix. Place on ice for 1h to fully lyse. After lysis, centrifuge at 4°C and 15000g for 25min. Aspirate the supernatant into a new tube, take an appropriate amount of protein and add 2× Loading Buffer to denature it. Lysis Buffer was purchased from Thermo Fisher Scientific (China) Co., Ltd., named NP40 Cell Lysis Buffer Native Buffer I, and the catalog number is FNN0021.

[0080] The specific method of Western-blot is as follows:

[0081] (1) Load 10 μl of sample and electrophoresis at 80 V for 30 min and 120 V for 1 h;

[0082] (2) Transfer: prepare the NC membrane and filter paper, transfer buffer, transfer electrophoresis tank, and ice in advance;

[0083] (3) After removing the glue, cut it into 8×4 cm pieces;

[0084] (4) Pour an appropriate amount of transfer buffer into the disk, soak a sponge and press it on the black plywood;

[0085] (5) Place two soaked filter sheets on the sponge, and then place the glue, membrane, and two filter sheets in sequence;

[0086] (6) Use a roller to remove bubbles, spread a sponge, press tightly and place in the transfer tank, and run at a constant current of 300 mA for 2 h;

[0087] (7) Prepare TBST and 5% milk, place the membrane with the transfer surface facing up in the milk, and block for 2 h;

[0088] (8) Prepare primary anti-MYC antibody (10 mL milk + 2 μl primary antibody), place the blocked membrane face-down inward in a 15 mL centrifuge tube, and add primary antibody;

[0089] (9) Tied to a vertical mixer at 4°C overnight;

[0090] (10) Wash the membrane 4 times with TBST, 10 min each time;

[0091] (11) Incubate with secondary antibody, same as primary antibody step, and wash the membrane (same as step 10);

[0092] (12) Place the film with the front side facing up on the card film, drop ECL (AB mixed in advance), and expose and develop in a closed manner.

[0093] The primary antibody was MYC antibody, purchased from ABmart with the catalog number M20002; the secondary antibody was Anti-lgG (H+L chain) (mouse) pAb-HRP, purchased from MBL with the catalog number 330.

[0094] Western-blot test results are as follows Figure 2 As shown in A, the results showed that the accumulation of ERF100-6A protein in the ERF100-6A overexpressing transgenic lines ERF100-OE12# and ERF100-OE19# was significantly increased compared with the control Fielder.

[0095] Example 3

[0096] Disease resistance testing

[0097] The erf100-6A mutant material in Example 1, the ERF100-6A overexpressing transgenic strain in Example 2 and the wild-type Fielder were respectively subjected to powdery mildew resistance phenotype identification. The specific operation is as follows:

[0098] (1) Wheat powdery mildew inoculation

[0099] Each group of wheat seeds were cultured in a light incubator with a photoperiod of 16h light / 8h dark and a temperature of 20°C. Wheat seedlings grown to the one-leaf stage were used for inoculation. The leaves were first sprayed with water mist, and then the E09 spores were used for full and uniform friction inoculation according to the experimental requirements.

[0100] (2) Phenotypic identification of wheat powdery mildew after inoculation

[0101] Three days after inoculation, the leaves were cut into 5 cm segments and stained with 0.6% Coomassie Brilliant Blue. For each inoculation, 10-15 leaf segments were taken from at least 10 seedlings for microcolony statistics, and the microcolony index was calculated according to Formula I.

[0102] Microcolony index = spores that developed microcolonies / total number of spores detected × 100% Formula I.

[0103] Furthermore, 7 days after inoculation, the leaves with the docking bacteria were photographed to identify the development of the Bgt colony, and the leaves were scanned using an ImageScanner III scanner, and the Bgt colony area was calculated using Image J software.

[0104] For the specific steps of disease resistance detection, please refer to the prior art (Zheng, Hongyuan; Dong, Lingli; Han, Xinyun; Jin, Huaibing; Yin, Cuicui; Han, Yali; Li, Bei; Qin, Huanju; Zhang, Jinsong; Shen, Qianhua; Zhang, Kunpu; Wang, Daowen. The TuMYB46L-TuACO3 module regulates ethylene biosynthesis in einkorn wheat defense to powdery mildew. New Phytologist (2020) 225: 2526-2541 doi: 10.1111 / nph.16305).

[0105] The results showed that the number of microcolonies in the erf100-6A mutant material increased significantly compared with the wild-type Fielder control 3 days after inoculation ( Figure 1 D and E), the colony area on wheat leaves increased significantly 7 days after inoculation, and the disease on the leaves became more severe ( Figure 1 B and C). It can be seen that ERF100-6A can regulate wheat powdery mildew resistance.

[0106] Compared with the wild-type Fielder control, the number of microcolonies in the ERF100-6A overexpressing transgenic strain was significantly reduced 3 days after inoculation ( Figure 2 D and E), analysis of the colony area of ​​wheat leaves 7 days after inoculation showed that the leaf disease of the ERF100-6A overexpressing transgenic line was significantly reduced ( Figure 2 The experimental results further verified that ERF100-6A can improve wheat disease resistance.

[0107] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A use of an ERF100-6A protein, a gene encoding the ERF100-6A protein or a derivative of the gene encoding the ERF100-6A protein in at least one of improving plant resistance to powdery mildew, cultivating powdery mildew-resistant transgenic plants and breeding powdery mildew-resistant plant varieties, wherein the ERF100-6A protein is at least one of the following proteins; 1) a protein having an amino acid sequence as shown in SEQ ID NO: 1; 2) A fusion protein formed by the protein described in item 1) and a tag sequence.

2. The application according to claim 1, characterized in that: The improving the resistance of plants to powdery mildew comprises reducing the number of spores and / or spore biomass of powdery mildew microcolonies infecting powdery mildew plants.

3. The application according to claim 1, characterized in that: The nucleotide sequence of the gene encoding the ERF100-6A protein is shown in SEQ ID NO:

2.

4. The use according to claim 1, characterized in that: The derivative of the coding gene includes at least one of the following: a recombinant expression vector containing the coding gene of the ERF100-6A protein or a recombinant bacterium containing the coding gene of the ERF100-6A protein and a gene expression cassette containing the coding gene of the ERF100-6A protein.

5. The use according to any one of claims 1 to 4, characterized in that: The plants include wheat.

6. A method for improving plant resistance to powdery mildew, characterized in that: The method is to increase the expression level of the ERF100-6A protein or the gene encoding the ERF100-6A protein according to claim 1 in plants.

7. The method according to claim 6, characterized in that: The method for increasing the expression level of ERF100-6A protein or the gene encoding the ERF100-6A protein in the plant is to transfer the gene encoding the ERF100-6A protein or the derivative of the gene encoding the gene into the plant for overexpression.

8. A method for breeding powdery mildew resistant plant varieties, characterized in that: Detecting the expression level of the ERF100-6A protein or the gene encoding the ERF100-6A protein according to claim 1 in the genome of a plant: Plants with upregulated expression of the ERF100-6A protein or the gene encoding the ERF100-6A protein are selected as breeding materials for breeding resistant plant varieties.

9. The method according to claim 8, characterized in that: The detection reagents include reagents for detecting ERF100-6A protein and / or reagents for detecting genes encoding ERF100-6A protein; The reagent for detecting ERF100-6A protein includes MYC antibody; The reagent for detecting the gene encoding the ERF100-6A protein comprises a forward primer qF having a nucleotide sequence as shown in SEQ ID NO:3 and a reverse primer qR having a nucleotide sequence as shown in SEQ ID NO:

4.

10. The method according to any one of claims 6 to 9, characterized in that: The plants include wheat.

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