Application of TaE3-3A protein or coding gene thereof in improving powdery mildew resistance of plants

Abstract

The invention provides application of TaE3-3A 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 application of a TaE3-3A protein or a coding gene of the TaE3-3A protein to 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, TaE3-3A gene silencing wheat and TaE3-3A gene overexpression wheat are respectively used as research objects, and phenotypes after powdery mildew inoculation are detected. Results show that TaE3-3A gene silencing wheat shows relatively high resistance, and TaE3-3A gene overexpression wheat is more sensitive to powdery mildew pathogenic bacteria. Therefore, the powdery mildew resistance of the plants can be improved through TaE3-3A gene silencing.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology, and specifically relates to application of TaE3-3A protein or its encoding gene 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 for controlling 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 a TaE3-3A protein or a gene encoding the TaE3-3A protein for use 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 a TaE3-3A protein or a gene encoding the TaE3-3A protein for use 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 TaE3-3A protein is at least one of the following proteins:

[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 nucleotide sequence of the gene encoding the TaE3-3A protein is shown in SEQ ID NO:2.

[0008] 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.

[0009] Preferably, the plant comprises wheat.

[0010] The invention provides a method for improving the resistance of plants to powdery mildew, which inhibits the expression of the TaE3-3A protein or the coding gene of the TaE3-3A protein in the plants.

[0011] Preferably, the method for inhibiting the expression of TaE3-3A protein in a plant comprises silencing the target region of the gene encoding the plant TaE3-3A protein using gene silencing technology;

[0012] The nucleotide sequence of the target region is shown in SEQ ID NO:3.

[0013] Preferably, the gene silencing technology includes BSMV-VIGS technology.

[0014] The present invention provides a method for breeding powdery mildew-resistant plant varieties, which comprises detecting the expression level of the TaE3-3A protein or the gene encoding the TaE3-3A protein in the genome of the plant:

[0015] Plants in which the expression level of the TaE3-3A protein or the gene encoding the TaE3-3A protein is downregulated are selected as breeding materials for breeding resistant plant varieties.

[0016] Preferably, the detection reagent comprises a forward primer qF having a nucleotide sequence as shown in SEQ ID NO:4 and a reverse primer qR having a nucleotide sequence as shown in SEQ ID NO:5.

[0017] Preferably, the plant comprises wheat.

[0018] Beneficial effects:

[0019] The present invention provides an application of a TaE3-3A protein or a gene encoding the TaE3-3A 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 TaE3-3A protein or a gene encoding the TaE3-3A protein or a derivative of the gene encoding can negatively regulate wheat powdery mildew resistance. The present invention uses TaE3-3A gene-silenced wheat and TaE3-3A gene-overexpressed wheat as research objects, respectively, and detects the phenotype after powdery mildew inoculation. The results show that compared with the wild-type Fielder control, the TaE3-3A gene-silenced strain has a very significantly reduced number of microcolonies 3 days after inoculation, and the disease resistance of wheat powdery mildew is significantly enhanced. The experimental results show that the TaE3-3A gene plays a negative regulatory role in the process of wheat resisting powdery mildew. Compared with the wild-type Fielder control, the TaE3-3A overexpressed transgenic strain has a very significantly increased number of microcolonies 3 days after inoculation. At the same time, analysis of the colony area of ​​wheat leaves 7 days after inoculation found that the leaf disease of the TaE3-3A overexpressing transgenic strain was significantly aggravated. Therefore, TaE3-3A gene silencing can improve the plant's resistance to powdery mildew, and TaE3-3A protein and its encoding gene will play an important role in breeding disease-resistant wheat varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of the test of the powdery mildew resistance of wheat TaE3-3A gene-silenced strains and wild-type Fielder materials; A is the change of gene expression in TaE3-3A gene-silenced strains, B is the leaf microcolony phenotype of TaE3-3A gene-silenced strains and wild-type Fielder materials inoculated with powdery mildew for 3 days, and C is the powdery mildew microcolony index statistical graph of TaE3-3A mutant materials and wild-type Fielder materials 3 days after inoculation with powdery mildew;

[0021] Figure 2 Figure 3 is a graph showing the results of the powdery mildew resistance test of wheat TaE3-3A overexpressing transgenic lines compared with the control wild-type Fielder; A is the results of the detection of TaE3-3A protein and coding genes in TaE3-3A overexpressing transgenic lines and wild-type Fielder, B is the microcolony phenotype of the leaves of TaE3-3A overexpressing transgenic lines and wild-type Fielder materials 3 days after inoculation with powdery mildew, C is the powdery mildew microcolony index statistical graph of TaE3-3A overexpressing transgenic lines and wild-type Fielder materials 3 days after inoculation with powdery mildew, D is the powdery mildew spore phenotype of TaE3-3A overexpressing transgenic lines and wild-type Fielder materials 7 days after inoculation with powdery mildew, and E is the powdery mildew spore area index statistical graph of TaE3-3A overexpressing transgenic lines and wild-type Fielder 7 days after inoculation with powdery mildew. DETAILED DESCRIPTION

[0022] The present invention provides a TaE3-3A protein or a gene encoding the TaE3-3A protein for use 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 TaE3-3A protein is at least one of the following proteins:

[0023] 1) Amino acid sequence such as SEQ ID NO: 1 (MAYRMVCAPQVIDLESERGHPHVHSESLIHNGNDLSDQGSQYTVRVVGNA MNAGLSDTQGYYNMSMNHPHQPVHSSPPNLGVDSGFVFPSNMYNPCMSTSMNRYVSHAQSFGLPLNQVVSGSMDGSTRNENVSETARGFIKRKNAAVAGTCHFVNGSASSSSSSHTSQNPT HRPWDPSFESNVFPNVTPFNPSEYHSHSTWPSVEGSSITGSNGFNLMAAHPESAQHGNYTFQSSHASQCFQPASTTWVSQAATGIVEGVPQWSYINAMSSVPGRFAHSGATEIVNGGFHEY QNGPSTISQGHLPYFHQHAVHSMQPHNPLDHTRVQVPYQQGHNNGILHSGVNHSGNRFHLSPRTPGLFSNSERSFGPPQHPFLVNPVNHRNLRILPPLQHGAIMDFSRLYEGSNVVDEHRD The protein shown by MRLDIDSMTYEELLALEEHIGDVNTGLAKSHIVDKLKTSLYVPGATCVSNQSSEPSTENEACIICQEEYEAEDCVGILDCGHRYHAVCLKQWLTVKNLCPICKTTALSAGRRS);

[0024] 2) A fusion protein formed by the protein described in item 1) and a tag sequence.

[0025] 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. The tag sequence is preferably connected to the 5' or 3' end of the TaE3-3A protein encoding gene.

[0026]

[0027] 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.

[0028] In the examples of the present invention, TaE3-3A gene-silenced wheat and TaE3-3A gene-overexpressed wheat were used as research objects, and the phenotypes after powdery mildew inoculation were detected. The results showed that compared with wild-type Fielder, TaE3-3A gene-silenced wheat showed higher resistance, and TaE3-3A gene-overexpressed wheat was more sensitive to powdery mildew pathogens. Therefore, TaE3-3A gene silencing can improve plant resistance to powdery mildew.

[0029] The invention provides a method for improving the resistance of plants to powdery mildew, which inhibits the expression of the TaE3-3A protein or the coding gene of the TaE3-3A protein in the plants.

[0030] In the present invention, the method for reducing the expression level of TaE3-3A protein in plants preferably comprises silencing the target region of the gene encoding the plant TaE3-3A protein by using gene silencing technology; the nucleotide sequence of the target region is shown in SEQ ID NO:3 (CTGCTCACCCAGAATCAGCACAGCATGGTAACTATACATTTCAATCAAGT CATGCTAGTCAGTGCTTTCAACCTGCCAGCACTACTTGGGTTTCCCAGGCT GCAACTGGAATTGTGGAAGGAGTAC). The gene silencing technology comprises BSMV-VIGS technology.

[0031] In the present invention, the specific method of BSMV-VIGS technology preferably uses the cloned Ta-E3-3A protein coding gene (nucleotide sequence as shown in SEQ ID NO: 2) as a template, and uses the primer BSMV-VIGS-TuE3-f as shown in the nucleotide sequence of SEQ ID NO: 8 (GATTCTTCTTCCGTTGCTAGCCTGCTCACCCAGAATCAGCAC) and the nucleotide sequence BSMV-VIGS-TuE3-r as shown in SEQ ID NO: 9 (TTTTTTTTTTTTTTAGCTAGCGTACTCCTTCCACAATTCCA) to amplify the silencing sequence shown in SEQ ID NO: 10 (GATTCTTCTTCCGTTGCTAGCCTGCTCACCCAGAATCAGCACAGCATGGT AACTATACATTTCAATCAAGTCATGCTAGTCAGTGCTTTCAACCTGCCAGC ACTACTTGGGTTTCCCAGGCTGCAACTGGAATTGTGGAAGGAGTACGCTA GCTAAAAAAAAAAAAAA). The silencing sequence is forward inserted into the γ vector (the γ part of the BSMV viral vector), and the obtained recombinant plasmid pCaBS-γ: TaE3as is obtained. pCaBS-α, pCaBS-β and the recombinant plasmid pCaBS-γ: TaE3as are linearized and transcribed in vitro using MluI, SpeI and MluI respectively, and the transcription products of pCaBS-α, pCaBS-β and pCaBS-γ: TaE3as are prepared into a mixed solution to inoculate the plant with the virus to obtain plants with reduced expression of the TaE3-3A gene or protein. The method of inserting the silencing sequence into the γ vector is preferably completed by enzyme digestion, preferably by double digestion and ligation with PacI and NotI. The pCaBS-α, pCaBS-β and γ vectors are from Wang Daowen Laboratory of Henan Agricultural University (see prior art: New Phytologist (2020) 225: 2526-2541 doi: 10.1111 / nph.16305). The reagents used to detect the recombinant plasmid pCaBS-γ:TaE3as include a forward primer pCa-γ-seq-f having a nucleotide sequence as shown in SEQ ID NO:11 (CACAGTTGTGGAATGCCATGCTC) and a reverse primer pCa-γ-seq-r having a nucleotide sequence as shown in SEQ ID NO:12 (CGAGCTCCTGCAGGACAGTC).The reagents for detecting the expression of the TaE3-3A gene in the plant include a forward primer RT-VIGS-E3-f having a nucleotide sequence as shown in SEQ ID NO:4 (CCACATCAGCCTGTTCATAGTT) and a reverse primer RT-VIGS-E3-r having a nucleotide sequence as shown in SEQ ID NO:5 (GCTCTGAGCATGAGAGACATATC). The plant preferably includes a plant with reduced expression of the TaE3-3A gene or protein.

[0032] The present invention provides a method for reducing plant resistance to powdery mildew by transferring the TaE3-3A protein or the coding gene of the TaE3-3A protein into the plant for overexpression. The overexpression method is preferably to transfect the host bacteria with a recombinant expression vector containing the coding gene of the TaE3-3A protein to obtain a recombinant bacterium, and to infect the host plant with the recombinant bacterium to obtain a plant with a high expression level of the TaE3-3A protein or the coding gene of the TaE3-3A protein. The types of the backbone vectors of the recombinant expression vector preferably include at least one of the following: plasmid, clay and phage. The construction method of the recombinant expression vector is preferably: seamlessly cloning the coding gene of the TaE3-3A protein into the backbone vector to obtain a recombinant expression vector. 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 the Wang Daowen research group of Henan Agricultural University (New Phytologist (2020) 225: 2526-2541 doi: 10.1111 / nph.16305). The host bacteria species of the recombinant bacteria preferably include bacteria and / or fungi. The bacteria preferably include Agrobacterium or Escherichia coli. The method for constructing the recombinant bacteria is preferably to transfect the recombinant vector into the host bacteria, and screen and verify to obtain the recombinant bacteria. In an embodiment of the present invention, the host bacteria is preferably Agrobacterium C58C1, purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd., with the product number ZC1504. The method of infection is preferably infection by the embryo stripping method. The method of transfection is preferably the freeze-thaw method. The expression level of the TaE3-3A protein is preferably detected by immunoblotting, and the reagent for detecting the TaE3-3A protein preferably includes HA antibodies.

[0033] The present invention provides a method for breeding powdery mildew-resistant plant varieties, which comprises detecting the expression level of the TaE3-3A protein or the gene encoding the TaE3-3A protein in the genome of the plant:

[0034] Plants in which the expression level of the TaE3-3A protein or the gene encoding the TaE3-3A protein is downregulated are selected as breeding materials for breeding resistant plant varieties.

[0035] In the present invention, the reagent for detecting the TaE3-3A protein preferably includes HA antibody, and the reagent for detecting the expression level of the gene encoding the TaE3-3A protein preferably includes a forward primer qF with a nucleotide sequence as shown in SEQ ID NO:4 and a reverse primer qR with a nucleotide sequence as shown in SEQ ID NO:5.

[0036] 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.

[0037] In any of the above-mentioned applications or methods, the plants silencing or overexpressing TaE3-3A protein include not only the first generation transgenic plants obtained by transforming the coding gene of TaE3-3A 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 commercial varieties, by conventional breeding techniques. The transgenic plants include seeds, callus tissues, complete plants and cells.

[0038] 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.

[0039] Example 1

[0040] Obtaining and testing TaE3-3A gene silenced plants

[0041] 1. Obtaining TaE3-3A gene silenced plants

[0042] The coding gene of the cloned Ta-E3-3A protein (nucleotide sequence as shown in SEQ ID NO: 2) was used as a template, and primers BSMV-VIGS-TuE3-f (SEQ ID NO: 8GATTCTTCTTCCGTTGCTAGCCTGCTCACCCAGAATCAGCAC) and BSMV-VIGS-TuE3-r (SEQ ID NO: 9TTTTTTTTTTTTTTAGCTAGCGTACTCCTTCCACAATTCCA) were used to amplify the silencing sequence shown in SEQ ID NO: 10. PCR reaction system: 10× Buffer 5.0 μl, dNTP 5.0 μl, 1.5 μl of upstream and downstream primers (concentration 10 uM), cDNA 1.0 μl, Taq enzyme 1.0 μl (1.0 U), and finally made up to 50.0 μl with water. PCR reaction program: 94°C 2min; then 98°C 10s, 60°C 30s, 68°C 1min, 33 cycles.

[0043] The silencing sequence was inserted between the restriction sites PacI and NotI of the γ vector pCaBS-γ (the γ part of the BSMV viral vector) by restriction ligation to obtain the recombinant plasmid pCaBS-γ: TaE3as.

[0044] The primers used to detect the recombinant plasmid pCaBS-γ:TaE3as are the forward primer pCa-γ-seq-f with a nucleotide sequence as shown in SEQ ID NO:11 (CACAGTTGTGGAATGCCATGCTC) and the reverse primer pCa-γ-seq-r with a nucleotide sequence as shown in SEQ ID NO:12 (CGAGCTCCTGCAGGACAGTC).

[0045] pCaBS-α, pCaBS-β and the recombinant plasmid pCaBS-γ:TaE3as were further linearized, and the linearized fragments were transcribed in vitro to obtain the transcription products of pCaBS-α, pCaBS-β and pCaBS-γ:TaE3as.

[0046] Subsequently, the transcription products were configured into infection premix for virus inoculation, and the second leaf of wheat was inoculated at the two-leaf stage. The inoculation method adopted the friction inoculation method, with pCaBS-α, pCaBS-β and pCaBS-γ linearized, and the infection premix configured with the transcription products was inoculated as a control.

[0047] 2. Detection of TaE3-3A gene silenced plants

[0048] 14 days after virus inoculation, the relative expression of wheat TaE3-3A gene was detected. The nucleotide sequence of the primer pair for detecting the relative expression of TaE3-3A is as follows:

[0049] The primer used to detect the expression of the TaE3-3AGS gene is a nucleotide sequence such as SEQ ID NO:4

[0050] The forward primer RT-VIGS-E3-f is shown in (CCACATCAGCCTGTTCATAGTT) and the reverse primer RT-VIGS-E3-r has a nucleotide sequence shown in SEQ ID NO: 5 (GCTCTGAGCATGAGAGACATATC).

[0051] The reaction system for the test is: 2×ChamQ Universal SYBRqPCRMasterMix 10.0μl

[0052] Primer 1 (10 μM) 0.4 μl

[0053] Primer 2 (10 μM) 0.4 μl

[0054] Template cDNA 1μl

[0055] ddH2O 8.2μl

[0056] The reaction procedure is:

[0057] Rep:1 95℃ 5min

[0058] Reps:40 95℃ 10s 60℃ 30s*

[0059] 2×ChamQ Universal SYBR qPCRMasterMix was purchased from Novozymes Biotech Co., Ltd. with the trade name of ChamQ Universal SYBR qPCRMasterMix and the product number of Q711-02.

[0060] The results showed that the expression of TaE3-3A gene in BSMV:TaE3-3Aas-1 and BSMV:TaE3-3Aas-2 was significantly downregulated compared with BSMV:EVC ( Figure 1 (A). This indicates that after virus inoculation, the expression of the TaE3-3A gene in the TaE3-3A gene-silenced plants BSMV:TaE3-3Aas-1 and BSMV:TaE3-3Aas-2 was silenced.

[0061] Example 2

[0062] Acquisition of TaE3-3A overexpression materials and detection of protein expression

[0063] 1) Construction of the recombinant vector pUbi::TaE3-3A-HA

[0064]

[0065] The TaE3-3A-HA gene with a nucleotide sequence as shown in SEQ ID NO: 13 was connected to the SacI site of the pUbi vector by the Infusion method to obtain the recombinant vector pUbi::TaE3-3A-HA, which was then sequenced and verified.

[0066] The primer pair used for sequencing was a forward primer pUbi-Seq-f having a nucleotide sequence as shown in SEQ ID NO: 14 (ATCCCCGGGTACCGAGCTC) and a reverse primer pUbi-Seq-r having a nucleotide sequence as shown in SEQ ID NO: 15 (GTCAGGGTGCAGAGCAGGCA).

[0067] Sequencing showed that there was no mutation in the TaE3-3A gene in the recombinant vector.

[0068] 2) Obtaining recombinant bacteria

[0069] The recombinant vector pUbi::TaE3-3A-HA was transferred into Agrobacterium C58C1 to obtain the recombinant bacteria pUbi::TaE3-3A-HA / C58C1. The primer pair for detecting the recombinant bacteria was the forward primer pUbi-Seq-f having a nucleotide sequence as shown in SEQ ID NO:14 and the reverse primer pUbi-Seq-r having a nucleotide sequence as shown in SEQ ID NO:15.

[0070] The specific conversion method is as follows:

[0071] 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;

[0072] 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.

[0073] 3) Obtaining pUbi::TaE3-3A-HA transgenic wheat

[0074] The recombinant bacteria pUbi::TaE3-3A-HA / C58C1 was used to infect the wild-type hexaploid spring wheat Fielder with good growth status, and genetic transformation was carried out by embryo stripping. After callus differentiation, the transgenic wheat lines with TaE3-3A were obtained by screening.

[0075] The method for wheat genetic transformation is as follows:

[0076] 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, put them in 15% NaClO solution and shake for about 10 min, wash them with sterile water 4 times and peel the embryos. Put 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.

[0077] 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 culture 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 callus was transferred to a differentiation medium. After 14 days, the plants were transferred to 1 / 2MS culture medium to allow them to root. After rooting, the TaE3-3A overexpression transgenic lines TaE3-3A-OE1 and TaE3-3A-OE2 were obtained.

[0078] 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).

[0079] 4) Gene level identification

[0080] The expression level of the gene encoding TaE3-3A protein in the wild-type hexaploid spring wheat Fielder and the above-mentioned TaE3-3A overexpression transgenic line was detected using the forward primer qF as shown in the nucleotide sequence of SEQ ID NO:16 (ATGGCATATCGAATGGTATGC) and the reverse primer qR as shown in the nucleotide sequence of SEQ ID NO:17 (CGTAATCGGGCACATCATAAG). The results showed that the expression level of the gene encoding TaE3-3A protein in the TaE3-3A overexpression transgenic line was higher than that in the wild-type hexaploid spring wheat Fielder ( Figure 2 (A).

[0081] The reaction system for detecting the expression level of the gene encoding the TaE3-3A protein is:

[0082] 2×ChamQ Universal SYBR qPCRMasterMix 10.0μl

[0083] Primer 1 (10 μM) 0.4 μl

[0084] Primer 2 (10 μM) 0.4 μl

[0085] Template cDNA 1μl

[0086] ddH2O 8.2μl

[0087] The reaction procedure is:

[0088] Rep:1 95℃ 5min

[0089] Reps:35 95℃ 30s 58℃ 30s 72℃ 1min30s

[0090] Rep:1 72℃ 10min

[0091] 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.

[0092] 5) Protein level identification

[0093] The crude proteins of wild-type hexaploid spring wheat Fielder and the above-mentioned TaE3-3A overexpressing transgenic lines TaE3-3A-OE1 and TaE3-3A-OE2 were extracted respectively, and the expression level of TaE3-3A protein was detected by Western-blot.

[0094] 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.

[0095] The specific method of Western-blot is as follows:

[0096] (1) Load 10 μl of sample and electrophoresis at 80 V for 30 min and 120 V for 1 h;

[0097] (2) Transfer: prepare the NC membrane and filter paper, transfer buffer, transfer electrophoresis tank, and ice in advance;

[0098] (3) After removing the glue, cut it into 8×4 cm pieces;

[0099] (4) Pour an appropriate amount of transfer buffer into the disk, soak a sponge and press it on the black plywood;

[0100] (5) Place two soaked filter sheets on the sponge, and then place the glue, membrane, and two filter sheets in sequence;

[0101] (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;

[0102] (7) Prepare TBST and 5% milk, place the membrane with the transfer surface facing up in the milk, and block for 2 h;

[0103] (8) Prepare primary anti-HA antibody (10 mL milk + 2 μl primary antibody), place the blocked membrane face inward in a 15 mL centrifuge tube, and add primary antibody;

[0104] (9) Tied to a vertical mixer at 4°C overnight;

[0105] (10) Wash the membrane 4 times with TBST, 10 min each time;

[0106] (11) Incubate with secondary antibody, same as primary antibody step, and wash the membrane (same as step 10);

[0107] (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.

[0108] The primary antibody was Anti-HA-tag mAb antibody, purchased from MBL, catalog number M180-3, and the secondary antibody was Anti-lgG (H+Lchain) (mouse) pAb-HRP, purchased from MBL, catalog number 330.

[0109] Western-blot test results are as follows Figure 2 As shown in A, the results showed that the accumulation of TaE3-3A protein in the TaE3-3A overexpressing transgenic lines TaE3-3A-OE1 and TaE3-3A-OE2 was significantly increased compared with the control Fielder.

[0110] Example 3

[0111] Disease resistance testing

[0112] The TaE3-3A gene silencing strain in Example 1, the TaE3-3A 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:

[0113] (1) Wheat powdery mildew inoculation

[0114] 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.

[0115] (2) Phenotypic identification of wheat powdery mildew after inoculation

[0116] 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.

[0117] Microcolony index = spores that developed microcolonies / total number of spores detected × 100%.

[0118] Seven days after inoculation, the leaves with the bacteria were photographed to identify the development of the Bgt colony. The leaves were scanned using an ImageScanner III scanner, and the Bgt colony area was calculated using Image J software.

[0119] For the specific steps of disease resistance detection, please refer to the prior art (New Phytologist (2020) 225: 2526-2541 doi: 10.1111 / nph.16305).

[0120] The results showed that the number of microcolonies in the TaE3-3A gene-silenced strain was significantly reduced compared with the wild-type Fielder control 3 days after inoculation ( Figure 1 In B and C), the disease resistance of wheat powdery mildew was significantly enhanced. The experimental results showed that the TaE3-3A gene played a negative regulatory role in the process of wheat resistance to powdery mildew.

[0121] Compared with the wild-type Fielder control, the number of microcolonies in the TaE3-3A overexpressing transgenic strain increased significantly 3 days after inoculation ( Figure 2 D and E). At the same time, analysis of the colony area on wheat leaves 7 days after inoculation showed that the colony area on the leaves of the TaE3-3A overexpressing transgenic strain increased, and the disease was significantly aggravated ( Figure 2The experimental results further verified that the TaE3-3A gene inhibits wheat powdery mildew resistance.

[0122] 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 a TaE3-3A protein or a gene encoding the TaE3-3A 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 TaE3-3A 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 nucleotide sequence of the gene encoding the TaE3-3A protein is shown in SEQ ID NO:

2.

3. 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.

4. The use according to any one of claims 1 to 3, characterized in that: The plants include wheat.

5. A method for improving plant resistance to powdery mildew, characterized in that: Inhibiting the expression of the TaE3-3A protein or the gene encoding the TaE3-3A protein according to claim 1 in a plant.

6. The method according to claim 5, characterized in that: The method for inhibiting the expression of TaE3-3A protein in plants comprises silencing the target region of the gene encoding the plant TaE3-3A protein using gene silencing technology; The nucleotide sequence of the target region is shown in SEQ ID NO:

3.

7. The method according to claim 6, characterized in that: The gene silencing technology includes BSMV-VIGS technology.

8. A method for breeding powdery mildew resistant plant varieties, characterized in that: Detecting the expression level of the TaE3-3A protein or the gene encoding the TaE3-3A protein according to claim 1 in the genome of a plant: Plants in which the expression level of the TaE3-3A protein or the gene encoding the TaE3-3A protein is downregulated are selected as breeding materials for breeding resistant plant varieties.

9. The method according to claim 8, characterized in that: The detection reagent includes a forward primer qF having a nucleotide sequence as shown in SEQ ID NO:4 and a reverse primer qR having a nucleotide sequence as shown in SEQ ID NO:

5.

10. The method according to any one of claims 5 to 9, characterized in that: The plants include wheat.

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