Wheat powdery mildew resistance gene TaGAP1 as well as encoding protein and application thereof

Abstract

The invention provides a wheat powdery mildew resistance gene TaGAP1 as well as an encoding protein and application thereof. The TaGAP1 is silenced through a BSMV-mediated instantaneous silencing technology, after powdery mildew E09 is inoculated, the number of powdery mildew colonies of a silenced plant is obviously increased, the powdery mildew grows and develops faster than that of a control plant, and it is indicated that the silent TaGAP1 reduces the resistance of wheat to powdery mildew; the bacterial colony area and biomass of powdery mildew of overexpressed TaGAP1 are remarkably reduced, and the resistance of wheat to powdery mildew is remarkably enhanced, so that TaGAP1 can be used as an effective gene resource for improving the powdery mildew resistance of wheat. Tobacco transient expression and wheat protoplast localization show that the TaGAP1 protein is localized in cytoplasm and cell nucleus. The wheat powdery mildew-resistant gene TaGAP1 provided by the invention provides a new gene resource for cultivating powdery mildew-resistant wheat varieties, and has important application value for widening the genetic basis of wheat and genetically improving the powdery mildew resistance of wheat.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and in particular relates to a wheat powdery mildew resistance gene TaGAP1, its encoded protein and application thereof. Background Art

[0002] Wheat (Triticum aestivum) is a very important dryland crop, and wheat powdery mildew is one of its main diseases, which can greatly reduce wheat yield and quality. Wheat powdery mildew is caused by Blumeria graminis forma specialis tritici, which mainly harms wheat and its closely related species. The fungus competes with crop leaves for nutrients and hinders their photosynthesis. Since the application of chemical agents has very limited control effect on the pathogen and will pollute food and the ecological environment, breeding wheat varieties with powdery mildew resistance is the most effective coping strategy. It is currently urgent to clone and study the efficient resistance genes carried by wheat and its closely related species to meet the needs of resistance breeding.

[0003] In plants, there are many proteins with C2 domains, which can be divided into at least four different groups according to their functions. Proteins containing C2 domains often participate in signal transduction processes by interacting with other molecules (Jambunathan and McNellis 2003), such as: C2 domains may be involved in phospholipid binding and play a role in regulating or mediating phospholipid signals. In addition, functional studies of small C2 domain proteins in plants have shown that they play a role in pollen reproduction as well as abiotic stress and plant defense responses. TaGAP1 protein does not contain any transmembrane domains, but contains a single C2 domain and belongs to a small C2 domain protein. Some C2 domain-containing proteins can bind to G proteins and affect their activity (Wang et al. 1999). In Caenorhabditis elegans, it was found that RGS proteins containing C2 domains can bind to the α subunit of G proteins (Sato et al. 2003). In addition, C2 domains have been found in several GAPs, including SynGAP (Pena et al. 2008); RasGAPs (Gawler et al. 1995), RhoGAPs (Hallam et al. 2002) and ArfGAPs (Jensen et al. 2009; Zhang et al. 2005). Studies have shown that GTPase-activating proteins containing C2 domains can participate in the activation function of GTPases. SynGAP requires its C2 domain to achieve GAP activity, and the C2 domain coordinates movement to the switch Il region of Rap to assist GTPase stimulation (Pena et al. 2008). OsGAP1 and AtGAP1, which encode GTPase-activating proteins, also contain a single C2 domain and can activate the GTPase activity of unconventional G proteins (Cheung et al. 2008). In this study, we obtained the wheat-specific small C2 domain protein GTPase-activating protein TaGAP1.

[0004] The C2 domain plays a role in controlling the subcellular localization of proteins. Studies have shown that OsGAP1 can activate the GTPase activity of OsYchF1 and activate different defense responses at different locations in the cell. OsGAP1 is mainly located in the cytoplasm and nucleus. Under biological stress, OsYchF1 can be transferred from the cytoplasm to the plasma membrane through the phospholipid binding ability of OsGAP1, activating the NTPase activity of OsYchF1 and activating the defense response (Cheung et al. 2010). Under salt stress, both OsGAP1 and OsYchF1 are localized in the cytoplasm, OsGAP1 activates the NTPase activity of OsYchF1, and the salt stress response is activated (Cheung et al. 2013). In addition, other single C2 domain proteins can also be transported between the cytoplasm and the plasma membrane. For example, the protein OsPBP1 that regulates rice pollen fertility has a calcium concentration-dependent phospholipid binding activity, which can regulate intracellular Ca 2+ In response to changes in concentration, the localization shifts to the plasma membrane (Yang et al. 2008). However, TaGAP1 in this study is mainly localized in the cytoplasm and nucleus (see Figure 4 , Figure 5). Single C2 domain proteins can participate in defense and stress responses in different species. AtBAP1 in Arabidopsis suppresses plant defense by inhibiting programmed cell death, an essential response of the hypersensitive response. Loss of BAP1 function enhances disease resistance to virulent bacterial and oomycete pathogens, and this enhanced resistance is mediated by salicylic acid, PAD4, and the disease resistance gene SNC1 (Yang et al. 2006; Yang et al. 2007). On the other hand, AtBAP1 or its homolog AtBAP2 can alleviate oxidative stress in yeast (Yang et al. 2007). OsGAP1 is involved in defense and high salt stress response in rice. High salt induces the expression of OsGAP1, the transmembrane subunit of V-ATPase OsVHA-a1, and the small GTPase OsRab11. OsVHA-a1 can directly interact with OsRab11 and OsGAP1. Under salt stress conditions, these interactions regulate the vesicular transport of OsVHA-a1 between the Golgi network TGN and the prevacuolar compartment PVC and the localization of OsGAP1 in the central vacuole. The interaction between OsRab11 and OsGAP1 promotes the vesicular transport of OsVHA-a1 to regulate the ion imbalance in the cytoplasm caused by high salt (Son et al. 2013). Overexpression of OsGAP1 enhances the resistance of rice to different subspecies of Xanthomonas Xoo with a broad spectrum of resistance, while increasing the basal transcription levels of three defense marker genes PR1, GRCWP and PBZ1 (Chern et al. 2005; Cheung et al. 2008). In addition, Arabidopsis transgenic for OsGAP1 enhanced its resistance to Pst DC3000 and the transcriptional levels of defense marker genes PR1, PR2, PDF1.2 and Thi2.1 (Thomma et al. 1998; Cheong et al. 2002; Cheung et al. 2008), indicating that OsGAP1 may be involved in multiple signaling pathways and confer broad-spectrum disease resistance (Cheung et al. 2008).

[0005] Arabidopsis AtGAP1, also known as C2 domain ABA-associated (CAR4) protein, is involved in abscisic acid (ABA) signaling. AtGAP1 (CAR4) mediates the recruitment of the PYR / PYL regulatory components of the ABA receptor (RCAR) to the plasma membrane in a calcium-dependent manner, during which transient interactions occur, thereby affecting the subcellular localization of PYR / PYL (Diaz et al. 2016; Rodriguez et al. 2014). At the same time, the interaction between PYR / PYL and pp2c can inhibit the dephosphorylation activity of sucrose nonfermenting kinase subfamily 2 (SnRK2s) activated by ABA, and AtGAP1 (CAR4) can promote the interaction between the two and positively regulate the ABA pathway. In addition, ABF transcription factors phosphorylated by SnRK2s can bind to ABA molecular response elements to regulate the transcription of defense genes in the ABA signaling pathway (Park et al. 2009; Ma et al. 2009; Yoon et al. 2020). The loss of the car gene can lead to reduced sensitivity to ABA, thereby inhibiting root growth. It is speculated that environmental stress may cause intracellular Ca 2+ CAR protein may mediate the oscillation of intracellular ABA and Ca 2+ Crosstalk between signals (Diaz et al. 2016; Rodriguez et al. 2014). In addition, AtGAP1 is also involved in the response to biotic stress. Overexpression of AtGAP1 enables Arabidopsis to more effectively resist the pathogen Pst DC3000 pressure and increase the expression level of PR1. Proteomic analysis of Arabidopsis plants overexpressing AtGAP1 after Pst DC3000 inoculation showed that AtGAP1 overexpression increased the cell wall thickness of mesophyll cells and reduced the stomatal aperture of leaves by increasing the accumulation of cell wall modification-related proteins before pathogen infection, thereby enhancing Arabidopsis resistance to infection by the pathogen Pst DC3000 (Cheng et al. 2022). In summary, a single C2 domain protein can regulate the initiation and process of plant immune responses and enhance the resistance of plants to adverse environments. Summary of the invention

[0006] The purpose of the present invention is to provide a wheat powdery mildew resistance gene TaGAP1, its encoding protein and application thereof.

[0007] The present invention is achieved through the following technical solutions:

[0008] The present invention relates to a wheat powdery mildew resistance gene TaGAP1. According to the analysis of the Ensembl Plants website, the full length of TaGAP1 is 513 bp, encoding a GTPase activating protein of 170aa, and the protein size is 18.98 kDa. The conserved domain of NCBI predicts that the protein belongs to the ArfGAP family and contains a C2 domain, wherein 6-150aa is the C2 domain.

[0009] The Ensembl Plants and NCBI databases revealed that TaGAP1 has three copies in wheat, located on chromosomes 2A, 2B, and 2D. The sequences of the three copies were compared using DNAMAN software, and the similarity between the three copies was as high as 98%.

[0010] The wheat powdery mildew resistance gene TaGAP1 involved in the present invention has a nucleotide sequence as shown in SEQ ID NO.1.

[0011] The amino acid sequence of the protein encoded by the wheat powdery mildew resistance gene TaGAP1 involved in the present invention is shown in SEQ ID NO.2.

[0012] The allele TaGAP1-1as of the wheat powdery mildew resistance gene TaGAP1 involved in the present invention has a nucleotide sequence in the coding region:

[0013] <120> >TraesCS2A01G501300.1

[0014]

[0015]

[0016] Its amino acid sequence is:

[0017] MLGHLAGLVKVRVTRGVNLAIRDLRSSDPYVVVRMGKQKLKTRVVRKSINPEWNDELTLSVEDPTIPVKLDVFDKDTFFDDPMGNAELDIGPLVEAATMRIQLQGVADGTVVKKLVPNRQNCLAEESAVYLSEGTVKQDVVLRLRNVECGEVELQLQWIDIPGSKGASGF

[0018] The allele TaGAP1-2as of the wheat powdery mildew resistance gene TaGAP1 of the present invention has a nucleotide sequence in the coding region:

[0019] <120> >TraesCS2D01G501800.2

[0020]

[0021]

[0022] Its amino acid sequence is:

[0023] MLGHLVGLVKVRVTRGVNLAIRDLRSSDPYVVVRMGKQKLKTRVVRKSINPEWNDELTLSIEDPTIPVKLDVFDKDTFFDDPMGNAELDIGPLVEAATMRIQLQGVADNSVVKKLVPNRQNCLAEESAIYLSEGTVKQDVVLRLRNVECGEVELQLQWIDIPGSKGASGF

[0024] The invention also relates to the application of wheat powdery mildew resistance gene TaGAP1 in wheat powdery mildew resistance breeding.

[0025] The present invention has the following advantages:

[0026] In this study, TaGAP1 was silenced through BSMV-mediated transient silencing technology. After inoculation with E09, it was found that the number of powdery mildew colonies in the silenced plants increased significantly, the growth and development of powdery mildew was faster than that of the control plants, the hyphal branching, length, and infection area were significantly higher than those of the control, and the expression level of disease-related proteins was downregulated, indicating that silencing TaGAP1 reduced wheat resistance to powdery mildew. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The bioinformatics analysis diagram of TaGAP1; (A) is the predicted conserved domain diagram of TaGAP1; (B) is the sequence alignment analysis diagram of three copies of TaGAP1;

[0028] Figure 2 is the phylogenetic tree diagram of TaGAP1;

[0029] Figure 3 is the transcription level analysis diagram of TaGAP1;

[0030] Figure 4 This is a diagram showing the tobacco subcellular localization of TaGAP1;

[0031] Figure 5 This is a diagram showing the wheat protoplast localization of TaGAP1;

[0032] Figure 6The figures are the phenotypic observation and silencing efficiency analysis diagrams of silencing TaGAP1 and inoculation with E09 in the non-compatible combination; (A) is the phenotypic observation diagram of silencing TaGAP1; (B) is the phenotypic diagram of the silenced plants after inoculation with powdery mildew E09; (C) is the transcription level analysis diagram of TaGAP1 in the silenced plants 24 h, 48 h, 72 h and 96 h after inoculation with E09;

[0033] Figure 7 The figures are the phenotypic observation and silencing efficiency analysis of TaGAP1 silenced in the affinity combination after inoculation with E09; (A) the phenotypic observation of TaGAP1 silenced; (B) the phenotypic figure of the silenced plants after inoculation with powdery mildew E09; (C) the transcription level analysis of TaGAP1 in the silenced plants 24h, 48h, 72h and 96h after inoculation with E09.

[0034] Figure 8 The histological observation and statistical analysis of powdery mildew in TaGAP1-silenced plants in the non-compatible combination; (A) is the observation of the growth of powdery mildew E09 in TaGAP1-silenced plants and control plants, C: conidia; PGT: primary germ tube; AP: appressorium; SH: secondary hyphae. Scale bar = 20 μm; (B) is the statistical analysis of the hyphae branches of E09 in silenced plants and control plants; (C) is the statistical analysis of the hyphae length of E09 in silenced plants and control plants; (D) is the statistical analysis of the infection area of ​​E09 in silenced plants and control plants;

[0035] Fig. 9 The histological observation and statistical analysis of powdery mildew in TaGAP1-silenced plants in the affinity combination; (A) is an observation of the growth of powdery mildew E09 in TaGAP1-silenced plants and control plants; C: conidia; PGT: primary germ tube; AP: appressorium; SH: secondary hyphae. The scale bars for 24hpi, 48hpi and 72hpi = 20μm, and the scale bar for 96hpi = 50μm; (B) is a statistical analysis of the hyphae branches of E09 in silenced plants and control plants; (C) is a statistical analysis of the hyphae length of E09 in silenced plants and control plants; (D) is a statistical analysis of the infection area of ​​E09 in silenced plants and control plants;

[0036] Fig.10 This is the transcription level analysis of the pathogenesis-related proteins TaPR1 and TaPR2 after silencing TaGAP1;

[0037] Fig.11 It is a diagram for identifying positive strains overexpressing TaGAP1; wherein, (A) is a PCR identification analysis diagram of TaGAP1-OE-T0 generation strains; (B) is a diagram for detecting the transcription level of TaGAP1 in TaGAP1-OE-T0 generation strains by qRT-PCR;

[0038] Fig.12 It is a diagram for identification of disease resistance of TaGAP1-OE-T1 generation strains; wherein, (A) is a diagram for PCR detection of positive plants of TaGAP1-OE-T1-L4 and TaGAP1-OE-T1-L6 strains; a diagram for phenotypic identification of positive plants after inoculation with powdery mildew E09; (B) is a diagram for biomass analysis of positive plants and wild-type plants after inoculation with E09; (C) is a diagram for transcription level of TaGAP1 in TaGAP1-T1-L4 overexpressing strains; (D) is a diagram for transcription level of TaGAP1 in TaGAP1-T1-L6 overexpressing strains. DETAILED DESCRIPTION

[0039] The present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments are only further descriptions of the present invention, but the protection scope of the present invention is not limited to the following embodiments.

[0040] Example

[0041] 1. Test materials and instruments

[0042] 1.1.1 Materials

[0043] Blumeria graminis: E09, provided by our laboratory;

[0044] Wheat materials: Jingshuang 16: used to propagate wheat powdery mildew; Xingmin 318 and Shuiyuan 11: used to detect the transcription level of TaGAP1 under non-affinity and affinity conditions and for BSMV-VIGS experiments; Fielder: used for transgenic experiments;

[0045] Tobacco materials: Nicotiana benthamiana: used for interaction verification experiments and subcellular localization;

[0046] Vector materials: BSMV: BSMV-PDS, BSMV-α, BSMV-β and BSMV-γ for BSMV-VIGS experiments, pCAMBIA-1302-GFP and pJIT163 for subcellular localization experiments; CUB for transgenic experiments.

[0047] Test strain material: Escherichia coli 5α Chemically Competent Cell, Agrobacterium GV3101 Chemically Competent Cell and EHA105 Chemically Competent Cell.

[0048] 1.1.2 Reagents

[0049] Endonucleases: BamHI, NotI, PacI, MluI, BssHII, SpeI (Takara)

[0050] Kits: Gel recovery kit (Magen), wheat protoplast preparation and transformation kit (Coolaber), Gold Medal super-endotoxin-free plasmid extraction kit (Kangwei Century), RiboMAX TM Large Scale RNA Production Systems (Promega), Reverse Transcription Kit (Vazyme), Plasmid Extraction Kit (Magen), RNA Extraction Kit (Magen), Ribo m7G Cap Analog

[0051] Reagents: MgCl2, acetosyringone, Ribo Gm7G Gap Analog (Human), ChamQ SYBR qPCR Master Mix (Vazyme), 2×Phanta Flash Master Mix (Vazyme), RNase inhibitor (yeasen),

[0052] 1.1.3 Instruments

[0053] Artificial climate incubator (Daskart), PCR instrument (C1000 Bio-Rad), electrophoresis instrument (JY-SCZ7 Junyi), fluorescence quantitative PCR instrument (Thermo), gel imager (Quantumst4 Wuzhou Dongfang), sterilizer, Olympus biological microscope (BX-53Olympus), spectrophotometer (NanoDrop 2000c Thermo), high-speed refrigerated centrifuge (ST16RThermo), shaker (IS-RDVS Jingqi).

[0054] 1.2 Test methods

[0055] 1.2.1 Propagation and preservation of wheat powdery mildew

[0056] Powdery mildew strain E09 was propagated and preserved on the wheat powdery mildew susceptible variety Jingshuang 16. Select the seeds of Jingshuang 16 with full grains, place them in clean water for germination, and sow them after the seeds turn white. Use round flower pots, sow about 10-12 seeds in each pot, cover with soil, put on a transparent plastic cover, tie the top with 6 layers of gauze leather rings (to avoid contamination by miscellaneous bacteria), and place them in an incubator at 16°C, 16h light and 8h dark alternating. The seedlings were inoculated with powdery mildew by shaking grafting, and the seedlings were inoculated when they grew to one leaf and one heart. Spray water on the seedlings to make the water droplets fall on the seedlings, shake off the seedlings attached with powdery mildew above the seedlings, and then spray water so that the powdery mildew can fall on the seedlings. After settling for a while, tie the gauze tightly and place them in an artificial climate incubator for culture (17°C, 75% humidity, 16h / 8h day and night light cycle). The preservation of wheat powdery mildew is carried out in test tube seedlings. The seedlings are cultured and inoculated with powdery mildew in the same manner as above. When powdery spores appear, they are moved to 4°C for storage.

[0057] 1.2.2 Bioinformatics analysis and transcriptional expression analysis of TaGAP1

[0058] Based on the transcriptomic data of wheat-powdery mildew interaction obtained in the laboratory, GTPase activating protein 1 (GAP1) was screened. The full-length sequence of TaGAP1 was obtained from the Ensembl Plants website (http: / / plants.ensembl.org / index.html) and the copy number of the gene in the wheat genome was analyzed. The protein sequences of different copies were compared using DNAMAN. The protein sequence was downloaded using Uniport (https: / / www.uniprot.org / ), and the phylogenetic tree of protein TaGAP1 and its homologs was constructed using MEGA7.0.

[0059] 1.2.3 Analysis of TaGAP1 transcriptional expression

[0060] 1.2.3.1 Sample collection

[0061] Wheat varieties Shuiyuan 11 and powdery mildew E09 form an affinity combination, and Xingmin 318 and E09 form an affinity combination. Mingxian 169 and Xingmin 318 with full grains were selected for planting. When they grew to the one-leaf and one-heart stage, they were inoculated with powdery mildew E09. Samples were collected at 0h, 6h, 12h, 24h, 48h, 72h, 96h and 120h. The sterilized steel beads were placed in a 2ml centrifuge tube in advance, and the collected samples were placed in the tube, quickly frozen in liquid nitrogen, and stored at -80℃. Three biological replicates were performed at each time point.

[0062] 1.2.3.2 RNA extraction

[0063] RNA was extracted using the HiPure Plant RNA Mini Kit (Magen) as follows:

[0064] (1) Preliminary preparation: Grind the collected samples into powder using a tissue grinder. Spray the clean bench with RNAse remover and sterilize with UV for 30 minutes. Wear a mask and gloves during the operation.

[0065] (2) Add 800 μL of Buffer RLC to the ground sample, mix the sample thoroughly, place it on ice for 3 min, and centrifuge it at 14,000 g for 5 min;

[0066] (3) Assemble the filter column, pipette 600 μL of supernatant into the filter column, centrifuge at 14,000 g for 2 min, and discard the filter column;

[0067] (4) Add 300 μL of anhydrous ethanol to the filtrate and mix by pipetting;

[0068] (5) Transfer the mixture in (4) to an RNA Mini Column filter column placed on a 2 ml collection tube, centrifuge at 12000 g for 1 min, and discard the filtrate;

[0069] (6) If the mixed solution in (4) exceeds 700 μL, repeat (5);

[0070] (7) Discard the filtrate, add 500 μL of Buffer RW1 to the filter column containing the sample, and centrifuge at 10,000 g for 1 min;

[0071] (8) Discard the filtrate, add 500 μL of Buffer RW2 to the filter column containing the sample, and centrifuge at 12000 g for 1 min;

[0072] (9) Discard the filtrate and repeat step (8);

[0073] (10) Discard the filtrate, centrifuge at 12000g for 2 min;

[0074] (11) Place the filter column in a 1.5 ml centrifuge tube, add 40 μL of RNase Free Water to the filter column, let stand for 2 min, and centrifuge at 12,000 g for 1 min. Repeat this process;

[0075] (12) Discard the filter column, check the quality, determine its concentration and store it at -80°C.

[0076] 1.2.3.3 Reverse transcription synthesis of cDNA

[0077] HiScript IIQ Select RT Super Mix for qPCR kit was used for reverse transcription synthesis of cDNA. Preliminary preparation: RNase Free PCR tubes, RNase Free pipette tips, etc. were placed in a clean room and sterilized by UV for 30 minutes. Masks and gloves were worn throughout the experiment.

[0078] (1) Add the following to a single PCR tube: RNA sample (1.5 μg), 4×g DNA wiper mix (4 μL), and RNase-free ddH2O (to 16 μL). Mix and centrifuge. The PCR program is 42°C for 2 min.

[0079] (2) After the PCR reaction, add 4 μL of 5×HiScript IIQ Select qRTSuperMixⅡ to the above PCR tube, mix and centrifuge. The PCR program is: 50°C, 15 min; 85°C, 5 s;

[0080] (3) After the reaction is completed, add 180 μL ddH2O to dilute and store at -20°C for later use.

[0081] 1.2.3.4 Real-time fluorescence quantitative PCR

[0082] Primer Premier 5.0 was used to design TaGAP1 specific quantitative primers. The designed primers were verified for amplification efficiency and specificity by qRT-PCR and the transcription level was detected. Masks and gloves were worn throughout the experimental operation. The reaction system was as follows: Figure 1 As shown:

[0083] Table 1

[0084]

[0085] The reaction procedure is shown in Table 2:

[0086] Table 2

[0087]

[0088] The experiments were performed biologically and technically in triplicate, and the data were analyzed using the comparative 2-ΔΔCt method.

[0089] 1.2.4 Construction of the vector for wheat gene TaGAP1

[0090] 1.2.4.1 Amplification and recovery of TaGAP1 gene

[0091] The protein sequence was downloaded from Uniport according to the accession number G8XUN9 in the proteome data, and the nucleotide sequence was searched on Ensembl Plants. The full-length primers of TaGAP1 were designed, and PCR amplification was performed using the following system. The detection and recovery of the target bands were performed using agarose gel electrophoresis, as shown in Table 3:

[0092] Table 3

[0093]

[0094] Recover the target band according to the steps of the agarose gel DNA recovery kit:

[0095] (1) Under UV conditions, cut the desired band, place it in a 2 ml centrifuge tube, and add 1 ml of Buffer GDP;

[0096] (2) Place in a 55°C water bath for 10-15 min, shaking every 5 min to accelerate the dissolution of the gel;

[0097] (3) Place the HiPure Gel Pure DNA Mini Column adsorption column in a centrifuge tube, pipette the gel in (2) into the adsorption column, and centrifuge at 12,000 g for 1 min.

[0098] (4) Pour off the centrifugal filtrate in the collection tube, add 300 μL of Buffer GDP to the adsorption column, let stand for 2 min, and centrifuge at 12,000 g for 1 min;

[0099] (5) Discard the filtrate, add 600 μL of Buffer DW2 to the adsorption column, and centrifuge at 12,000 g for 1 min.

[0100] (6) Discard the filtrate, add 300 μL of Buffer DW2 to the adsorption column, and centrifuge at 12,000 g for 1 min.

[0101] (7) Discard the filtrate and centrifuge at 12000g for 2 min;

[0102] (8) After centrifugation, open the cover of the adsorption column and air dry it for 5-10 minutes to evaporate the residual ethanol;

[0103] (9) Place the adsorption column in a 1.5 ml centrifuge tube, add 20 μL of Elution Buffer, let stand for 2 min, centrifuge for 1 min, repeat this process, and store at -20 °C until use.

[0104] 1.2.4.2 Connection and transformation of recovered product and carrier

[0105] The empty vector was digested according to the restriction site, and the uncut vector was used as a control to detect whether the vector was in a linear state. The recovered product was recombined with the linearized vector. 50-100ng / μL of the recovered product, 50-100ng / μL of the digested vector, and 5μL of the recombinase were added to a 0.2ml tube in sequence, and ddH2O was added to 10μL. Mix gently, centrifuge for a few seconds, and react in a PCR instrument at 50℃ for 15min. After the PCR is completed, a transformation experiment was performed. After all 10μL of PCR products were added to 50μL of the competent large intestine, ice bathed for 30min, stood in a 42℃ water bath for 45sec, stood on ice for 2min, added 700μL of LB medium (without antibiotics), and placed in a shaker at 200rpm for 1h. Centrifuge at 4500rpm for 5min, discard the supernatant, aspirate the remaining liquid to blow the bacterial block, apply the sample to the surface of the LB plate with the gun tip, and place it upside down in a 37℃ incubator for 12-16h. Select a single colony for colony PCR detection. The PCR reaction procedure is shown in Table 4:

[0106] Table 4

[0107]

[0108] Agarose gel electrophoresis was used to detect the PCR product to confirm whether the target band was amplified. According to the electrophoresis results, the colonies corresponding to the target DNA bands of the expected size were selected, and the correct colonies were picked up with a sterilized white pipette tip and suspended in liquid LB medium (containing antibiotics), and shaken at 37°C 200rpm for 12-16h before continuing the subsequent experiments.

[0109] 1.2.4.3 Extraction of small amount of recombinant vector

[0110] The shaken bacterial solution was used for plasmid extraction. The extraction steps are as follows:

[0111] Add the bacterial solution several times into a 2 ml centrifuge tube, centrifuge at 10000g for 1 min, and discard the supernatant.

[0112] Add 250 μL of Solution I (RNase A added in advance and stored at 4°C) to resuspend the aggregated bacterial cells; then add 250 μL of Solution II, gently invert the sample 3-5 times to ensure sufficient mixing, and let it stand for 2 minutes;

[0113] Continue to add 350 μL of Solution III, gently invert the sample 3-5 times to mix Solution III thoroughly, observe the appearance of floccules in the sample and the sample is white, centrifuge at 13000g for 10 min; install the filter column in advance, draw the supernatant in step (4) into the filter column, and centrifuge at 13000g for 1 min;

[0114] Discard the filtrate, pipette 500 μL HBC Buffer into the filter column, and centrifuge at 13000 g for 1 min; discard the filtrate, add 700 μL DNA Wash Buffer (to remove impurities), and centrifuge at 13000 g for 1 min. Repeat this process;

[0115] Discard the filtrate, simmer at 13000g for 2 min, and dry at room temperature for 5-7 min to evaporate the remaining ethanol;

[0116] Insert the filter column containing DNA into a 1.5 ml centrifuge tube, pipette 40 μL of Elution Buffer (to elute DNA) into the filter column, let it stand for 2 min, centrifuge at 13000 g for 1 min, repeat this process, and store at -20°C for later use.

[0117] 1.2.4.4 Sequencing and alignment of recombinant plasmids

[0118] 8 μL of the above plasmid was pipetted into a PCR tube, and the universal primer of the plasmid was pipetted into a PCR tube, and the sample was sent to Qingke Biotechnology Co., Ltd. for sequencing analysis. The obtained sequencing results were compared with the sequence of the target genome using the NCBI alignment tool. Through the comparison analysis, if the sequenced sequence is completely consistent with the sequence of the target gene, the related vector of TaGAP1 was successfully constructed.

[0119] 1.2.5 Barley streak mosaic virus-mediated gene silencing

[0120] 1.2.5.1 Construction of gene silencing vectors

[0121] Select two specific fragments from the TaGAP1 gene, design specific primers, use high-fidelity enzyme 2×PhantaFlash Master Mix (Dye Plus) to amplify the two fragments, construct them into BSMV:γ vector, and obtain BSMV:TaGAP1-1as and BSMV:TaGAP1-2as recombinant plasmids. For specific construction methods, refer to 1.2.4;

[0122] 1.2.5.2 BSMV vector linearization

[0123] The recombinant viral vectors BSMV:α, BSMV:β, BSMV:γ, BSMV:TaGAP1-1as, BSMV:TaGAP1-2as and BSMV:PDS were linearized. The specific system is as follows:

[0124] The following BSMV:α, BSMV:γ enzyme digestion system, PCR program 37°C 2h, 65°C 15min, as shown in Table 5:

[0125] Table 5

[0126]

[0127] The following enzyme digestion systems of BSMV: TaGAP1-1as, BSMV: TaGAP1-2as and BSMV: PDS, and PCR program of 50°C for 2h, 65°C for 15min, are shown in Table 6:

[0128] Table 6

[0129]

[0130] The following BSMV:β enzyme digestion system, PCR program 37°C 2h, 65°C 15min, as shown in Table 7:

[0131] Table 7

[0132]

[0133] After the PCR reaction is completed, the uncut vector is used as a control to detect whether the vector is linearized and stored at -20°C for later use. 1.2.5.3 In vitro transcription of BSMV vector linearization product

[0134] Preliminary preparation: sterilize the clean bench and the gun tips and PCR tubes in advance with ultraviolet light. Perform all experimental operations on ice (to ensure that the samples are kept at low temperatures). Wear a mask to avoid cross contamination. The gun tips and PCR tubes used in the experiment are enzyme-free (to avoid contamination by exogenous enzymes). Use an in vitro transcription kit for in vitro transcription. Add the reagents shown in Table 8 to the RNAase-free PCR tube in sequence:

[0135] Table 8

[0136]

[0137]

[0138] Mix gently, centrifuge briefly, and react at 37℃ for 1h. After the reaction, pipette 1μL of the product into a PCR tube and add 5μL of RNAase-Free Water. Use agarose gel electrophoresis to detect the PCR product to determine whether the in vitro transcription is effective. The remaining product is stored at -80℃ for subsequent experiments.

[0139] 1.2.5.4 BSMV virus inoculation

[0140] Select Shuiyuan 11 and Xingmin 318 wheat seeds with full grains, and plant 15 seeds in each pot. Wheat with two leaves and one heart was used for the BSMV virus inoculation experiment;

[0141] Divide the products in 1.2.5.3 into three combinations: control group: MOCK, BSMV: α+β+PDS, BSMV: α+β+γ, experimental group: BSMV: α+β+TaGAP1-1as, BSMV: α+β+TaGAP1-2as; mix each combination product with 800 μL of FES buffer in a PE glove, gently blow and mix, and divide it into several drops for inoculation;

[0142] Inoculate 5 pots of wheat for each combination. Wear small rubber gloves during inoculation and rub the mixture on the second leaf of the wheat. After inoculation, place the plants in an artificial climate box at 25°C, keep them away from light and moisturized overnight, then adjust to a normal light cycle and culture them under high humidity.

[0143] About 10-12 days after inoculation, pay close attention to see if the virus is present. Before inoculation, cut off the leaves that are not poisonous and conduct an inoculation experiment on the leaves that are poisonous.

[0144] 1.2.5.5 Inoculation of wheat powdery mildew and collection and processing of samples

[0145] Use a marker to mark the edges of the third and fourth leaves of the infected plants to ensure that the inoculation location can be clearly identified to facilitate subsequent sampling and phenotypic observation. Refer to 2.2.1 for powdery mildew inoculation;

[0146] Quantitative and histological samples were collected at different time points (24h, 48h, 72h and 96h) after docking with bacteria, and the quantitative samples were quick-frozen in liquid nitrogen. RNA extraction and reverse transcription refer to 2.2.3.2;

[0147] 9 days after inoculation, observe whether the control group is fully diseased. After the disease is fully developed, observe the phenotype of the silent plants and take photos;

[0148] Leaves used for histological observation need to be cut into small pieces of 2-3 cm and placed in a decolorizing solution (ethanol: glacial acetic acid = 1:1). After the decolorizing solution changes color, replace it and repeat several times to ensure the decolorizing effect. Finally, the leaves are transparent.

[0149] After decolorization is completed, remove the decolorization solution and add chloral hydrate to soak for 12 hours;

[0150] Rinse the leaves several times with distilled water, immerse the leaves completely in Coomassie Brilliant Blue dye solution for 15 minutes, and then continue to wash with distilled water for 3-5 times to remove the dye solution;

[0151] Prepare slides and observe the hyphae length, hyphae branching and colony area of ​​powdery mildew under a microscope, and record at least 50 infection points at each time point.

[0152] 1.2.6 Subcellular localization in tobacco

[0153] 1.2.6.1 Construction of tobacco subcellular localization vector

[0154] TaGAP1 was constructed into pCAMBIA1302-GFP, and the empty vector of pCAMBIA1302-GFP was used as a control. For the specific construction method, refer to 1.2.4;

[0155] 1.2.6.2 Transformation of Agrobacterium and transient overexpression in tobacco

[0156] In the clean bench, 1 μg of plasmid was added to the competent Agrobacterium, gently mixed, ice bathed for 15 min, frozen in liquid nitrogen for 5 min, heated in a 37°C water bath for 5 min, ice bathed for 5 min, added 600 μL of LB, and cultured at 28°C and 180 rpm for 2-3 h;

[0157] Centrifuge at 4500 rpm for 5 min, discard the supernatant, blow the bacterial block with the remaining liquid, apply it on the surface of LB plate (containing resistance) with a sterile nozzle, and invert and culture at 28°C for 2-3 days;

[0158] Select the colonies of the appropriate size, use a sterile pick to pick up the colonies and place them in liquid LB medium (containing antibiotics), shake at 28°C 200rpm for 12-16h;

[0159] The cultured Agrobacterium was subjected to bacterial liquid PCR, and the positive bacterial liquid was stored in glycerol stock at -80°C for future use and then used for subsequent experiments.

[0160] 1.2.6.3 Tobacco transient overexpression

[0161] 2 ml of the Agrobacterium solution tested by PCR was taken and centrifuged at 4500 rpm for 5 min;

[0162] Prepare 10 mM magnesium chloride and AS buffer, discard the supernatant, add 1 ml of 10 mM magnesium chloride, centrifuge at 4500 rpm for 5 min, and repeat; wherein, the AS buffer is shown in Table 9:

[0163] Table 9

[0164]

[0165]

[0166] Discard the supernatant, adjust the OD value of Agrobacterium to OD600 = 0.6 with AS buffer, and incubate at 28°C in the dark for 2-3 h;

[0167] Select healthy tobacco for tobacco injection. First, gently puncture the back of the tobacco with a needle, inject the bacterial solution into the area, place it in a 25℃ incubator in the dark and keep moist overnight, then transfer it to a normal light cycle for 2-3 days, and observe the expression under an Olympus biological microscope.

[0168] 1.2.7 Subcellular localization in wheat protoplasts

[0169] 1.2.7.1 Construction of wheat protoplast localization-related vectors

[0170] TaGAP1 was constructed into pJIT163-GFP, and the empty vector of pJIT163-GFP was used as a control. For the specific construction method, refer to 1.2.4.

[0171] 1.2.7.2 Large-scale extraction of recombinant plasmid

[0172] The colonies that passed the PCR test were selected and suspended in LB (containing antibiotics), and cultured at 37°C with a shaker at 200 rpm for 12-16 hours, and then transferred to the pre-prepared 500 ml LB liquid culture medium at a ratio of 1:100, and cultured at 37°C with a shaker at 200 rpm for 2-3 hours. The plasmid was extracted according to the plasmid extraction kit of Kangwei Century Company. The specific steps are as follows: Among them, the LB culture medium is shown in Table 10:

[0173] Table 10

[0174] Reagents Mass / Volume Yeast Extract (YE) 5g Peptone 10g NaCl 10g <![CDATA[ddH2O]]> 1L

[0175] Centrifuge the shaken bacterial solution several times at 12000g for 10 min to collect the bacteria and discard the supernatant;

[0176] Add 12 ml of Buffer P1 (add RNAase A in advance and store at 4°C) to the bacteria in (1) and mix thoroughly; add 12 ml of Buffer P2, gently invert and mix, and let stand for 5 minutes to allow for sufficient reaction; add 12 ml of Buffer P3, gently invert and mix until a flocculent precipitate appears and is white, let stand for 5 minutes to ensure sufficient reaction, centrifuge at 12000g for 10 minutes, and aspirate the supernatant into a new centrifuge tube;

[0177] Add isopropanol (the volume is 0.3 times the volume of the supernatant) and mix thoroughly by inversion;

[0178] Install the adsorption column in advance, pipette 2 ml of Buffer ES into the adsorption column, centrifuge at 12000 g for 2 min, and discard the filtrate;

[0179] Transfer the mixed solution in (3) to the adsorption column in small amounts several times, centrifuge at 12000g for 2 min, and discard the filtrate;

[0180] Pipette 10 ml of Buffer PW containing anhydrous ethanol into the adsorption column, centrifuge at 12000 g for 2 min, discard the filtrate, and repeat this process;

[0181] Add 10 ml of Endo-Free Buffer PW, centrifuge at 12000 g for 5 min, discard the filtrate, and dry in a clean bench for several minutes;

[0182] Install the adsorption column in a centrifuge tube, add 800 μL of Endo-Free Buffer EB, let stand for 5 min, centrifuge at 12000 g for 2 min, repeat this process, transfer the plasmid to a 1.5 ml centrifuge tube for subsequent experiments, and store at -20 °C.

[0183] 1.2.7.3 Preparation and transformation of wheat protoplasts

[0184] Select and plant 11 seeds of water source, cultivate them under weak light at 25℃ until they have one leaf and one heart, and then conduct subsequent experiments;

[0185] Prepare enzymatic solution (weigh 0.3g cellulase, 0.06g pectinase and 16ml enzyme solution in a conical flask, place in a 55°C water bath for 10min, mix 2-3 times during the process; after cooling, add the following ingredients: 200μL 10% bovine serum albumin, 7.14μL β-mercaptoethanol, 10μL Amp, and dilute to 20ml with ddH2O);

[0186] Use a knife to cut the wheat leaves into small pieces of about 1 mm in size and pour them into a conical flask, and place them in a vacuum chamber away from light for 30 minutes;

[0187] Transfer the conical flask to a 25℃ shaker at 40 rpm for 2 hours, then place it at 25℃ for 4 hours. Use a yellow pipette with a scissor tip to draw a drop of cells for microscopic examination. If the cells are round and shiny, continue with the subsequent experiment.

[0188] Rinse the 200-mesh cell sieve with 4°C W5 solution, repeat three times, wherein the W5 solution is shown in Table 11:

[0189] Table 11

[0190] Reagents Mass / Volume NaCl 9g <![CDATA[CaCl2]]> 18.4g KCl 3.75g MES 0.42g <![CDATA[ddH2O]]> 1L

[0191] Adjust pH to 5.7 with NaOH and sterilize by high pressure.

[0192] Use a cell sieve to filter the lysate into a new centrifuge tube, centrifuge at 100g for 10 min at 4°C with a horizontal rotor, and use a 3-speed pipette to aspirate the supernatant.

[0193] Gently add 4℃ W5 solution into the protoplasts to evenly disperse the protoplasts, centrifuge at 100g for 5 min in a horizontal rotor centrifuge, and discard the supernatant;

[0194] Add 10 ml of 4°C W5 solution to the protoplasts, place on ice for 30 min, centrifuge at 100 g for 5 min in a horizontal rotor centrifuge, and discard the supernatant;

[0195] Slowly add 2 ml of 4°C MMG, gently shake the centrifuge tube to evenly disperse the protoplasts, and place on ice. At this point, the protoplast preparation is complete. The MMG solution is shown in Table 12:

[0196] Table 12

[0197] Reagents Mass / Volume Mannitol 7.285g MgCl2 0.305g MES 0.085g <![CDATA[ddH2O]]> 1L

[0198] Adjust pH to 5.7 with NaOH and sterilize by high pressure.

[0199] 15 μg of plasmid, 165 μL of protoplasts, and 150 μL of PEG transformation solution were added to a 2 ml centrifuge tube, gently mixed, and placed in the dark at 25° C. for 30 min. The PEG transformation solution is shown in Table 13:

[0200] Table 13

[0201] Reagents Mass / Volume Mannitol 0.036g PEG4000 0.4g <![CDATA[1M CaCl2]]> 100μL <![CDATA[ddH2O]]> To 1ml

[0202] Slowly and gently add 4℃ precooled W5 solution into the centrifuge tube, shake gently to evenly disperse the protoplasts, let stand for 2 minutes, discard the supernatant, and repeat;

[0203] Slowly add 600 μL of pre-cooled W5 solution into the centrifuge tube, gently rotate the centrifuge tube to mix thoroughly, and place it in a 25°C incubator in the dark for 16-18 hours; observe the expression of the target protein under a microscope.

[0204] 1.2.8 Creation of wheat transgenic and gene editing materials

[0205] TaGAP1 was constructed into the CUB vector. The specific construction method is as shown in 1.2.4. At the same time, the plasmid was transferred into the competent cells of EHA105. After the colonies grew up, the positive colonies were marked after PCR detection. The plates were sent to the transgenic platform of the State Key Laboratory of Stress Biology of Arid Crops of Northwest Agriculture and Forestry University for Agrobacterium-mediated wheat genetic transformation. The wheat recipient variety was Fielder. For the creation of gene editing materials, three copies of the sequence were sent to the Shandong Academy of Agricultural Sciences to construct the gene editing vector of TaGAP1 and perform wheat genetic transformation.

[0206] 1.2.8.1 Molecular identification of T0 plants

[0207] Transplant the seedlings of the T0 generation plants into pots for cultivation. When the seedlings grow to a certain size, collect the leaves. One portion is used as a DNA sample for PCR detection of positive plants, and one portion is used as an RNA sample for qRT-PCR screening of transgenic positive plants with high expression levels. Refer to 1.2.3.2 for RNA extraction, reverse transcription, and real-time fluorescence quantitative PCR. The DNA extraction steps are as follows:

[0208] Add steel balls to the centrifuge tube containing the leaves and use a grinder to crush the leaves into powder;

[0209] Add 750 μL of CTAB extract solution that has been heated in a 65°C water bath to the DNA sample, gently mix the sample, and place it in a 65°C water bath for 1 hour, mixing every 20 minutes;

[0210] Among them, the CTAB extract is shown in Table 14:

[0211] Table 14

[0212] Reagents Mass / Volume CTAB 20g 0.5M Tris-HCl (PH = 8.0) 200ml 0.5M EDTA (PH = 8.0) 40ml NaCl 81.8g 0.2% mercaptoethanol 2ml <![CDATA[ddH2O]]> 1L

[0213] 750 μL of nucleic acid extraction reagent (25:24:1) was pipetted into the sample, the sample was gently mixed, and then centrifuged at 12000 rpm at 4°C for 10 min;

[0214] Pipette the supernatant into a new centrifuge tube, do not aspirate the precipitate, pipette 750 μL of nucleic acid extraction reagent (24:1) into the sample, gently mix the sample, and centrifuge at 12000 rpm and 4°C for 10 min;

[0215] Pipette the supernatant into a new centrifuge tube, add 750 μL -20℃ isopropanol to the supernatant, and place it at -20℃ for 16-24 hours to ensure sufficient precipitation;

[0216] Centrifuge at 12000 rpm for 10 min at 4°C and discard the supernatant;

[0217] Add 500 μL of 75% ethanol to the precipitated sample and wash twice, and evaporate the residual ethanol under ventilation conditions;

[0218] Add 50 μL of ddH2O to dissolve the DNA and store at -20°C until use.

[0219] 1.2.8.2 Phenotypic identification and biomass detection of T1 plants

[0220] Plant seeds with high expression and positive plant families, and when they grow to one leaf and one heart, inoculate with powdery mildew E09. Observe the disease of the plants 7-10 days after inoculation. After Fielder is fully diseased, observe and take photos to record the phenotype.

[0221] Biomass detection: Collect biomass samples 7 days after inoculation, and extract DNA according to the method in 1.2.8.1. Use the extracted DNA as a template, dilute the DNA concentration to the same concentration, and use primers Bgtβ-tublin and TaEF for fluorescence quantitative PCR; bring the Ct value into the standard curve to obtain the Bgtβ-tublin and TaEF copy numbers, respectively, Bgt / Wheat = Bgtβ-tublin copy number / TaEF copy number.

[0222] 1.3 Results and Analysis

[0223] 1.3.1 Bioinformatics analysis of TaGAP1

[0224] The full length of TaGAP1 was 513 bp, encoding a 170aa GTPase activating protein with a protein size of 18.98 kDa, as shown in Table 15; the conserved domain of NCBI predicted that the protein belonged to the ArfGAP family and contained a C2 domain, of which 6-150aa was the C2 domain ( Figure 1 -A). The Ensembl Plants and NCBI databases revealed that TaGAP1 has three copies in wheat, located on chromosomes 2A, 2B, and 2D. The sequences of the three copies were compared using DNAMAN software, and the similarity between the three copies was as high as 98% ( Figure 1 -B).

[0225] Table 15

[0226]

[0227]

[0228] The GAP1 protein sequences in wheat and closely related species were compared and analyzed, and the phylogenetic tree was constructed using MEGA7.0 software using the neighbor-joining method. The phylogenetic tree results showed that TaGAP1 was most closely related to Urartu wheat and most distantly related to maize, millet, and sorghum (see Figure 2). The phylogenetic tree of TaGAP1 was constructed using MEGA7.0 software using the neighbor-joining method. Hv: Hordeum vulgare, Os: Rice, Oryza sativa, At: Arabidopsis thaliana, Arabidopsis thaliana, Zm: Zea mays, Zea mays, Tu: Triticum urartu, At: Aegilops tauschii, Aegilops tauschii, Ta: Triticum aestivum, Si: Foxtail millet, Setaria italica, Ph: Panicum spp., Panicum hallii, Sb: Sorghum bicolor.

[0229] 1.3.2 Analysis of TaGAP1 transcriptional expression

[0230] To explore whether TaGAP1 is involved in the interaction between wheat and powdery mildew, qRT-PCR was used to analyze the transcriptional expression profile of TaGAP1 in wheat and powdery mildew compatible (Shuiyuan 11 / E09) and non-compatible combinations (Xingmin 318 / E09). The results showed that in the compatible combination, the expression level of TaGAP1 was downregulated as a whole, which could be downregulated by 2 times; in the non-compatible combination, the expression level of TaGAP1 was significantly upregulated as a whole, reaching a peak at 72 hours, and the expression level reached 4 times (see Figure 3 ). Therefore, it is speculated that TaGAP1 may be involved in wheat resistance to powdery mildew as a positive regulatory factor. Figure 3 It can be seen that the standard deviation represents the mean ± standard error of three independent samples. -ΔΔCt Relative quantification method, expression levels were normalized to TaEF-1α. Asterisks indicate significant differences by t-test (*, P < 0.05; **, P < 0.01).

[0231] 1.3.3 TaGAP1 is localized in the cytoplasm and nucleus

[0232] To clarify the subcellular localization of TaGAP1, a TaGAP1-GFP fusion vector was constructed, and pCAMBIA1302-GFP was used as an empty vector control. The results of transient overexpression in tobacco showed that the empty vector GFP was localized in the cytoplasm, cell membrane and nucleus, while the fluorescence of TaGAP1-GFP was mainly detected in the cytoplasm and nucleus (e.g. Figure 4 ).

[0233] To further clarify the subcellular localization of TaGAP1, TaGAP1 was connected to pJIT163-GFP to construct a TaGAP1-GFP fusion vector, and the pJIT163-GFP empty vector was used as a control. TaGAP1-GFP and GFP empty vectors were transferred into wheat protoplasts by PEG-mediated transformation. After culturing in the dark for 16-19 hours, the cells were observed under a fluorescence microscope. The results showed that GFP was localized in the cytoplasm, cell membrane and nucleus of wheat cells, while TaGAP1-GFP was mainly localized in the cytoplasm and nucleus (such as Figure 5 In summary, TaGAP1 is localized in the cytoplasm and nucleus.

[0234] 1.3.4 Transient silencing of TaGAP1 reduces wheat resistance to powdery mildew

[0235] 1.3.4.1 Phenotypic observation of transient silencing of TaGAP1

[0236] To clarify the function of TaGAP1 in wheat resistance to powdery mildew, BSMV-VIGS was used to transiently silence two fragments of TaGAP1, BSMV: TaGAP1-1as and BSMV: TaGAP1-2as. Ten days after infection, obvious photobleaching stripes appeared on the leaves of the positive control group BSMV: TaPDS, indicating successful infection, while obvious chlorotic spots were observed on the leaves of the blank control group MOCK, the negative control group: BSMV:γ and the experimental groups: BSMV: TaGAP1-1as, BSMV: TaGAP1-2as (see Figure 6 -A. Figure 7 -A). The silenced plants and control plants were inoculated with powdery mildew E09, and samples for silencing efficiency detection and histological observation were collected 24h, 48h, 72h, and 96h after inoculation. The phenotypes were observed 10-12d after inoculation. In the non-compatible combination, no powdery spores appeared on the leaves of MOCK and BSMV:γ plants, while a small amount of powdery spores appeared on the leaves of the silenced plants (see Figure 6 -B); the leaves of the affinity combination MOCK and BSMV:γ and the silenced plants all had white powdery spores, but the white powdery spores on the leaves of the silenced plants were significantly more than those in the control group ( Figure 7 -B). The results of qRT-PCR silencing efficiency test showed that the expression level of TaGAP1 in the silenced plants was significantly lower than that in the control group. In the non-affinity combination, the silencing efficiency of the leaves of the silenced plants was greater than 50%; in the affinity combination, the silencing efficiency was between 49% and 72% (see Figure 6 -C. Figure 7 -C), demonstrating that TaGAP1 silencing was successful and the VIGS experimental results were reliable.

[0237] 1.3.4.2 Histological observation of wheat powdery mildew

[0238] Histological observation results showed that in the non-compatible combination, at 24 hpi, there was no significant difference in hyphal growth between TaGAP1-silenced plants and control plants; at 48 hpi, 72 hpi and 96 hpi, hyphal growth in silenced plants was significantly faster than that in control plants (see Figure 8 -A), and statistical analysis also showed that the colony area and hypha length were significantly greater than the control group, hypha branching had no significant difference at 48hpi, but was significantly greater than the control group at 72hpi and 96hpi (see Figure 8 BD). In the affinity combination, the growth of powdery mildew in the silenced plants was also significantly greater than that in the control plants (see Fig. 9 -A). Statistical analysis results showed that the colony area, hyphae length and hyphae branching of powdery mildew at each sampling point were significantly higher than those of the control plants (see Fig. 9 BD).

[0239] 1.3.4.3 Analysis of transcriptional expression profiles of pathogenesis-related protein (PR) genes

[0240] The results of qRT-PCR detection of the transcriptional expression levels of TaPR1 and TaPR2 genes showed that in the non-compatible combination, the expression levels of TaPR1 and TaPR2 in the TaGAP1-silencing plants were significantly decreased; in the compatible combination, the expression level of TaPR1 was significantly decreased only at 24 hpi, and at 72 hpi, it was significantly down-regulated in the BSMV:TaGAP1-2as plants; at 24 hpi and 72 hpi, the expression level of TaPR2 was significantly down-regulated in the BSMV:TaGAP1-2as plants and BSMV:TaGAP1-1as plants (see Fig.10 ).

[0241] 1.3.5 Overexpression of TaGAP1 enhances wheat resistance to powdery mildew

[0242] To further study the disease resistance function of TaGAP1 in the interaction between wheat and powdery mildew, a TaGAP1-overexpressing wheat line was created and the following experiments were carried out.

[0243] 1.3.5.1 Obtaining transgenic wheat plants

[0244] The plasmid Cub-TaGAP1 was transferred into Agrobacterium-mediated transgenic technology and sent to the transgenic platform of the National Key Laboratory of Crop Stress Resistance and High-efficiency Production of Northwest Agriculture and Forestry University. PCR detection was used to screen T0 generation positive plants, and a total of 12 positive plants from L1 to L12 were obtained (see Fig.11-A). qRT-PCR was further used to detect the expression level of TaGAP1 in the positive strains. The expression levels of 11 strains were significantly higher than Fielder, among which L4 and L6 had the highest expression levels, reaching 14.87 times and 19.89 times, respectively (see Fig.11 -B). Therefore, L4 and L6 were subcultured and the T1 generation plants were tested for disease resistance.

[0245] 1.3.5.2 Disease resistance testing of TaGAP1 transgenic overexpressing strains

[0246] The phenotypes of the T1 strains of the L4 and L6 families overexpressing TaGAP1 were observed 7 days after inoculation with powdery mildew E09. The number of powdery mildew colonies in the L4 and L6 strains was significantly less than that in the Fielder powdery mildew (see Fig.12 -A), and the biomass is also significantly lower than Fielder (see Fig.12 -B). qRT-PCR results showed that the expression level of TaGAP1 in L4 and L6 strains was about 7-25 times that of Fielder (see Fig.12 -C, D). In summary, overexpression of TaGAP1 enhanced wheat resistance to powdery mildew.

[0247] In summary: G protein signal transduction in plants plays a key role in mediating plant immune responses. GAPs can regulate the activity of G proteins and promote GTP hydrolysis of G proteins. GAPs are believed to promote the conversion of active GTPase-GTP forms to GTPase-GDP forms. Based on its ability to regulate GTPase proteins and other domains, GAPs are directly or indirectly involved in various cellular physiological processes.

[0248] In this study, TaGAP1 was silenced by BSMV-mediated transient silencing technology. After inoculation with E09, the number of powdery mildew colonies in the silenced plants increased significantly (see Figure 6 , Figure 7 ), the growth and development of powdery mildew was faster than that of the control plants, and the hyphae branching, length and infection area were significantly higher than those of the control plants (see Figure 8 , Fig. 9 ), the expression of pathogenesis-related proteins was downregulated, indicating that silencing TaGAP1 reduced wheat resistance to powdery mildew. Overexpression of TaGAP1 enhanced wheat resistance to powdery mildew (see Fig.12 ), clarify the disease resistance function of TaGAP1 in the interaction between wheat and powdery mildew, but subsequent experiments are needed to analyze the expression levels of key genes in related signaling pathways in transgenic plants overexpressing TaGAP1 and to clarify the signaling pathways in which they are involved.

[0249] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which will not affect the essence of the present invention.

Claims

1. Wheat powdery mildew resistance gene TaGAP1, characterized in that Analysis of the Ensembl Plants website showed that TaGAP1 is 513 bp in length and encodes a GTPase-activating protein of 170 aa with a protein size of 18.98 kDa.

2. The wheat powdery mildew resistance gene TaGAP1 according to claim 1, characterized in that Its nucleotide sequence is shown as SEQ ID NO.

1.

3. The protein encoded by the wheat powdery mildew resistance gene TaGAP1 according to claim 1, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

4. Use of the wheat powdery mildew resistance gene TaGAP1 as claimed in claim 1 in wheat powdery mildew resistance breeding.

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