Wheat powdery mildew resistance gene TaGAP1, its coding protein and application thereof

Abstract

The application provides a wheat powdery mildew resistance gene TaGAP1, a coding protein thereof and application thereof; the application silences TaGAP1 through a BSMV-mediated transient silencing technology, and after inoculation of powdery mildew E09, it is found that the number of powdery mildew colonies of the silenced plant is obviously increased, and the growth and development of the powdery mildew is faster than that of the control plant, which indicates that silencing TaGAP1 reduces the resistance of wheat to powdery mildew; and the area and biomass of the powdery mildew colonies of the overexpressed TaGAP1 are significantly reduced, and the resistance of the wheat to the powdery mildew is significantly enhanced, which indicates that TaGAP1 can be used as an effective gene resource for improving the resistance of wheat to powdery mildew. Tobacco transient expression and wheat protoplast positioning show that the TaGAP1 protein is located in the cytoplasm and the nucleus. The wheat powdery mildew resistance gene TaGAP1 provided by the application provides a new gene resource for cultivating wheat varieties resistant to powdery mildew, and has important application value for widening the genetic basis of wheat and genetically improving the resistance of wheat to powdery mildew.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant genetic engineering; in particular, it relates to a wheat powdery mildew resistance gene TaGAP1, the encoded protein thereof and the application thereof. BACKGROUND

[0002] Wheat (Triticum aestivum) is a very important dryland crop, and wheat powdery mildew is one of the major diseases that can greatly reduce the yield and quality of wheat. Wheat powdery mildew is caused by Blumeria graminis forma specialis tritici, which mainly harms wheat and its closely related species, and competes with the crop leaves for nutrients and hinders their photosynthesis. Since the control effect of applying chemical agents on the pathogen is very limited, and it can pollute the grain and the ecological environment, therefore, cultivating wheat varieties with powdery mildew resistance is the most effective countermeasure. Cloning and studying the high-efficiency resistance genes carried by wheat and its closely related species to meet the needs of resistance breeding is currently the most urgent task.

[0003] In plants, there are many proteins with C2 domain, which can be divided into at least four different groups according to their functions. C2 domain-containing proteins often participate in signal transduction processes through interactions with other molecules (Jambunathan and McNellis 2003), such as: C2 domain may be involved in phospholipid binding, playing a role in regulating or mediating phospholipid signals. In addition, the study of small C2 domain proteins in plants shows that they play a role in pollen reproduction and non-biological stress and plant defense response. TaGAP1 protein does not contain any transmembrane domain, but contains a single C2 domain belonging to small C2 domain proteins. Some C2 domain-containing proteins can bind to G proteins and affect their activity (Wang et al. 1999). It was found in Caenorhabditis elegans that RGS proteins containing C2 domain can bind to the alpha subunit of G protein (Sato et al. 2003). In addition, C2 domain was also 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 C2 domain-containing GTPase-activating proteins can be involved in the activation function of GTPase. 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 encoding GTPase-activating protein also contains a single C2 domain and can activate the GTPase activity of unconventional G proteins (Cheung et al. 2008). In this study, we obtained a small C2 domain protein GTPase-activating protein TaGAP1 specific to wheat.

[0004] C2 domain plays a role in controlling the subcellular localization of proteins. Studies have shown that OsGAP1 can activate the GTPase activity of OsYchF1, activating different defense responses at different locations in the cell. OsGAP1 is mainly localized in the cytoplasm and nucleus, and under biological stress, through the phospholipid binding ability of OsGAP1, OsYchF1 can be transferred from the cytosol to the plasma membrane, activating the NTPase activity of OsYchF1, and the defense response is activated (Cheung et al. 2010). While under salt stress, both OsGAP1 and OsYchF1 are localized in the cytoplasm, and 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 transport between the cytoplasm and the plasma membrane, such as the protein OsPBP1 that regulates rice pollen fertility has calcium concentration-dependent phospholipid binding activity, which can respond to changes in intracellular Ca 2+ concentration, and the localization is switched to the plasma membrane (Yang et al. 2008). TaGAP1 in this study is mainly localized in the cytoplasm and nucleus (see Figure 4 、 Figure 5). Single C2 domain proteins can be involved in defense and stress responses in different species. AtBAP1 in Arabidopsis thaliana suppresses plant defense by inhibiting programmed cell death, a necessary response of hypersensitive response. Loss of BAP1 function enhances disease resistance to toxic bacteria and oomycete pathogens, and this enhanced resistance is mediated by salicylic acid, PAD4 and 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 responses in rice. High salt induces the expression of OsGAP1, the transmembrane subunit of V-ATPase OsVHA-a1 and small GTPase OsRab11, and OsVHA-a1 can directly interact with OsRab11 and OsGAP1. Under salt stress conditions, these interactions regulate the vesicle trafficking of OsVHA-a1 between the trans-Golgi network (TGN) and prevacuolar compartment (PVC) and the localization of OsGAP1 in the central vacuole. The interaction of OsRab11 with OsGAP1 promotes the vesicle trafficking of OsVHA-a1 to regulate the ion imbalance in the cytoplasm caused by high salinity (Son et al. 2013). Overexpression of OsGAP1 enhances resistance to different races of Xanthomonas oryzae pv. oryzae (Xoo) in rice, with broad-spectrum resistance, and increases the basal transcriptional levels of three defense marker genes PR1, GRCWP and PBZ1 (Chern et al. 2005; Cheung et al. 2008). In addition, OsGAP1 -transformed Arabidopsis enhances 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). This suggests that OsGAP1 can be involved in multiple signaling pathways, conferring broad-spectrum disease resistance (Cheung et al. 2008).

[0005] Arabidopsis AtGAP1, also known as C2 domain ABA-related (CAR4) protein, is involved in abscisic acid (ABA) signaling. AtGAP1 (CAR4) mediates the PYR / PYL regulatory components of ABA receptors (RCAR) to be recruited to the plasma membrane in a calcium-dependent manner, during which a transient interaction occurs, 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 the sucrose non-fermenting kinase 2 (SnRK2) family activated by ABA, and AtGAP1 (CAR4) can promote the interaction between the two, positively regulating the ABA pathway. In addition, the ABF transcription factor phosphorylated by SnRK2 can bind to the ABA molecule response element to regulate the transcription of defense genes in the ABA signal pathway (Park et al. 2009; Ma et al. 2009; Yoon et al. 2020). The deletion of the car gene can cause the sensitivity to ABA to decrease, thereby inhibiting the growth of the root system. It is speculated that environmental stress can cause the oscillation of the intracellular Ca 2+ level, and the CAR protein can mediate the crosstalk between intracellular ABA and Ca 2+ signals (Diaz et al. 2016; Rodriguez et al. 2014). In addition, AtGAP1 is also involved in biotic stress response, and overexpression of AtGAP1 enables Arabidopsis to more effectively resist Pst DC3000 stress and increase the expression level of PR1. Proteomic analysis of AtGAP1 overexpressing Arabidopsis plants after Pst DC3000 inoculation showed that AtGAP1 overexpression increased the thickness of the cell wall of mesophyll cells by increasing the accumulation of cell wall modification-related proteins before pathogen infection, reduced the stomatal pore size of leaves, and enhanced the resistance of Arabidopsis to Pst DC3000 infection (Cheng et al. 2022). In summary, a single C2 domain protein can regulate the initiation and process of plant immune response and enhance the resistance of plants to adverse environments. SUMMARY

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

[0007] The present application is realized by the following technical solutions:

[0008] The present application relates to a wheat powdery mildew resistance gene TaGAP1, and the full-length 513bp of TaGAP1 is analyzed through an Ensembl Plants website, encodes a GTPase-activating protein with 170aa, and the protein size is 18.98kDa. The protein is predicted to belong to the ArfGAP family by the conserved domain of NCBI, and contains a C2 domain, wherein 6-150aa is the C2 domain.

[0009] It is found through searching in Ensembl Plants and NCBI databases that TaGAP1 has three copies in wheat, which are located on 2A, 2B and 2D chromosomes respectively. The sequences of the three copies are aligned through DNAMAN software, and the similarity among the three copies is as high as 98%.

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

[0011] The protein encoded by the wheat powdery mildew resistance gene TaGAP1 involved in the present application has an amino acid sequence as shown in SEQ ID NO. 2.

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

[0013] <120>>TraesCS2A01G501300.1

[0014]

[0015]

[0016] The amino acid sequence is as follows:

[0017] MLGHLAGLVKVRVTRGVNLAIRDLRSSDPYVVVRMGKQKLKTRVVRKSINPEWNDELTLSVEDPTIPVKLDVFDKDTFFDDPMGNAELDIGPLVEAATMRIQLQGVADGTVVKKLVPNRQNCLAEESAVYLSEGTVKQDVVLRLRNVECGEVELQLQWIDIPGSKGASGF

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

[0019] <120>>TraesCS2D01G501800.2

[0020]

[0021]

[0022] The amino acid sequence is:

[0023] MLGHLVGLVKVRVTRGVNLAIRDLRSSDPYVVVRMGKQKLKTRVVRKSINPEWNDELTLSIEDPTIPVKLDVFDKDTFFDDPMGNAELDIGPLVEAATMRIQLQGVADNSVVKKLVPNRQNCLAEESAIYLSEGTVKQDVVLRLRNVECGEVELQLQWIDIPGSKGASGF

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

[0025] The application has the following advantages:

[0026] In the present application, TaGAP1 is silenced by BSMV-mediated transient silencing technology, and after inoculation of E09, it is found that the number of powdery mildew colonies of the silenced plants is obviously increased, the growth and development of the powdery mildew are faster than those of the control plants, the mycelium branching, length and infection area are all significantly higher than those of the control, and the expression amount of the pathogenesis-related protein is down-regulated, which indicates that silencing TaGAP1 reduces the resistance of the wheat to the powdery mildew. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a bioinformatics analysis diagram of TaGAP1, wherein (A) is a conserved domain prediction diagram of TaGAP1, and (B) is a sequence alignment analysis diagram of three copies of TaGAP1.

[0028] Figure 2 is a phylogenetic evolution tree diagram of TaGAP1.

[0029] Figure 3 is a transcription level analysis diagram of TaGAP1.

[0030] Figure 4 is a tobacco subcellular localization display diagram of TaGAP1.

[0031] Figure 5 is a wheat protoplast localization display diagram of TaGAP1.

[0032] Figure 6Fig. 1 is a phenotype observation and silencing efficiency analysis diagram of silencing TaGAP1 after inoculation of E09 in non-compatible combination; wherein, (A) is an observation diagram of the phenotype of silencing TaGAP1 after inoculation; (B) is a phenotype diagram of the silencing plant after inoculation of E09; (C) is an analysis diagram of the transcription level of TaGAP1 in the silencing plant after inoculation of E09 for 24 h, 48 h, 72 h and 96 h;

[0033] Figure 7 Fig. 2 is a phenotype observation and silencing efficiency analysis diagram of silencing TaGAP1 after inoculation of E09 in compatible combination; wherein, (A) is an observation diagram of the phenotype of silencing TaGAP1 after inoculation; (B) is a phenotype diagram of the silencing plant after inoculation of E09; (C) is an analysis diagram of the transcription level of TaGAP1 in the silencing plant after inoculation of E09 for 24 h, 48 h, 72 h and 96 h.

[0034] Figure 8 Fig. 3 is a histological observation and statistical analysis diagram of E09 in the TaGAP1 silencing plant in non-compatible combination; wherein, (A) is an observation diagram of the growth of E09 in the TaGAP1 silencing plant and the control plant; C: conidium; PGT: primary germ tube; AP: appressorium; SH: secondary hypha. Scale bar = 20 μm; (B) is a statistical analysis diagram of the hyphal branching of E09 in the silencing plant and the control plant; (C) is a statistical analysis of the hyphal length of E09 in the silencing plant and the control plant; (D) is a statistical analysis diagram of the infection area of E09 in the silencing plant and the control plant;

[0035] Figure 9 Fig. 4 is a histological observation and statistical analysis diagram of E09 in the TaGAP1 silencing plant in compatible combination; wherein, (A) is an observation diagram of the growth of E09 in the TaGAP1 silencing plant and the control plant; C: conidium; PGT: primary germ tube; AP: appressorium; SH: secondary hypha. Scale bar = 20 μm at 24 hpi, 48 hpi and 72 hpi, and scale bar = 50 μm at 96 hpi; (B) is a statistical analysis diagram of the hyphal branching of E09 in the silencing plant and the control plant; (C) is a statistical analysis of the hyphal length of E09 in the silencing plant and the control plant; (D) is a statistical analysis diagram of the infection area of E09 in the silencing plant and the control plant;

[0036] Figure 10 Fig. 5 is an analysis diagram of the transcription level of the pathogenesis-related proteins TaPR1 and TaPR2 after silencing TaGAP1;

[0037] Figure 11 Fig. 6 is a positive line identification diagram of overexpressing TaGAP1; wherein, (A) is a PCR identification analysis diagram of the TaGAP1-OE-T0 generation line; (B) is a diagram of detecting the transcription level of TaGAP1 in the TaGAP1-OE-T0 generation line by qRT-PCR.

[0038] Figure 12 Figure 1 is a disease resistance identification chart of TaGAP1-OE-T1 generation lines; wherein, (A) is a PCR detection chart of positive plants of TaGAP1-OE-T1-L4 and TaGAP1-OE-T1-L6 lines; a phenotype identification chart of the positive plants after inoculation with powdery mildew E09; (B) is a biomass analysis chart of the positive plants and wild type plants after inoculation with E09; (C) is a chart of the transcription level of TaGAP1 in the overexpression TaGAP1-T1-L4 line; (D) is a chart of the transcription level of TaGAP1 in the overexpression TaGAP1-T1-L6 line. DETAILED DESCRIPTION

[0039] The application will be described in detail below with specific examples. It should be noted that the following examples are only further illustrations of the application, and the protection scope of the application is not limited to the following examples.

[0040] EXAMPLE

[0041] 1. Test materials and instruments

[0042] 1.1.1 Materials

[0043] Wheat powdery mildew: E09, provided by the laboratory;

[0044] Wheat materials: Jingshuang 16: used for propagating wheat powdery mildew; Xingmin 318 and Shuiyuan 11: used for detecting the transcription level of TaGAP1 under non-compatible and compatible, and used for BSMV-VIGS experiment, Fielder: used for transgenic experiment;

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

[0046] Vector materials: BSMV: BSMV-PDS, BSMV-a, BSMV-b and BSMV-g for BSMV-VIGS experiment, pCAMBIA-1302-GFP and pJIT163 for subcellular localization experiment, CUB for transgenic experiment.

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

[0048] 1.1.2 Reagents

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

[0050] Kit: Gel recovery kit (Magen), wheat protoplast preparation and transformation kit (Coolaber), gold super-excess endotoxin-free plasmid large extraction kit (Kangwei Century), RiboMAX TM Large Scale RNA Production Systems (Promega), reverse transcription kit (Vazyme), plasmid small extraction kit (Magen), RNA extraction kit (Magen), Ribo m7G Cap Analog

[0051] Reagent: MgCl2, acetyl-syringone, Ribo Gm7G Gap Analog (human grass), ChamQ SYBR qPCR Master Mix (Vazyme), 2x Phanta Flash Master Mix (Vazyme), RNase inhibitor (yeasen),

[0052] 1.1.3 Instruments

[0053] Artificial climate incubator (DASGARD), PCR instrument (C1000 Bio-Rad), electrophoresis instrument (JY-SCZ7 Junyi), fluorescent quantitative PCR instrument (Thermo), gel imaging instrument (Quantumst4 Wuzhou Oriental), sterilization pot, Olympus biological microscope (BX-53 Olympus), spectrophotometer (NanoDrop 2000c Thermo), high-speed refrigerated centrifuge (ST16R Thermo), shaking bed (IS-RDVS Jingqi).

[0054] 1.2 Test method

[0055] 1.2.1 Propagation and preservation of wheat powdery mildew

[0056] E09 of Erysiphe graminis is used for propagation and preservation on wheat variety Jingshuang 16. Select Jingshuang 16 seeds with full grains, place them in clean water to germinate, and sow them after the seeds turn white. Use round pots, sow about 10-12 seeds per pot, cover them with soil, and cover them with a transparent plastic cover. Tighten the top with 6 layers of gauze and leather (to prevent contamination by other bacteria), and place them in a 16°C, 16h light and 8h dark alternating incubator. Inoculate the seedlings with Erysiphe graminis using the shaking method when they reach the one-leaf-one-heart stage. Spray water on the seedlings to make water droplets fall on them, and shake the seedlings with the Erysiphe graminis attached to them. Then spray water to allow the Erysiphe graminis to fall on the seedlings, and tighten the gauze. Place them in an artificial climate incubator (17°C, 75% humidity, 16h / 8h day / night light cycle). The preservation of wheat powdery mildew is carried out by using test tube seedlings for preservation, and the above-mentioned steps are used to cultivate seedlings and inoculate powdery mildew. When the powdery spores appear, they are moved to 4°C for preservation.

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

[0058] According to the previous transcriptomic data of the interaction between wheat and Erysiphe graminis, 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 aligned using DNAMAN. The protein sequences were downloaded from Uniport (https: / / www.uniprot.org / ), and the phylogenetic tree of protein TaGAP1 and its homologs was constructed using MEGA7.0.

[0059] 1.2.3 Transcriptional expression analysis of TaGAP1

[0060] 1.2.3.1 Collection of samples

[0061] Wheat variety Shuiliang 11 and Erysiphe graminis E09 form an affinity combination, and Xingmin 318 and E09 form an affinity combination. Select Mingxian 169 and Xingmin 318 seeds with full grains, and inoculate them with Erysiphe graminis E09 when they reach the one-leaf-one-heart stage. Select 0h, 6h, 12h, 24h, 48h, 72h, 96h, and 120h time points for sampling. Put sterilized steel balls into 2ml centrifuge tubes in advance, and put the collected samples into the tubes. Freeze them in liquid nitrogen and store them at -80°C. Each time point has 3 biological replicates.

[0062] 1.2.3.2 Extraction of RNA

[0063] RNA extraction was performed using HiPure Plant RNA Mini Kit kit (Magen), and the steps were as follows:

[0064] (1) Preliminary work: grind the collected samples using a tissue grinder into powder. Spray the clean bench with RNAse to remove, ultraviolet sterilization for 30 min, and wear a mask and gloves during operation;

[0065] (2) Add 800 μL of Buffer RLC to the ground sample, mix the sample thoroughly, and stand on ice for 3 min, centrifuge at 14000g for 5 min;

[0066] (3) Assemble the filter column, and aspirate 600 μL of supernatant into the filter column, centrifuge at 14000g for 2 min, and discard the filter column;

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

[0068] (5) Transfer the mixed solution in (4) to the RNA Mini Column filter column on the 2 ml collection tube, centrifuge at 12000g 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, and add 500 μL of Buffer RW1 to the sample-containing filter column, and centrifuge at 10000g for 1 min;

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

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

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

[0074] (11) Place the filter column in a 1.5 ml centrifuge tube, and add 40 μL of RNase Free Water to the filter column, stand for 2 min, and centrifuge at 12000g for 1 min. Repeat once;

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

[0076] 1.2.3.3cDNA reverse transcription synthesis

[0077] The reverse transcription synthesis of cDNA was performed using HiScript IIQ Select RT Super Mix for qPCR kit. The RNase Free PCR single tube, RNase Free gun head, etc. were placed in super clean and ultraviolet sterilized for 30 min, and the whole experiment was performed with masks and gloves.

[0078] (1) Add RNA sample (1.5 μg), 4×gDNA wiper mix (4 μL), RNase-free ddH2O (to 16 μL) to the PCR single tube. Mix and centrifuge, and the PCR program is 42℃, 2 min;

[0079] (2) After the PCR reaction, add 4 μL of 5×HiScript IIQ Select qRT SuperMix II to the above-mentioned PCR single tube, mix and centrifuge, and the PCR program is 50℃, 15 min; 85℃, 5 s;

[0080] (3) After the reaction, dilute with 180 μL of ddH2O, and store at -20℃ for standby.

[0081] 1.2.3.4 Real-time fluorescent quantitative PCR

[0082] The TaGAP1 specific quantitative primer was designed by Primer Premier 5.0. The amplification efficiency and specificity of the designed primer were verified by qRT-PCR, and the transcription level was detected. The whole experimental operation process was performed with masks and gloves, and the reaction system was as shown in Table 1: Figure 1

[0083] Table 1

[0084]

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

[0086] Table 2

[0087]

[0088] The experiment was repeated three times biologically and technically, and the data was analyzed by comparative 2-ΔΔCt method.

[0089] 1.2.4 Construction of wheat gene TaGAP1 vector

[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 primer of TaGAP1 was designed, and the PCR amplification was carried out using the following system. The target band was detected and recovered by agarose gel electrophoresis, as shown in Table 3:

[0092] Table 3

[0093]

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

[0095] (1) Under ultraviolet conditions, cut the required band and put it into a 2 ml centrifuge tube, and add 1 ml of Buffer GDP;

[0096] (2) 55℃ water bath for 10-15 min, shake once every 5 min to accelerate the dissolution of the gel;

[0097] (3) Put the HiPure Gel Pure DNA Mini Column adsorption column on the centrifuge tube, and suck the gel in (2) into the adsorption column, centrifuge at 12000g for 1 min;

[0098] (4) Discard the centrifugal filtrate in the collection tube, add 300 μL of Buffer GDP to the adsorption column, stand for 2 min, centrifuge at 12000g for 1 min;

[0099] (5) Discard the filtrate, add 600 μL of Buffer DW2 to the adsorption column, centrifuge at 12000g for 1 min;

[0100] (6) Discard the filtrate, add 300 μL of Buffer DW2 to the adsorption column, centrifuge at 12000g for 1 min;

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

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

[0103] (9) Put the adsorption column in a 1.5 ml centrifuge tube, add 20 μL of Elution Buffer to it, stand for 2 min, centrifuge for 1 min, repeat once, and store at -20℃ for standby.

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

[0105] The empty vector is digested according to the enzyme cutting site, and the uncut vector is used as a control to detect whether the vector is in a linear state. The recovered product is recombined with the linearized vector, and 50-100 ng / μL of the recovered product, 50-100 ng / μL of the digested vector, and 5 μL of the recombination enzyme are sequentially added to a 0.2 ml tube, and ddH2O is added to 10 μL. Mix gently, centrifuge for a few seconds, and react in a PCR instrument at 50°C for 15 min. After the PCR is completed, a transformation experiment is performed. 10 μL of the PCR product is added to 50 μL of E. coli competent cells, and the mixture is incubated on ice for 30 min, incubated in a 42°C water bath for 45 sec, and incubated on ice for 2 min. 700 μL of LB medium (without antibiotics) is added, and the mixture is incubated in a 200 rpm shaker for 1 h. The mixture is centrifuged at 4500 rpm for 5 min, and the supernatant is discarded. The residual liquid is blown to the bacterial mass, and the sample is coated on the surface of an LB plate with a gun head. The plate is inverted and placed in a 37°C incubator for 12-16 h. A single colony is selected for colony PCR detection, and the PCR reaction program is shown in Table 4:

[0106] Table 4

[0107]

[0108] The PCR product is detected by agarose gel electrophoresis to confirm whether the target band is amplified. According to the electrophoresis result, the colony corresponding to the target DNA band of the expected size is selected, and the correct colony is picked up with a sterilized white gun head and suspended in liquid LB medium (containing antibiotics). After incubation at 37°C and 200 rpm for 12-16 h in a shaker, the subsequent experiment is continued.

[0109] 1.2.4.3 Small-scale extraction of the recombined vector

[0110] The above shaken bacterial solution is subjected to plasmid extraction, and the extraction steps are as follows:

[0111] The bacterial solution is added to a 2 ml centrifuge tube multiple times, centrifuged at 10000 g for 1 min, and the supernatant is discarded.

[0112] 250 μL of Solution I (Rnaase A is added in advance and stored at 4°C) is added to resuspend the aggregated bacterial cells; then 250 μL of Solution II is added, and the sample is gently inverted 3-5 times to ensure thorough mixing, and the sample is allowed to stand for 2 min.

[0113] 350 μL of Solution III is added, the sample is gently inverted 3-5 times to ensure that Solution III is thoroughly mixed, and the sample is observed to have a white state with flocculation; centrifugation is performed at 13000 g for 10 min; a filter column is installed in advance, and the supernatant in step (4) is absorbed into the filter column, and centrifugation is performed at 13000 g for 1 min.

[0114] Discard the filtrate, and pipette 500 μL HBC Buffer into the filter column, centrifuge at 13000 g for 1 min; discard the filtrate, and pipette 700 μL of DNA Wash Buffer (remove impurities), centrifuge at 13000 g for 1 min, repeat once;

[0115] Discard the filtrate, centrifuge at 13000 g for 2 min, dry at room temperature for 5-7 min to volatilize the residual ethanol;

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

[0117] 1.2.4.4 Sequencing and alignment of recombinant plasmids

[0118] Pipette 8 μL of the above plasmid into a PCR tube, and pipette the universal primer of the plasmid into the PCR tube, and send it to Qikang Biotechnology Co., Ltd. for sequencing analysis. Use the NCBI alignment tool to align and analyze the obtained sequencing results with the sequence of the target genome. Through alignment analysis, if the sequencing sequence is completely consistent with the sequence of the target gene, the related vector construction of TaGAP1 is successful.

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

[0120] 1.2.5.1 Construction of gene silencing-related vectors

[0121] Two specific fragments were selected from the TaGAP1 gene, specific primers were designed, and high-fidelity enzyme 2x PhantaFlash Master Mix (Dye Plus) was used to amplify the two fragments and construct them into BSMV:γ vectors to obtain BSMV:TaGAP1-1as and BSMV:TaGAP1-2as recombinant plasmids. The specific construction method is referred to 1.2.4;

[0122] 1.2.5.2 Linearization of BSMV vectors

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

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

[0125] Table 5

[0126]

[0127] The following BSMV: TaGAP1-1 as, BSMV: TaGAP1-2as and BSMV: PDS enzyme cutting system, PCR program 50°C 2h, 65°C 15min, see Table 6:

[0128] Table 6

[0129]

[0130] The following BSMV: β enzyme cutting system, PCR program 37°C 2h, 65°C 15min, see Table 7:

[0131] Table 7

[0132]

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

[0134] Preparation: The ultraclean workbench and the gun head and PCR tube used were sterilized with ultraviolet light in advance, all experimental operations were carried out on ice (to ensure that the sample was at low temperature), a mask was worn to avoid cross contamination, and the gun head and PCR tube used in the experiment were enzyme-free (to avoid contamination by exogenous enzymes). The in vitro transcription kit was used for in vitro transcription, and the reagents shown in Table 8 were added to the RNAase-free PCR tube in turn:

[0135] Table 8

[0136]

[0137]

[0138] Gently mix, centrifuge, and PCR reaction at 37°C for 1h. After the reaction, 1μL of the product was taken into a PCR tube and 5μL of RNAase-Free Water was added, and agarose gel electrophoresis was used to detect the PCR product to determine whether the in vitro transcription was effective. The remaining product was stored at -80°C for subsequent experiments.

[0139] 1.2.5.4 Inoculation of BSMV virus

[0140] The seeds of the water source 11 and the Xingmin 318 wheat were selected and planted in each flowerpot, with 15 seeds per flowerpot. The BSMV virus inoculation experiment used two-leaf-one-heart wheat;

[0141] The product in 1.2.5.3 is divided into three groups on average: control group: MOCK, BSMV: a+PDS, BSMV: a+PDS, experimental group: BSMV: a+TaGAP1-1as, BSMV: a+TaGAP1-2as; each combination product is mixed with 800 μL of FES buffer in a PE glove, gently blow and mix, and inoculated in several drops;

[0142] Each combination inoculates 5 pots of wheat, wears a small rubber glove when inoculating, dips the mixture to inoculate and rub the second leaf of wheat. After inoculation, it is placed in a 25℃ artificial climate box for overnight light protection and moisture, and then adjusted to normal photoperiod and high humidity for culture;

[0143] After about 10-12 days of inoculation, pay close attention to observe whether it is toxic. The leaves without toxicity are cut off before inoculation, and the leaves with toxicity are inoculated for inoculation experiment.

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

[0145] Use a marker pen to mark the edges of the third and fourth leaves of the toxic plant to ensure that the inoculation position can be clearly identified, which is convenient for subsequent sampling and phenotype observation. Refer to 2.2.1 for the inoculation of powdery mildew;

[0146] Quantitative and histological samples are collected at different time points (24h, 48h, 72h and 96h) after inoculation, and quantitative samples are quickly frozen in liquid nitrogen. RNA extraction and reverse transcription refer to 2.2.3.2;

[0147] After 9 days of inoculation, observe whether the control group is fully diseased. After full disease, observe the phenotype of the silenced plant and take pictures;

[0148] The leaves for histological observation need to be cut into 2-3 cm segments and placed in decolorizing solution (ethanol: glacial acetic acid = 1:1). After the decolorizing solution changes color, replace it repeatedly to ensure the decolorizing effect, and finally the leaves become transparent;

[0149] After decolorization, remove the decolorizing solution and soak in chloral hydrate for 12h;

[0150] Rinse the leaves with distilled water several times, immerse the leaves in Coomassie brilliant blue dye for 15min, and continue to wash with distilled water for 3-5 times after staining to remove the dye;

[0151] Make a film, and observe the mycelium length, mycelium branching and colony area of powdery mildew under a microscope. At least 50 infection points are recorded at each time point.

[0152] 1.2.6 Tobacco subcellular localization

[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. The specific construction method was referred to 1.2.4;

[0155] 1.2.6.2 Agrobacterium transformation and tobacco transient overexpression

[0156] In the clean bench, 1 μg of plasmid was added to the Agrobacterium competent cells, 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, and then 600 μL of LB was added. The mixture was incubated at 28°C and 180 rpm for 2-3 h;

[0157] Centrifugation was performed at 4500 rpm for 5 min, and the supernatant was discarded. The bacterial pellet was blown and hit with the residual liquid, and then it was streaked on the surface of an LB plate (containing antibiotics) under sterile conditions. The plate was inverted and incubated at 28°C for 2-3 d;

[0158] A colony of appropriate size was selected and picked with a sterile pipette tip, and then it was inoculated in liquid LB medium (containing antibiotics) in a shaking incubator at 28°C and 200 rpm for 12-16 h.

[0159] Bacterial liquid PCR was performed on the cultured Agrobacterium, and the glycerol bacteria of positive detection were stored for later use. The glycerol bacteria were stored at -80°C for standby and subsequent experiments.

[0160] 1.2.6.3 Tobacco transient overexpression

[0161] The Agrobacterium liquid with correct PCR detection was centrifuged at 4500 rpm for 5 min.

[0162] 10 mM magnesium chloride and AS buffer were prepared, the supernatant was discarded, 1 ml of 10 mM magnesium chloride was added, centrifugation was performed at 4500 rpm for 5 min, and the above steps were repeated once. The AS buffer is shown in Table 9:

[0163] Table 9

[0164]

[0165]

[0166] The supernatant was discarded, and the OD value of the Agrobacterium was adjusted to OD600 = 0.6 with AS buffer. The mixture was incubated at 28°C in the dark for 2-3 h.

[0167] Healthy tobacco is selected for tobacco injection. First, a needle is used to lightly prick the back of the tobacco, and the bacteria solution is injected into the area. The tobacco is placed in a 25℃ incubator overnight in the dark and moistened. After 2-3 days of normal photoperiod cultivation, the expression is observed under an Olympus biological microscope.

[0168] 1.2.7 Subcellular localization of wheat protoplasts

[0169] 1.2.7.1 Construction of a wheat protoplast localization-related vector

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

[0171] 1.2.7.2 Large-scale extraction of recombinant plasmid

[0172] The colonies that are detected by PCR are suspended in LB (containing antibiotics), and cultured at 37℃ for 12-16h on a shaker at 200rpm. Then, the colonies are transferred into 500ml of LB liquid medium prepared in advance at a ratio of 1:100, and cultured at 37℃ for 2-3h on a shaker at 200rpm. The plasmid is extracted according to the plasmid extraction kit of Kangweishiji Company, and the specific steps are as follows: the LB medium is shown in Table 10.

[0173] Table 10

[0174] Reagent Mass / volume Yeast extract (YE) 5g Peptone 10g NaCl 10g ddH2O 1L

[0175] The bacterial liquid is centrifuged at 12000g for 10min to collect the bacterial bodies, and the supernatant is discarded.

[0176] To the bacterial bodies of (1), 12ml of Buffer P1 (RNAase A is added in advance, and stored at 4℃) is added, and mixed thoroughly. 12ml of Buffer P2 is added, and mixed gently by inversion. After standing for 5min, the reaction is complete. 12ml of Buffer P3 is added, and mixed uniformly by inversion. After standing for 5min to ensure complete reaction, the mixture is centrifuged at 12000g for 10min, and the supernatant is transferred to a new centrifuge tube.

[0177] Isopropyl alcohol (0.3 times the volume of the supernatant) is added, and mixed uniformly by inversion.

[0178] The adsorption column is installed in advance, and 2ml of Buffer ES is absorbed in the adsorption column, and centrifuged at 12000g for 2min. The filtrate is discarded.

[0179] The mixed solution in (3) is transferred to the adsorption column in small amounts and multiple times, and centrifuged at 12000g for 2min. The filtrate is discarded.

[0180] 10 ml of Buffer PW containing anhydrous ethanol was sucked into the adsorption column, centrifuged at 12000 g for 2 min, the filtrate was discarded, and the operation was repeated once;

[0181] 10 ml of Endo-Free Buffer PW was added, centrifuged at 12000 g for 5 min, the filtrate was discarded, and the operation was repeated once, and the column was dried in the clean bench for several minutes;

[0182] The adsorption column was installed in a centrifuge tube, 800 μL of Endo-Free Buffer EB was added, and the operation was repeated once, and the plasmid was transferred to a 1.5 ml centrifuge tube for subsequent experiments, and was stored at -20°C.

[0183] 1.2.7.3 Preparation and transformation of wheat protoplasts

[0184] The seeds of water source 11 were selected and planted, and the subsequent experiments were carried out when the plants grew to one leaf and one heart under weak light at 25°C;

[0185] The enzyme solution was prepared (0.3 g of cellulase, 0.06 g of pectinase and 16 ml of enzyme solution were weighed in a conical flask, and were mixed for 2-3 times in a 55°C water bath for 10 min; after cooling, the following ingredients were added: 200 μL of 10% bovine serum albumin, 7.14 μL of β-mercaptoethanol, 10 μL of Amp, and ddH2O was added to 20 ml) ;

[0186] The wheat leaves were cut into small pieces of about 1 mm in size using a knife and were poured into a conical flask, and were treated in the dark for 30 min under vacuum;

[0187] After the conical flask was transferred to a 25°C shaker at 40 rpm for 2 h, it was left standing at 25°C for 4 h, and a drop of microscope was sucked using a sharp yellow gun head, and if the cells were round and bright, the subsequent experiment was continued;

[0188] The 200 μm cell sieve was rinsed with W5 solution at 4°C, and the operation was repeated three times, wherein the W5 solution is shown in Table 11:

[0189] Table 11

[0190] Reagent Mass / volume NaCl 9g CaCl2 18.4g KCl 3.75g MES 0.42g ddH2O 1L

[0191] NaOH was used to adjust the pH to 5.7, and was autoclaved.

[0192] The lysate was filtered into a new centrifuge tube using a cell sieve, and was centrifuged at 100 g for 10 min using a 4°C horizontal rotor centrifuge, and the speed was increased and decreased by 3, and the supernatant was sucked using a sharp gun head;

[0193] Add 4℃ W5 solution to the protoplasts gently, and make the protoplasts evenly dispersed. Centrifuge at 100g for 5min, and discard the supernatant.

[0194] Add 10ml 4℃ W5 solution to the protoplasts, and place in an ice bath for 30min. Centrifuge at 100g for 5min, and discard the supernatant.

[0195] Slowly add 2ml 4℃ MMG to the centrifuge tube, and gently shake the centrifuge tube to make the protoplasts evenly dispersed. Place on ice until the protoplast preparation is complete. The MMG solution is shown in Table 12:

[0196] Table 12

[0197] Reagent Mass / volume Mannitol 7.285g MgCl2 0.305g MES 0.085g ddH2O 1L

[0198] Adjust the pH to 5.7 with NaOH, and autoclave.

[0199] Add 15μg plasmid, 165μL protoplasts, and 150μL PEG transformation solution to a 2ml centrifuge tube, and mix gently. Place in a 25℃ dark place for 30min. The PEG transformation solution is shown in Table 13:

[0200] Table 13

[0201] Reagent Mass / volume Mannitol 0.036g PEG 4000 0.4g 1 M CaCl2 100 μL ddH2O To 1 ml

[0202] Slowly add 4℃ pre-cooled W5 solution to the centrifuge tube, and gently shake to make the protoplasts evenly dispersed. Let stand for 2min, discard the supernatant, and repeat once.

[0203] Slowly add 600μL pre-cooled W5 solution to the centrifuge tube, and gently shake the centrifuge tube to mix thoroughly. Place in a 25℃ incubator in the dark for 16-18h. Observe the expression of the target protein under a microscope.

[0204] 1.2.8 Creation of Transgenic and Gene Edited Wheat Materials

[0205] Construct TaGAP1 into CUB vector, and the specific construction method is referred to 1.2.4. At the same time, transform the plasmid into EHA105 competent cells. After the colonies grow, mark the positive colonies after PCR detection. Send the plate to the transgenic platform of the State Key Laboratory of Stress Biology of Arid Crops in Northwest Agriculture and Forestry University for Agrobacterium-mediated wheat genetic transformation, and the wheat recipient variety is Fielder. For the creation of gene edited materials, send three copies of the sequence to Shandong Academy of Agricultural Sciences to construct a gene editing vector of TaGAP1 and perform wheat genetic transformation.

[0206] 1.2.8.1 Molecular Identification of T0 Plants

[0207] The seedlings of T0 generation plants were transplanted into flower pots for culture. When the seedlings grew to a certain extent, the leaves were collected. One part was used as a DNA sample for PCR detection of positive plants, and one part was used as an RNA sample for qRT-PCR screening of transgenic positive plants with high expression. The steps of RNA extraction, reverse transcription and real-time fluorescent quantitative PCR were carried out according to 1.2.3.2. The DNA extraction steps are as follows:

[0208] Steel balls were added to the centrifuge tube containing the leaves, and the leaves were crushed into powder using a grinder;

[0209] 750 μL of 65°C water bath CTAB extract was added to the DNA sample, and the sample was mixed gently and then placed in a 65°C water bath for 1 h, with mixing every 20 min;

[0210] The CTAB extract is shown in Table 14:

[0211] Table 14

[0212] Reagent Mass / volume CTAB 20g 0.5 M Tris-HCl (PH=8.0) 200ml 0.5 M EDTA (PH=8.0) 40ml NaCl 81.8g 0.2% Mercaptoethanol 2ml ddH2O 1L

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

[0214] The supernatant was transferred to a new centrifuge tube without sucking in the precipitate, and 750 μL of nucleic acid extraction reagent (24:1) was added to the sample, which was mixed gently and then centrifuged at 12000 rpm at 4°C for 10 min;

[0215] The supernatant was transferred to a new centrifuge tube, 750 μL of -20°C isopropanol was added to the supernatant, and it was placed in a -20°C refrigerator for 16-24 h to ensure complete precipitation;

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

[0217] 500 μL of 75% ethanol was added to the precipitated sample and washed twice, and the residual ethanol was allowed to evaporate under ventilation;

[0218] 50 μL of ddH2O was added to dissolve the DNA, which was stored at -20°C for later use.

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

[0220] Seeds of high expression and positive plant families were planted, and when they grew to one leaf and one heart, they were inoculated with powdery mildew E09. The disease condition of the plants was observed 7-10 days after inoculation. After Fielder was fully diseased, the phenotype was observed and photographed;

[0221] Biomass detection: After 7 days of inoculation, biomass samples were collected, and DNA was extracted according to the method in 1.2.8.1. The extracted DNA was used as a template, and the DNA concentration was diluted to the same concentration. Fluorescent quantitative PCR was performed using primers Bgt β-tublin and TaEF; the Ct value was brought into the standard curve to obtain the Bgt β-tublin and TaEF copy numbers, respectively, and Bgt / Wheat = Bgt β-tublin copy number / TaEF copy number.

[0222] 1.3 Results and analysis

[0223] 1.3.1 Bioinformatics analysis of TaGAP1

[0224] The full-length 513 bp of TaGAP1 was analyzed by Ensembl Plants website, which encodes a GTPase-activating protein of 170 aa, with a protein size of 18.98 kDa, as shown in Table 15; NCBI's conserved domain predicts that the protein belongs to the ArfGAP family, containing a C2 domain, of which 6-150 aa is a C2 domain Figure 1 -A). It was found by searching in Ensembl Plants and NCBI databases that TaGAP1 has three copies in wheat, located on chromosomes 2A, 2B, and 2D. By using DNAMAN software to align the sequences of the three copies, the similarity between the three copies was very high, up to 98% Figure 1 -B).

[0225] Table 15

[0226]

[0227]

[0228] The sequences of GAP1 proteins in wheat and related species were analyzed by alignment, and the Neighbor-Joining method was used to construct a phylogenetic tree using MEGA7.0 software. The results of the phylogenetic tree showed that TaGAP1 had the closest genetic relationship with Ural wheat and the farthest genetic relationship with corn, millet, and millet (see Figure 2). The phylogenetic tree of TaGAP1 was constructed by using MEGA 7.0 software with the neighbor-joining method. Hv: barley, Hordeum vulgare, Os: rice, Oryza sativa, At: Arabidopsis thaliana, Zm: maize, Zea mays, Tu: Triticum urartu, At: Aegilops tauschii, Ta: wheat, Triticum aestivum, Si: foxtail millet, Setaria italica, Ph: Panicum hallii, Sb: Sorghum bicolor.

[0229] 1.3.2 Transcriptional expression analysis of TaGAP1

[0230] To explore whether TaGAP1 is involved in the interaction between wheat and powdery mildew, the transcriptional expression profile of TaGAP1 in wheat and powdery mildew compatible (Shuiyuan 11 / E09) and non-compatible (Xingmin 318 / E09) combinations was analyzed by qRT-PCR. The results showed that in the compatible combination, the expression level of TaGAP1 was down-regulated by 2-fold in general; in the non-compatible combination, the expression level of TaGAP1 was significantly up-regulated, reaching the peak at 72h, and the expression level reached 4-fold (see Figure 3 ). Therefore, it is speculated that TaGAP1 may act as a positive regulatory factor to participate in the resistance of wheat to powdery mildew. From Figure 3 It can be seen that: the standard deviation represents the mean ± standard error of three independent samples. Data analysis was performed by 2 -ΔΔCt Relative quantification method, the expression level was normalized to TaEF-1a. The asterisk indicates the significance of t-test (*, P < 0.05; **, P < 0.01).

[0231] 1.3.3 Subcellular localization of TaGAP1

[0232] To clarify the subcellular localization of TaGAP1, TaGAP1-GFP fusion vector was constructed, and pCAMBIA1302-GFP was used as the 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 TaGAP1-GFP was mainly detected in the cytoplasm and nucleus (as shown in Figure 4 ).

[0233] To further confirm the subcellular localization of TaGAP1, TaGAP1 was connected to pJIT163-GFP to construct TaGAP1-GFP fusion vector, with pJIT163-GFP empty vector as control. PEG-mediated transformation was used to transform TaGAP1-GFP and GFP empty vector into wheat protoplasts, which were cultured in dark for 16-19 h, and then observed under fluorescence microscope. The results showed that GFP was located in cytoplasm, cell membrane and nucleus of wheat cells, while TaGAP1-GFP was mainly located in cytoplasm and nucleus (as shown in FIG. 1C). Figure 5 In summary, TaGAP1 was located in cytoplasm and nucleus.

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

[0235] 1.3.4.1 Phenotype observation of transient silencing of TaGAP1

[0236] To confirm the function of TaGAP1 in wheat resistance to powdery mildew, two fragments of BSMV-VIGS transiently silencing TaGAP1, BSMV:TaGAP1-1as and BSMV:TaGAP1-2as, were used. After 10 days of inoculation, obvious light bleaching stripes appeared on the leaves of the positive control group BSMV:TaPDS, indicating successful inoculation, while obvious chlorosis spots were observed on the leaves of the blank control group MOCK, the negative control group BSMV:γ, and the test groups BSMV:TaGAP1-1as and BSMV:TaGAP1-2as (see FIG. 2A). Figure 6 -A, Figure 7 -A). The silencing plants and control plants were inoculated with powdery mildew E09, and samples for silencing efficiency detection and histological observation were collected at 24 h, 48 h, 72 h and 96 h after inoculation. After 10-12 days of inoculation, the phenotype was observed. In the non-compatible combination, no powdery spores appeared on the leaves of the plants of MOCK and BSMV:γ, while a small amount of powdery spores appeared on the leaves of the silencing plants (see FIG. 2B); in the compatible combination, powdery spore piles appeared on the leaves of the plants of MOCK, BSMV:γ and the silencing plants, but the powdery spores on the leaves of the silencing plants were significantly more than those of the control groups (see FIG. 2B). Figure 6 Figure 7 qRT-PCR silencing efficiency detection results showed that the expression of TaGAP1 in the silencing plants was significantly lower than that in the control groups. In the non-compatible combination, the silencing efficiency of the leaves of the silencing plants was more than 50%; in the compatible combination, the silencing efficiency was 49%-72% (see FIG. 2C-C), proving that the silencing of TaGAP1 was successful, and the results of VIGS experiment were reliable. Figure 6 Figure 7

[0237] 1.3.4.2 Histological observation of wheat powdery mildew ​​​

[0238] Histological observation results showed that in the non-compatible combination, there was no significant difference in mycelial growth between TaGAP1 silenced plants and control plants at 24hpi; at 48hpi, 72hpi and 96hpi, the growth and development of mycelium in the silenced plants were significantly faster than in the control plants (see Figure 8 -A), and statistical analysis also showed that the colony area and mycelial length were significantly greater than the control, and the mycelial branching was not significantly different at 48hpi, but significantly greater than the control at 72hpi and 96hpi (see Figure 8 B-D). In the compatible combination, the growth of powdery mildew in the silenced plants was also significantly greater than in the control plants (see Figure 9 -A). Statistical analysis results showed that the colony area, mycelial length and mycelial branching of powdery mildew at each sampling point were significantly higher than in the control plants (see Figure 9 B-D).

[0239] 1.3.4.3 Transcript expression profile analysis of pathogenesis-related protein (PR) gene

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

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

[0242] To further study the disease resistance function of TaGAP1 in the interaction between wheat and powdery mildew, TaGAP1 overexpression wheat lines were created, and the following tests were conducted.

[0243] 1.3.5.1 Obtaining of transgenic wheat plants

[0244] The plasmid Cub-TaGAP1 was transformed into Agrobacterium competent cells by Agrobacterium-mediated transgenic technology, and was sent to the Transgenic Platform of the State Key Laboratory of Crop Stress Resistance and High Efficiency Production, Northwest A&F University. PCR detection was used to screen T0 positive plants, and a total of 12 positive plants L1-L12 were obtained (see Figure 11-A). Further qRT-PCR was used to detect the expression level of TaGAP1 in the positive lines. The expression levels in 11 lines were significantly higher than those in Fielder, with L4 and L6 showing the highest expression levels, reaching 14.87-fold and 19.89-fold, respectively (see [link to qRT-PCR]). Figure 11 -B). Therefore, L4 and L6 were subsequently subcultured, and the disease resistance of the T1 generation plants was tested.

[0245] 1.3.5.2 Disease resistance detection of TaGAP1 transgenic overexpression lines

[0246] Phenotypic observation was performed 7 days after inoculation of the T1 generation of TaGAP1 transgenic overexpression L4 and L6 families with powdery mildew E09. The number of powdery mildew colonies in the L4 and L6 lines was significantly less than that in Fielder powdery mildew (see...). Figure 12 -A), and the biomass was also significantly lower than that of Fielder (see -A). Figure 12 -B). qRT-PCR results showed that the expression levels of TaGAP1 in each line of L4 and L6 were approximately 7-25 times that of Fielder (see...). Figure 12 -C, D). In conclusion, overexpression of TaGAP1 enhances wheat resistance to powdery mildew.

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

[0248] This study silenced TaGAP1 using BSMV-mediated transient silencing technology. After inoculation with E09, the number of powdery mildew colonies in the silenced plants was found to be significantly increased (see...). Figure 6 , Figure 7 The powdery mildew grew and developed faster than the control plants, with significantly higher mycelial branching, length, and infection area (see...). Figure 8 , Figure 9 Downregulation of disease-related proteins indicates that silencing TaGAP1 reduces wheat resistance to powdery mildew. Conversely, overexpression of TaGAP1 enhances wheat resistance to powdery mildew (see...). Figure 12 This study aims to clarify the disease resistance function of TaGAP1 in the interaction between wheat and powdery mildew. However, further experiments are needed to analyze the expression levels of key genes in related signaling pathways in transgenic plants that overexpress TaGAP1 and to identify the signaling pathways involved.

[0249] The specific embodiments of the present application are described above. It needs to be understood that the present application is not limited to the specific embodiments described above, and various modifications or changes can be made by those skilled in the art within the scope of the claims, which do not affect the essence of the present application.

Claims

1. A wheat powdery mildew resistance gene TaGAP1 use in wheat powdery mildew resistance breeding, characterized in that, Wheat powdery mildew resistance genes TaGAP1 The full length of TaGAP1 of 513 bp was analyzed by Ensembl Plants website, encoding a GTPase-activating protein of 170 aa; wherein the protein size is 18.98 kDa, and the nucleotide sequence is shown as SEQ ID NO. 1.

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