CN111118042B - Powdery mildew-resistant grape calcium-dependent protein kinase gene VpCDPK9 and application thereof - Google Patents

Abstract

The patent application of the present invention discloses the isolated powdery mildew-resistant grape calcium-dependent protein kinase gene VpCDPK9 and its application. The present application relates to the field of genetic engineering, especially to the field of grape disease resistance breeding. The calcium-dependent protein kinase gene VpCDPK9 was isolated from the genome of the disease-resistant Chinese wild East Huadong grape Baihe-35-1 by gene cloning technology, and the gene was transferred into powdery mildew susceptible grape plants to obtain powdery mildew-resistant transgenic plants Grape plants, improve the resistance of grapes to powdery mildew.

Description

Powdery mildew resistant grape calcium-dependent protein kinase gene VpCDPK9 and its application

technical field

The patent application of the present invention belongs to the technical field of plant stress resistance gene identification and genetic engineering, in particular to the isolation and identification of powdery mildew-resistant grape calcium-dependent protein kinase genes, and more specifically relates to the anti-stress gene isolated from Chinese wild Huadong grape Baihe-35-1. Powdery mildew calcium-dependent protein kinase gene VpCDPK9 and its application against powdery mildew.

Background technique

Powdery mildew is an important fungal disease that seriously harms grape production, often resulting in severe yield reduction or even no harvest, resulting in huge economic losses. In order to prevent and control the disease, pesticides need to be sprayed 20-30 times a year in production, which seriously affects the edible safety of grape products, threatens human health, and pollutes the ecological environment (Gadoury et al., 2012). At present, grape breeders are increasingly aware that it is an important way to fundamentally solve this problem to explore and utilize the disease resistance genes of wild grapes as soon as possible and actively carry out molecular breeding of grapes for precise disease resistance.

Calcium ions are important second messengers in eukaryotic cells. Calcium-dependent protein kinases (CDPKs) are a special class of calcium signal decoding proteins in plants, which can independently complete the decoding and transmission of calcium signals. When plant cells are under stress, the receptor protein on the cell membrane recognizes the stress signal and activates the calcium ion channel rapidly, and the large influx of calcium ions in the apoplast space leads to an increase in the concentration of calcium ions in the cytoplasm. The EF-hand domain at the carboxyl terminus of CDPKs binds to calcium ions to induce conformational change of CDPKs, exposing its kinase active center. At this time, CDPKs can interact with and phosphorylate substrate proteins, which can activate or stabilize downstream substrate proteins. Sexual regulation. The above process converts calcium signals (cytoplasmic calcium ion concentration oscillation) into phosphorylation events, and realizes the transmission and processing of stress signals. Therefore, CDPKs proteins play an important regulatory role in plant growth and development and response to stress (Sheen, 1996) .

Calcidium-dependent protein kinases (CDPKs) exist in the grape genome as gene families. However, so far, there are few studies on grape calcium-dependent protein kinases (CDPKs), and no reports and studies on grape calcium-dependent protein kinases with anti-powder mildew function. This may be due to the scarcity of powdery mildew-resistant grape varieties, most of which are susceptible to powdery mildew. For example, the Eurasian grape variety Pinot Noir, from which the grape genome was first sequenced, is known for its delicacy and is used in many of the world's finest wines, such as Romanée-Conti. However, most Eurasian grape varieties, including this grape, are susceptible to diseases such as powdery mildew (Amrine et al., 2015; Gao et al., 2016; Yin et al., 2017).

Wild East China grape (Vitis pseudoreticulata) is a unique wild grape species in my country. The East China grape line Baihe-35-1 preserved in the wild grape germplasm resource garden of Northwest Agriculture and Forestry University showed high resistance to grape powdery mildew, downy mildew and other major grape diseases, but its specific genetic basis (resistance disease genes) is currently unclear (Gao et al., 2016; Yin et al., 2017). In this paper, the calcium-dependent protein kinase gene VpCDPK9 of the susceptible variety Pinot Noir grape (reference genome) was used as a reference to design specific primers, and cloned from the genome cDNA library of East China grape line Baihe-35-1 by gene cloning technology. The isolated powdery mildew-resistant calcium-dependent protein kinase gene provides genetic resources and molecular markers for precise molecular breeding of grape powdery mildew resistance, which has important theoretical and practical significance.

SUMMARY OF THE INVENTION

The patent application of the present invention applies for the calcium-dependent protein kinase (CDPKs) gene VpCDPK9, which was isolated and obtained from the wild Huadong grape Baihe-35-1 in China, and proved that it has the function of resisting powdery mildew. The present application also relates to the application of VpCDPK9 for grape powdery mildew resistance use.

In one embodiment, the isolated powdery mildew-resistant grape calcium-dependent protein kinase (CDPKs) gene VpCDPK9 of the present application has the nucleotide sequence shown in SEQ ID NO: 1:

One aspect of the patent application of the present invention relates to an isolated powdery mildew-resistant grape calcineurin kinase, which is encoded by the isolated powdery mildew-resistant calcineurin kinase gene VpCDPK9 described in the present application, said calcineurin kinase having SEQ ID NO. : the amino acid sequence shown in 13.

One aspect of the patent application of the present invention relates to the use of the isolated powdery mildew-resistant grape calcium-dependent protein kinase gene VpCDPK9 in the creation of powdery mildew-resistant transgenic grape varieties.

One aspect of the patent application of the present invention relates to a method for producing powdery mildew-resistant transgenic grape plants, characterized in that the isolated powdery mildew-resistant grape calcium-dependent protein kinase gene VpCDPK9 described in the present application is introduced into the grape plants and stabilized Express.

Therefore, one aspect of the patent application of the present invention also relates to a method for improving the resistance to powdery mildew of a grape plant, characterized in that the isolated powdery mildew-resistant grape calcium-dependent protein kinase gene VpCDPK9 described in the present application is introduced into the grape plant thing.

The patent application of the present invention provides the real-time fluorescence quantitative PCR detection primers of the Huadong grapebaihe-35-1 calcium-dependent protein kinase gene VpCDPK9 and the primers of the internal reference gene VpActin1, and the primer sequences are as follows:

VpCDPK9-qF: 5'ATGGGGAATAACTGTGTGGGA 3' (SEQ ID NO:2)

VpCDPK9-qR: 5' CCTCTTCGGTGTGGGTGTTGG 3' (SEQ ID NO: 3)

VpActin1-qF: 5' GTGCTGGATTCTGGTGATGGT 3' (SEQ ID NO: 4)

VpActin1-qR: 5'TCCCGTTCAGCAGTAGTGGTG 3' (SEQ ID NO:5)

The VpCDPK9 gene was detected in the leaves of different leaf ages of grape Baihe-35-1 in East China, under biotic stress (infection of powdery mildew), abiotic stress (salt, low temperature, high temperature), exogenous hormone treatment (salicylic acid (SA), Expression after abscisic acid (ABA), methyl jasmonate (MeJA) and ethephon (Eth). The results showed that the expression of VpCDPK9 gene was induced by powdery mildew, high temperature, salt and various hormone treatments.

The patent application of the present invention constructed a 35S::VpCDPK9-GFP plant expression vector for the first time, and introduced it into tobacco mesophyll cell protoplasts by PEG-Ca ²⁺ -mediated transformation to study the subcellular localization of VpCDPK9 protein. The results showed that the GFP-tagged VpCDPK9 full-length protein could co-localize with mCherry-tagged endoplasmic reticulum-resident protein AtCML5, Golgi-resident protein AtEMP12, and oil body proteins AtCOL3 and Atα-DOX1, but not with peroxisomes. Colocalization of the guide peptide PTS1.

The patent application of the present invention has created a transgenic seedless white grape line with overexpression of the Huadong grape Baihe-35-1 calcium-dependent protein kinase gene VpCDPK9. After PCR detection and Western Blot detection, it was confirmed that the exogenous fusion protein could be expressed at a low level in the transgenic grape leaves. After transgenic grape lines and non-transgenic grape lines of the same age were transplanted to an artificial climate incubator for half a year, the leaves were inoculated with the highly pathogenic grape powdery mildew En NAFU1, and trypan blue was used at different time points after inoculation. Staining, diaminobenzidine staining and aniline blue staining were used to detect the growth of powdery mildew mycelium and the defense response of grapes. The average number of conidia produced by a single powdery mildew colony was counted, the content of defense-related plant hormones ethylene and salicylic acid, and the content of the core defense molecule hydrogen peroxide and its scavenging factor anthocyanin were determined. The results showed that VpCDPK9 transgenic grape leaves infected with powdery mildew could produce higher levels of ethylene and salicylic acid than wild-type grape leaves, resulting in massive accumulation of hydrogen peroxide; Accumulation in the surrounding epidermal cells causes hydrogen peroxide to only spread to mesophyll cells, causing extensive necrosis of mesophyll tissue, resulting in leaf yellowing, premature senescence, and even shedding; thus limiting the growth of powdery mildew mycelium and the production of conidia, improving Powdery mildew resistance of grapes.

The beneficial effects of the patent application of the present invention:

(1) The patent application of the present invention obtained the isolated powdery mildew-resistant grape calcium-dependent protein kinase gene VpCDPK9 from Huadong grape Baihe-35-1 by cloning technology. This is the first isolated powdery mildew-resistant grape calcium-dependent protein kinase gene reported so far. By transferring this gene into the infected European grape variety Seedless White, and comparing the response of VpCDPK9 transgenic grape lines and control grape plants (wild-type Seedless white) to powdery mildew infection and the growth and reproduction status of the corresponding powdery mildew It was confirmed that VpCDPK9 transgenic plants showed strong ability to inhibit the growth of powdery mildew mycelium and conidial reproduction, indicating that VpCDPK9 gene is a calcium-dependent protein kinase gene against powdery mildew, which can improve the resistance of grapes to powdery mildew.

(2) The VpCDPK9 gene for powdery mildew resistance in the patent application of the present invention can improve the genetic trait of grape powdery mildew resistance and improve the disease resistance of cultivated grape varieties against powdery mildew.

Description of drawings

Figure 1 is the expression pattern of VpCDPK9 gene in grape leaves of different leaf ages. The left side shows the growth of leaves of different ages on the same branch; the right panel shows the relative expression levels of VpCDPK9 in leaves of different ages. This analysis uses VpActin1 as an internal reference gene. Means and standard deviations are from three biological replicates and three technical replicates.

Figure 2 is the expression pattern of VpCDPK9 gene under different stress and hormone treatment conditions. The type of stress treatment and different sampling time points are marked in the figure. This analysis uses VpActin1 as an internal reference gene. Means and standard deviations are from three biological replicates and three technical replicates.

Figure 3 is the subcellular localization of VpCDPK9 full-length protein in tobacco mesophyll cell protoplasts. mCherry-tagged AtCML5 belongs to endoplasmic reticulum resident protein, AtEMP12 is a Golgi resident protein, AtCOL3 and Atα-DOX1 belong to oil body proteins, and PTS1 is a peroxisome localization guide peptide.

Figure 4 is a Western Blot identification of VpCDPK9 transgenic grape lines overexpressing. Anti-GFP indicates that a mouse-derived GFP monoclonal antibody was used to recognize YFP-tagged protein during western blotting.

Figure 5 is an analysis of powdery mildew resistance of VpCDPK9 transgenic grape lines overexpressing. A. The growth of powdery mildew on leaves of wild-type (WT) and transgenic grape lines after inoculation with powdery mildew EnNAFU1 for 20 days. B. Histochemical staining was used to detect the growth of powdery mildew and the defense reaction of grape cells; trypan blue staining was used to identify the growth of powdery mildew mycelium and conidia production on leaves of wild-type and transgenic grapes after inoculation; Hydrogen peroxide accumulation on leaves of wild-type and transgenic grapes after inoculation; callose accumulation on leaves of wild-type and transgenic grapes after inoculation was identified by aniline blue staining. C. The average number of conidia produced by each single colony on leaves of wild-type and transgenic grapes 20 days after inoculation. D. The amount of ethylene released from leaves of wild-type and transgenic grapes after inoculation for 20 days. E. Contents of free salicylic acid in leaves of wild-type and transgenic grapes 20 days after inoculation. F. The hydrogen peroxide content of wild-type and transgenic grape leaves after inoculation for 20 days. G. The accumulation of anthocyanins in wild-type and transgenic grape leaves after inoculation for 20 days. * indicates significant difference compared with wild type, ** indicates extremely significant difference compared with wild type (Student's t-test, *P<0.05, **P<0.01). Detailed ways

The patent application of the present invention is described in further detail below in conjunction with the embodiments and the accompanying drawings:

Example 1: Cloning and expression analysis of the VpCDPK9 calcium-dependent protein kinase gene VpCDPK9 of Huadong grape Baihe-35-1

The total RNA of Baihe-35-1 grape leaves was extracted by OMEGA Plant RNA Kit. The extraction steps were carried out according to the instructions of the kit. According to the PrimeScript™ RT reagentKit with gDNA Eraser (Perfect Real Time) reverse transcription kit of TaKaRa company, RNA reverse transcription was performed to synthesize the first-strand cDNA, and the method was referred to the kit instructions. According to the VviCDPK9 gene sequence identified in the Pinot Noir grape genome published in NCBI, specific primers were designed using Vector NTI software, upstream primer: VpCDPK9-F: 5'ATGGGGAATAACTGTGTGGGA 3' (SEQ ID NO: 2), downstream primer: VpCDPK9- R: 5'ATAGACTGGTAGCGGCTGCCTA 3'(SEQ ID NO:6), using Baihe-35-1 leaf cDNA as template, PCR amplification was carried out with TAKARA's high-fidelity enzyme PrimeSTAR HS DNA Polymerase, and the specific amplification system was: 0.5 μL HS Taq, 6.0 μL 5X PCR buffer, 3.0 μL dNTP, 1.0 μL cDNA template, 1.0 μL Forward-primer, 1.0 μL Reverse-primer, 17.5 μL ddH ₂ O. The PCR amplification program was as follows: pre-denaturation at 98°C for 10s, cycle parameters were denaturation at 98°C for 10s, annealing at 57°C for 10s, extension at 72°C for 1min 30s, 34 cycles, and full extension at 72°C for 10min. PCR reaction products were detected by electrophoresis in a 1% agarose gel, imaged and photographed under a UV gel imaging system. After photographing, a single target band was cut out and recovered with a gel recovery kit from Genstar, and then ligated into the cloning vector pMD19-T to construct a pMD19T-VpCDPK9 plasmid, which was then transformed into E. coli competent cells. Pick white single clones from the transformed competent cells, and culture them at 37°C in a constant temperature shaker at 180rpm/min for 16-18h. The clones identified as positive by bacterial liquid PCR were sent to Beijing Aoke Yangling Sequencing Department for sequencing verification. The acid sequence and deduced amino acid sequence are shown in the Sequence Listing. The cloned VpCDPK9 gene sequence was analyzed. The full-length coding sequence of the gene was 1743 bp and encoded 580 amino acids. The nucleotide sequence similarity between the VpCDPK9 gene and its homologous gene VviCDPK9 (XM_002264404) in the susceptible grape Pinot Noir reference genome is 99.5%, and there are 9 single nucleotide differences, which lead to non-identical 4 amino acid sites. Synonymous mutation.

The inventors used real-time fluorescence quantitative PCR technology to detect the VpCDPK9 gene in the leaves of different leaf ages of Huadong grape Baihe-35-1, biotic stress (infection of powdery mildew), abiotic stress (salt, low temperature, high temperature), exogenous hormone treatment ( Expression after salicylic acid (SA), abscisic acid (ABA), methyl jasmonate (MeJA) and ethephon (Eth). The sampling of leaves of different leaf ages is shown in Figure 1. The specific methods of stress and hormone treatment are as follows:

Powdery mildew treatment: The grape powdery mildew EnNAFU1 ( E rysiphen ecator NAFU1) (Gao et al. 2016) was inoculated on the healthy leaves of Huadong grape Baihe-35-1 in greenhouse pots, and after treatment 0, 24, 48, 72, 96 , 120, 144 and 168h to collect the inoculated leaves, and then rapidly freeze in liquid nitrogen to extract RNA.

Salt stress treatment: Potted Baihe-35-1 grape plants grown under normal conditions were irrigated with saline (300 mM NaCl solution), and samples were taken at 0, 0.5, 2, 4, 8, 12, 24 and 48 h after watering, respectively. RNA was extracted after nitrogen flash freezing.

Low temperature stress treatment: The potted Baihe-35-1 grape plants grown under normal conditions were placed in an environment of 4°C for low temperature stress treatment. RNA was extracted after nitrogen flash freezing.

High temperature stress treatment: The potted Baihe-35-1 grape plants grown under normal conditions were placed at 42 °C for high temperature stress treatment. RNA was extracted after nitrogen flash freezing.

Phytohormone treatment: The leaves of potted Baihe-35-1 grape plants grown under normal conditions were evenly sprayed with 50 μM abscisic acid (ABA), 50 μM salicylic acid (SA), 50 μM methyl jasmonate (MeJA) and 50 μM ethephon ( Eth), and samples were taken at 0, 0.5, 2, 4, 8, 12, 24 and 48 h after spraying, and RNA was extracted after rapid freezing in liquid nitrogen.

According to the VpCDPK9 gene sequence, the following real-time quantitative PCR detection primers were designed:

VpCDPK9-qF: 5'ATGGGGAATAACTGTGTGGGA 3' (SEQ ID NO:2)

VpCDPK9-qR: 5' CCTCTTCGGTGTGGGTGTTGG 3' (SEQ ID NO: 3)

VpActin1-qF: 5' GTGCTGGATTCTGGTGATGGT 3' (SEQ ID NO: 4)

VpActin1-qR: 5'TCCCGTTCAGCAGTAGTGGTG 3' (SEQ ID NO:5)

RT-qPCR experiments were performed on a Bio-Rad IQ5 real-time quantitative PCR instrument using TaKaRa's real-time quantitative PCR kit. The reaction system was: SYBR Premix Ex Taq II 10.5 μL, cDNA template 1.0 μL, Forward-primer 0.8 μL, Reverse-primer 0.8 μL, ddH ₂ O 7.4 μL. PCR amplification program: pre-denaturation at 95°C for 3 min, 40 cycles (95°C for 30s, 58°C for 30s). After PCR cycling, the temperature was kept at 50°C for 1 min, and then gradually increased by 0.5°C every 10 seconds for melting curve analysis. The relative expression levels of genes were analyzed using the IQ5 software normalized expression method. Three biological replicates and three technical replicates were performed for each treatment.

The results showed that the expression of VpCDPK9 gene was higher in mature leaves and lower in young leaves and yellow leaves (Fig. 1); at the early stage of powdery mildew infection (24-120h), the up-regulated expression was strongly induced; under abiotic stress The VpCDPK9 gene showed obvious response to NaCl stress and high temperature stress, and also responded to low temperature stress, but the up-regulation range was not high; under exogenous hormone treatment, VpCDPK9 gene showed different degrees of up-regulation, among which Eth and MeJA were the most responsive. strong (Figure 2). These results suggest that VpCDPK9 gene may play an important role in participating in pathogenic bacteria, salt and temperature stress.

Example 2: Subcellular localization analysis of VpCDPK9 of East China grape Baihe-35-1 calcium-dependent protein kinase gene

PCR一步定向克隆试剂盒(无缝克隆)通过同源重组反应在合适的摩尔比例下连接到含有GFP标签的植物过表达载体pBI221上，构建融合过表达载体35S::VpCDPK9-GFP。将35S::VpCDPK9-GFP质粒与35S::AtCML5-mCherry、35S::AtEMP12-mCherry、35S::AtCLO3-mCherry、35S::Ata-DOX1-mCherry和35S::PTS1-mCherry等质粒等量混合均匀，通过PEG-Ca²⁺介导的转化法倒入烟草叶肉细胞原生质体中(Zhao等人，2016)，采用OLYMPUS BX63正置荧光显微镜观察GFP标记的VpCPDK9与mCherry标记的细胞器驻留蛋白是否共定位。结果表明，VpCDPK9可同时定位于内质网、高尔基体和油体，但不定位于过氧化物酶体(图3)。Design gene-specific primers with XbaI and KpnI restriction sites, upstream primer: VpCDPK9-GFP-F: 5'TCTGATCAAGAGACA TCTAGA ATGGGGAATAACTGTGTGGGA 3' (SEQ ID NO:7), downstream primer: VpCDPK9-GFP-R:5 'GCCCTTGCTCACCAT GGTACC ATAGACTGGTAGCGGCTGCCT 3' (SEQ ID NO: 8) (the underlined font indicates the restriction site), and the coding sequence of VpCDPK9 was amplified using the pMD19T-VpCDPK9 plasmid as a template, using

PCR one-step directional cloning kit (seamless cloning) was connected to the plant overexpression vector pBI221 containing the GFP tag by homologous recombination reaction at an appropriate molar ratio, and the fusion overexpression vector 35S::VpCDPK9-GFP was constructed. Mix equal amounts of 35S::VpCDPK9-GFP plasmid with 35S::AtCML5-mCherry, 35S::AtEMP12-mCherry, 35S::AtCLO3-mCherry, 35S::Ata-DOX1-mCherry and 35S::PTS1-mCherry plasmids Homogeneous, poured into tobacco mesophyll cell protoplasts by PEG-Ca ²⁺ -mediated transformation (Zhao et al., 2016), and observed whether GFP-tagged VpCPDK9 and mCherry-tagged organelle-resident proteins were observed using an OLYMPUS BX63 upright fluorescence microscope. Colocalization. The results showed that VpCDPK9 could simultaneously localize to the endoplasmic reticulum, Golgi apparatus and oil body, but not to the peroxisome (Figure 3).

Example 3: Obtainment and identification of transgenic seedless white line with overexpression of VpCDPK9 calcium-dependent protein kinase gene VpCDPK9 in East China grape Baihe-35-1

PCR一步定向克隆试剂盒(无缝克隆)通过同源重组反应在合适的摩尔比例下连接到含有YFP标签的植物过表达载体C15上(Wang等人，2007)，构建融合过表达载体35S::VpCDPK9-YFP。将35S::VpCDPK9-YFP质粒通过电转化的方法转入农杆菌感受态细胞GV3101中，以C15空载体作为对照。将转化后的感受态细胞活化后均匀涂在有卡那霉素(50mg/L)、庆大霉素(25mg/L)和利福平(25mg/L)的LBA固体培养基平板上。待平板长出单克隆后进行菌落PCR检测，将检测为阳性的单菌落扩繁后保存菌液。Design gene-specific primers with BamHI restriction site, upstream primer: VpCDPK9-C15-F: 5'TCTGATCAAGAGACA GGATCC ATGGGGAATAACTGTGTGGGA 3' (SEQ ID NO: 9), downstream primer: VpCDPK9-C15-R: 5'GCCCTTGCTCACCAT GGATCC ATAGACTGGTAGCGGCTGCCT 3' (SEQ ID NO: 10) (the underlined font indicates the restriction site), and the coding sequence of VpCDPK9 was amplified using the pMD19T-VpCDPK9 plasmid as a template, using

PCR one-step directional cloning kit (seamless cloning) was ligated to the plant overexpression vector C15 containing YFP tag (Wang et al., 2007) by homologous recombination reaction at an appropriate molar ratio to construct a fusion overexpression vector 35S:: VpCDPK9-YFP. The 35S::VpCDPK9-YFP plasmid was transformed into Agrobacterium competent cell GV3101 by electrotransformation, and the C15 empty vector was used as a control. The transformed competent cells were evenly spread on LBA solid medium plates with kanamycin (50mg/L), gentamicin (25mg/L) and rifampicin (25mg/L) after activation. Colony PCR was carried out after single clones were grown on the plate, and the positive single colonies were propagated and stored.

Take out the 35S::VpCDPK9-YFP/GV3101 bacterial solution stored in the refrigerator at -80 °C, thaw it in a 4 °C refrigerator, and inoculate 200 μL of the bacterial solution in the LBA liquid medium with antibiotics at 180 rpm/min. Incubate at 28°C for 18-20h to activate Agrobacterium. Inoculate 100 μL of the activated bacterial liquid into 20 ml LBA liquid medium containing antibiotics, 180 rpm/min, and 28 °C for expansion culture for 14-16 h. Pour it into a sterile centrifuge tube on an ultra-clean workbench, centrifuge at 5500rpm/min for 10min, leave the precipitate, remove the supernatant, add an equal volume of 1/2 MS solution containing 100μM acetosyringone to resuspend, 180rpm/min, 28°C Incubate at a constant temperature for 1-2 hours, and after measuring the concentration on a UV-Vis spectrophotometer, dilute the concentration to OD 600=0.5-0.8 in a clean bench for later use.

The bacterial liquid was poured into a sterile culture bottle, and the transformed receptor material (nucleus white grape embryogenic callus) was put into the culture bottle containing the infection solution and soaked for 20min, and the culture bottle was gently shaken during the soaking. , in order to make the bacterial liquid fully contact with the callus. The infected embryogenic callus was placed on sterile filter paper to absorb excess Agrobacterium. Then, it was placed on two layers of filter paper in a sterile glass dish (wetted with 1/2 MS liquid medium supplemented with 100 μM AS), and incubated at 26° C. for 48 h in the dark. After co-cultivation, the embryogenic callus was washed three times with sterile water, then sterilized with MS solution supplemented with 200 mg/L cephalosporin (Cef) and carbenicillin (Carb) for 10 min, and finally with sterile water. Rinse with water 3 times. The embryogenic callus after sterilization was placed on sterile filter paper to absorb excess sterile water, and then the problastoid mass was inoculated on KCC medium (KBN+200mg/L Carb+200mg/LCef) for 3 weeks. Delay screening, and then transfer to X3CC medium (X3+200mg/L Carb+200mg/L Cef) to delay screening for one week. After one month of delayed selection, the embryogenic callus was transferred to the resistance selection medium containing different concentrations of Basta, cultivated in a dark environment at 26°C, and subcultured once every 4-6 weeks, the concentration of Basta in the medium required for subculture. Gradually increase from 5mg/L to 20mg/L until resistant somatic embryos germinate. After the germination of the resistant somatic embryos, the cotyledon embryos were inoculated on GM medium (MS+15g/L sucrose+1g/L activated carbon+3g/L phytogel), cultivated in the light until the cotyledons turned green, and then inoculated in the rooting medium (MS+1mg/L IBA+30g/L sucrose+7g/L agar) to grow seedlings. After the roots of the resistant plants have developed and grown to the mouth of the culture bottle, they will be transplanted to the artificial climate room to strengthen the seedlings for 1-2 months, and then transplanted to the greenhouse.

In order to test whether the line that grows into seedlings after resistance screening is a 35S::VpCDPK9-YFP transgenic line, the genomic DNA of the resistant plants is extracted by the traditional CTAB method: about 0.1 g of leaves are taken and ground in a 1.5 mL centrifuge tube with Rod grinding, add 650 μL CTAB buffer, place in a 65°C oven for 30 min, take out and stand at room temperature, add an equal volume of chloroform isoamyl alcohol, mix well, centrifuge at 12,000 rpm/min for 10 min, take about 400 μL of supernatant, add double volume of pre- Cold absolute ethanol, precipitation at -20°C for 30 min, centrifugation at 12000 rpm/min for 10 min, pour off the absolute ethanol, and dry naturally. Add 20 μL of distilled water to dissolve. Universal primers were designed using the nucleotide sequences on the reporter gene YFP and promoter 35S (upstream YFP: 5' CAGGGTCAGCTTGCCGTAG 3' (SEQ ID NO: 11), downstream 35SA: 5' TCCTTCGCAAGACCCTTCCTCTAT 3' (SEQ ID NO: 12)) Resistant plants were tested for PCR levels. Among the 129 Basta-resistant transgenic lines obtained, 109 strains could amplify the target band.

In order to test whether the exogenous fusion gene in PCR-positive strains could be translated into protein normally, Western Blot was performed on the above-mentioned positive strains. Take 100 mg of fresh leaves and put them in liquid nitrogen to grind the samples, and the ground samples are stored in liquid nitrogen. Add protein extract (pH 8.0, 1M Tris-HCl: 10% SDS: 50% glycerol: B-mercaptoethanol = 2:4:4:1) to just cover the sample, mix well, and then bath in boiling water for 5 minutes, until the sample recovers After reaching room temperature, centrifuge at 13,000 rpm/min for 5 min, and take the supernatant as the extract containing protein. Prepare SDS-PAGE separation gel with a concentration of 8%. After the separation gel is solidified, prepare a stacking gel with a concentration of 4%. After the stacking gel is solidified, mix the loading buffer and the protein extraction solution. For gel electrophoresis, the migration process in the stacking gel needs to be electrophoresed at 70V for about 30 minutes, then adjusted to 90V and electrophoresis for 3 hours. When the blue line is still about 0.5cm from the bottom, the electrophoresis is stopped. Add the prepared transfer buffer to the glass bath. Cut a PVDF membrane of suitable size and soak it in a small amount of methanol for half a minute to activate. Cut the excess part of the SDS-PAGE gel. Fix the glue and membrane in the order of blackboard, sponge, filter paper, glue, membrane, filter paper, sponge, and whiteboard. After fixing the splint, place it in the transfer buffer. Transfer the membrane at 100V for 1h, and place the electrophoresis tank in an ice bath. After the membrane transfer is completed, take out the PVDF membrane, turn the side in contact with the gel up, and wash with TBST buffer on a destaining shaker for 10 min. Prepare 15 mL of TBST membrane blocking solution dissolved in 0.5% skimmed milk powder, shake well one hour in advance, and put the PVDF membrane into the blocking solution and incubate for 90 min. Add murine GFP monoclonal antibody diluted 2000 times and incubate overnight at 4°C. Wash 5 times with 20ml TBST, 10min each time. Add 5000-fold diluted HRP-labeled goat anti-mouse polyclonal antibody, incubate for one hour at room temperature, and then wash with TBS for 3 times, 5 min each time. The PVDF membrane was taken out, developed with a developing solution from Millipore, chemiluminescence imaging was performed with a BIO-RAD gel imaging system, and photographed and recorded. Finally, the PVDF membrane was incubated in Ponceau staining solution to stain the total protein, and then photographed and recorded with a Canon SLR camera. The results showed that in most of the transgenic lines, the VpCDPK9-YFP fusion protein could be expressed normally, but the expression level was low (Fig. 4).

Example 4: Identification of powdery mildew resistance of transgenic white seedless white lines overexpressing the VpCDPK9 calcium-dependent protein kinase gene of Huadong grape Baihe-35-1

After 35S::VpCDPK9-YFP transgenic grape lines and non-transgenic grape lines of the same age were transplanted to an artificial climate incubator for half a year, the leaves were inoculated with En NAFU1, a highly pathogenic grape powdery mildew. A small piece of leaf was cut 10 days after inoculation and stained with trypan blue, diaminobenzidine and aniline blue. First, 20 days after inoculation with powdery mildew, on the leaves of the wild-type seedless white strain, the powdery mildew had formed white velvety hyphae and large single colonies, but on the leaves of the VpCDPK9-YFP transgenic grape line, there was no obvious white powder. Colonies were produced, but a large number of necrotic plaques existed and the leaves turned yellow as a whole (Fig. 5A). Using trypan blue staining to observe the growth of mycelium and count the number of conidia, it was found that compared with the powdery mildew on the leaves of wild-type grapes, the growth of mycelium on the transgenic grape strain was inhibited, the density of mycelium was lower, and the number of conidia was lower. Yield was reduced (Figure 5B, 5C). After measuring the free salicylic acid and ethylene in leaf tissue at 20 days after inoculation by ultra-high performance liquid chromatography and gas chromatography, respectively, it was found that there were more ethylene accumulation and higher levels of salicylic acid synthesis in the leaves of the transgenic lines ( 5D, 5E). At the same time, the hydrogen peroxide and anthocyanin contents were determined by spectrophotometric analysis, and it was found that higher levels of hydrogen peroxide and anthocyanins were accumulated in the leaves of the transgenic lines, indicating that the leaves of the transgenic grapes were in a state of high oxidative stress. (Fig. 5F, 5G). Aniline blue staining showed that more calluses were accumulated in the leaves of the transgenic lines, and these calluses were mainly accumulated in the epidermal cells around the pierced epidermal cells (Fig. 5B). Therefore, we speculated that overexpression of VpCDPK9-YFP would promote the synthesis of ethylene and salicylic acid under powdery mildew-induced conditions, and activate downstream signals leading to hydrogen peroxide accumulation and callose accumulation; The corpus callosum prevented the diffusion of hydrogen peroxide from the stinged epidermal cells to the surrounding epidermal cells, causing it to diffuse mainly to the mesophyll cells, causing extensive necrosis of the mesophyll tissue. The necrosis of grape mesophyll restricts the nutrient supply of grape powdery mildew, inhibits its mycelial growth and conidia production, and improves the grape powdery mildew resistance to a certain extent.

references

Amrine KCH, Blanco-Ulate B, Riaz S, Pap D, Jones L, Figueroa-Balderas R, Walker MA, Cantu D (2015) Comparative transcriptomics of Central Asian Vitisvinifera accessions reveals distinct defense strategies againstpowdery.Hortic Res-England 2.doi :10.1038/hortres.2015.37

Gadoury DM, Cadle-Davidson L, Wilcox WF, Dry IB, Seem RC, Milgroom MG (2012) Grapevine powdery mildew (Erysiphe necator): a fascinating system for the study of the biology,ecology and epidemiology of an obligate biotroph.MolPlant Pathol 13 (1):1-16.doi:10.1111/j.1364-3703.2011.00728.x

Gao YR,Han YT,Zhao FL,Li YJ,Cheng Y,Ding Q,Wang YJ,Wen YQ(2016)Identification and utilization of a new Erysiphe necator isolate NAFU1 to quickly evaluate powdery mildew resistance in wild Chinese grapevine speciesusing detached leaves.Plant Physiol Biochem 98:12-24.doi:10.1016/j.plaphy.2015.11.003

Sheen J(1996) Ca2+-dependent protein kinases and stress signal transduction in plants. Science 274(5294):1900-1902.doi:10.1126/science.274.5294.1900

Wang W, Devoto A, Turner JG, Xiao S(2007) Expression of the membrane-associated resistance protein RPW8 enhances basal defense against biotrophicpathogens. Mol Plant Microbe Interact 20(8):966-976.doi:10.1094/MPMI-20- 8-0966

Yin X, Liu RQ, Su H, Su L, Guo YR, Wang ZJ, Du W, Li MJ, Zhang X, Wang YJ, LiuGT, Xu Y (2017) Pathogen development and host responses to Plasmopara viticolain resistant and susceptible grapevines: an ultrastructural study. Hortic Res-England 4. doi: 10.1038/hortres.2017.33

Zhao FL, Li YJ, Hu Y, Gao YR, Zang XW, Ding Q, Wang YJ, Wen YQ (2016) A highly efficient grapevine mesophyll protoplast system for transient gene expression and the study of disease resistance proteins. Plant Cell Tiss Org 125(1 ):43-57.doi:10.1007/s11240-015-0928-7

sequence listing

<110> Northwest A&F University

<120> Grape calcium-dependent protein kinase gene VpCDPK9 against powdery mildew and its application

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1743

<212> DNA

<213> East China Grape (Vitis pseudoreticulata)

<400> 1

atggggaata actgtgtggg atccatggtt cccgagcatg gattgtttga atcaatttct 60

aattcaattt ggtggacgag agcctcggag tgtatgactt ctcattctac cggagaaggt 120

gtcagcgaaa cgcaaagtaa agagcagaaa tctcctcctc ctgttcaaaa caagcctcca 180

gaagaggtgg agctaataaa tccgccagag gtaatgaaga taacaaagga agagacgaaa 240

ccaacaccca caccgaagag gccactcctt atgaagaggt tgccaagtgc tggacttgag 300

gtggacttgg ttttgaaaaa caaaactgat catttgaagg aacactataa tttggggcgg 360

aagcttgggc acggccagtt tgggacaact tttctatgtg tagagaaaga aacaggcaaa 420

gagtacgctt gcaaatccat tgcaaaaagg aagctgctga caagagatga cattgaagac 480

gtgaggaggg aaatccagat aatgcatcac ttggcgggcc actccaatat catctccatc 540

aagggagctt atgaggatgc ggtggcagtt catcttgtca tggaattgtg tacggggggt 600

gagctttttg ataggattgc caagcgaggc cattatacag aaagaaaggc agctcagctt 660

gcaaggacta taattggtgt tgtagaagcc tgccactctt taggggtcat gcaccgggac 720

cttaagcctg agaattttct ttttgtcaat gagcaagagg aatcacttct taagacaata 780

gactttgggt tgtccgtatt cttcaagcca ggggagattt tcactgatgt ggttggaagc 840

ccatattatg tggcacctga agttctgaga aagtgttatg gtccagaagc agatgtttgg 900

agtgttgggg tgatcattta tattctctta agtggggtgc ctccattttg ggcagaaagt 960

gagcaagaga tatttcaaga agtactgcat ggtgatctta acttctcatc agatccatgg 1020

cctcatatct ctgaaagtgc taaggacttg attaggagaa tacttgtcag agatcccaag 1080

aaacgcctaa ctgcccatga agtcctgtgt cacccttgga ttcaggttga tggagtagct 1140

cctgacaaaa cccttgattc tgcagtcata agtcgcttga agcagttttc agccatgaac 1200

aagcttaaga aaatggctct tagagtgatt gctgagaatc tctccgaaga agaaattgct 1260

ggattgaaag aaatgttcaa gattattgat acagacaata gtggtcagat tacttttgaa 1320

gaactcaagg ctggactaaa aagatttggc gctaatctta atgaagctga aatttatgat 1380

ctaatgcagg ctgcagatgt tgataatagt ggaacaatcg attatgggga gttcatagct 1440

gcaacattcc atctaaacaa aattgagaga gaagatcacc tatttgctgc tttttcctac 1500

tttgacaaag atggaagtgg ctatatcact ccagatgagc tccaaaaagc ctgtgaggag 1560

tttgggatgg aggatgtcca tttggaagaa atgatccaag aagttgacca ggacaatgat 1620

ggcctcatag attataatga gtttgtggca atgatgcaga aaggaaacaa taatgatttt 1680

ggtaagaagg gtctggagaa tggtattagc tttgggttta ggcagccgct accagtctat 1740

tga 1743

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 2

atggggaata actgtgtggg a 21

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 3

cctcttcggt gtgggtgttg g 21

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence

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gtgctggatt ctggtgatgg t 21

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence

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tcccgttcag cagtagtggt g 21

<210> 6

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 6

atagactggt agcggctgcc ta 22

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<211> 42

<212> DNA

<213> Artificial Sequence

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tctgatcaag agacatctag aatggggaat aactgtgtgg ga 42

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<212> DNA

<213> Artificial Sequence

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gcccttgctc accatggtac catagactgg tagcggctgc ct 42

<210> 9

<211> 42

<212> DNA

<213> Artificial Sequence

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tctgatcaag agacaggatc catggggaat aactgtgtgg ga 42

<210> 10

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<212> DNA

<213> Artificial Sequence

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gcccttgctc accatggatc catagactgg tagcggctgc ct 42

<210> 11

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 11

cagggtcagc ttgccgtag 19

<210> 12

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 12

tccttcgcaa gacccttcct ctat 24

<210> 13

<211> 580

<212> PRT

<213> East China Grape (Vitis pseudoreticulata)

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Met Gly Asn Asn Cys Val Gly Ser Met Val Pro Glu His Gly Leu Phe

1 5 10 15

Glu Ser Ile Ser Asn Ser Ile Trp Trp Thr Arg Ala Ser Glu Cys Met

20 25 30

Thr Ser His Ser Thr Gly Glu Gly Val Ser Glu Thr Gln Ser Lys Glu

35 40 45

Gln Lys Ser Pro Pro Pro Val Gln Asn Lys Pro Pro Glu Glu Val Glu

50 55 60

Leu Ile Asn Pro Pro Glu Val Met Lys Ile Thr Lys Glu Glu Thr Lys

65 70 75 80

Pro Thr Pro Thr Pro Lys Arg Pro Leu Leu Met Lys Arg Leu Pro Ser

85 90 95

Ala Gly Leu Glu Val Asp Leu Val Leu Lys Asn Lys Thr Asp His Leu

100 105 110

Lys Glu His Tyr Asn Leu Gly Arg Lys Leu Gly His Gly Gln Phe Gly

115 120 125

Thr Thr Phe Leu Cys Val Glu Lys Glu Thr Gly Lys Glu Tyr Ala Cys

130 135 140

Lys Ser Ile Ala Lys Arg Lys Leu Leu Thr Arg Asp Asp Ile Glu Asp

145 150 155 160

Val Arg Arg Glu Ile Gln Ile Met His His Leu Ala Gly His Ser Asn

165 170 175

Ile Ile Ser Ile Lys Gly Ala Tyr Glu Asp Ala Val Ala Val His Leu

180 185 190

Val Met Glu Leu Cys Thr Gly Gly Glu Leu Phe Asp Arg Ile Ala Lys

195 200 205

Arg Gly His Tyr Thr Glu Arg Lys Ala Ala Gln Leu Ala Arg Thr Ile

210 215 220

Ile Gly Val Val Glu Ala Cys His Ser Leu Gly Val Met His Arg Asp

225 230 235 240

Leu Lys Pro Glu Asn Phe Leu Phe Val Asn Glu Gln Glu Glu Ser Leu

245 250 255

Leu Lys Thr Ile Asp Phe Gly Leu Ser Val Phe Phe Lys Pro Gly Glu

260 265 270

Ile Phe Thr Asp Val Val Gly Ser Pro Tyr Tyr Val Ala Pro Glu Val

275 280 285

Leu Arg Lys Cys Tyr Gly Pro Glu Ala Asp Val Trp Ser Val Gly Val

290 295 300

Ile Ile Tyr Ile Leu Leu Ser Gly Val Pro Pro Phe Trp Ala Glu Ser

305 310 315 320

Glu Gln Glu Ile Phe Gln Glu Val Leu His Gly Asp Leu Asn Phe Ser

325 330 335

Ser Asp Pro Trp Pro His Ile Ser Glu Ser Ala Lys Asp Leu Ile Arg

340 345 350

Arg Ile Leu Val Arg Asp Pro Lys Lys Arg Leu Thr Ala His Glu Val

355 360 365

Leu Cys His Pro Trp Ile Gln Val Asp Gly Val Ala Pro Asp Lys Thr

370 375 380

Leu Asp Ser Ala Val Ile Ser Arg Leu Lys Gln Phe Ser Ala Met Asn

385 390 395 400

Lys Leu Lys Lys Met Ala Leu Arg Val Ile Ala Glu Asn Leu Ser Glu

405 410 415

Glu Glu Ile Ala Gly Leu Lys Glu Met Phe Lys Ile Ile Asp Thr Asp

420 425 430

Asn Ser Gly Gln Ile Thr Phe Glu Glu Leu Lys Ala Gly Leu Lys Arg

435 440 445

Phe Gly Ala Asn Leu Asn Glu Ala Glu Ile Tyr Asp Leu Met Gln Ala

450 455 460

Ala Asp Val Asp Asn Ser Gly Thr Ile Asp Tyr Gly Glu Phe Ile Ala

465 470 475 480

Ala Thr Phe His Leu Asn Lys Ile Glu Arg Glu Asp His Leu Phe Ala

485 490 495

Ala Phe Ser Tyr Phe Asp Lys Asp Gly Ser Gly Tyr Ile Thr Pro Asp

500 505 510

Glu Leu Gln Lys Ala Cys Glu Glu Phe Gly Met Glu Asp Val His Leu

515 520 525

Glu Glu Met Ile Gln Glu Val Asp Gln Asp Asn Asp Gly Leu Ile Asp

530 535 540

Tyr Asn Glu Phe Val Ala Met Met Gln Lys Gly Asn Asn Asn Asp Phe

545 550 555 560

Gly Lys Lys Gly Leu Glu Asn Gly Ile Ser Phe Gly Phe Arg Gln Pro

565 570 575

Leu Pro Val Tyr

580

Claims (3)

1. Use of the isolated powdery mildew-resistant grape calcium-dependent protein kinase gene VpCDPK9 shown in SEQ ID NO: 1 in the production of powdery mildew-resistant grape varieties. 2. A method for producing a powdery mildew-resistant grape plant, characterized in that the isolated powdery mildew-resistant grape calcium-dependent protein kinase gene VpCDPK9 shown in SEQ ID NO: 1 is introduced into the grape plant and stably expressed. 3. A method for improving the powdery mildew resistance of grape plants, characterized in that the isolated powdery mildew-resistant grape calcium-dependent protein kinase gene VpCDPK9 shown in SEQ ID NO: 1 is introduced into the grape plants.

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