CN111197035B - Powdery mildew-resistant grape calcium-dependent protein kinase gene VpCDPK13 and application thereof - Google Patents

Abstract

The patent application of the invention discloses a Chinese wild east China grape Baihe-35-1 calcium dependent protein kinase gene VpCDPK13 and application thereof. The application relates to the field of genetic engineering, in particular to the field of grape disease-resistant breeding. The calcium-dependent protein kinase gene VpCDPK13 is obtained by separating the disease-resistant Chinese wild east China grape Baihe-35-1 plant genome through a gene cloning technology, and the gene is transferred into a powdery mildew susceptible grape plant to obtain a powdery mildew resistant transgenic grape plant, so that the powdery mildew resistance of the grape is improved.

Description

Powdery mildew-resistant grape calcium-dependent protein kinase gene VpCDPK13 and application thereof

Technical Field

The invention belongs to the technical field of plant stress resistance gene identification and genetic engineering, in particular relates to separation and identification of a powdery mildew resistant grape calcium dependent protein kinase gene, and more particularly relates to a powdery mildew resistant grape calcium dependent protein kinase gene VpCDPK13 separated from wild east China grape Baihe-35-1 and application thereof in resisting powdery mildew.

Background

Powdery mildew is an important fungal disease seriously damaging grape production, often causing serious yield reduction and even top harvest, and causing huge economic loss. In order to prevent and control the disease, pesticides are required to be sprayed for 20-30 times every year in production, so that the edible safety of grape products is seriously influenced, the human health is threatened, and the ecological environment is polluted (Gadoury et al, 2012). At present, grape breeders more and more strongly recognize that the disease resistance genes of wild grapes are exploited and utilized as soon as possible, and the accurate disease resistance molecular breeding of grapes is actively developed, so that the method is an important way for fundamentally solving the problem.

Calcium ions are an important second messenger in eukaryotic cells. Calcium Dependent Protein Kinases (CDPKs) are a specific class of calcium signaling decoding proteins in plants that independently accomplish decoding and transmission of calcium signaling. When plant cells are stressed, receptor proteins on cell membranes can quickly activate calcium ion channels after recognizing stress signals, and calcium ions in apoplast spaces flow in a large quantity to increase the concentration of calcium ions in cytoplasm. The EF-hand structural domain at the carboxyl terminal of the CDPKs is combined with calcium ions to promote the CDPKs to generate conformational change and expose the kinase active center, and at the moment, the CDPKs can interact with substrate proteins and phosphorylate the substrate proteins to regulate and control the activity or stability of downstream substrate proteins. The above process converts calcium signals (oscillation of cytoplasmic calcium ion concentration) into phosphorylation events, which enables transmission and processing of stress signals, and thus, CDPKs proteins play an important role in plant growth and development and stress response (Sheen, 1996).

Calcium Dependent Protein Kinases (CDPKs) exist as a family of genes in the grape genome. However, to date, there have been few studies on the calcium-dependent protein kinases of Vitis vinifera (CDPKs), and there have been no reports or studies on the calcium-dependent protein kinases of Vitis vinifera having anti-powdery mildew activity. This is probably due to the scarcity of powdery mildew resistant grape varieties, most of which are susceptible to powdery mildew. For example, the eurasian grape variety, black bino grape (PinotNoir), used for the first sequencing of grape genome, is well known for its great proficiency and is used in many vintage wines around the world (such as roman god). However, most European and Asian grape varieties, including the grape, are susceptible to diseases such as powdery mildew (Amrine et al, 2015; Gao et al, 2016; Yin et al, 2017).

Wild Vitis vinifera (Vitis pseudoreticulata) is a wild grape species in China. The Baihe-35-1 of the east China grape strain stored in the wild grape germplasm resource garden of northwest agriculture and forestry science and technology university has higher resistance to main grape diseases such as grape powdery mildew, downy mildew and the like, but the specific genetic basis (disease resistance gene) is not clear at present (Gao et al, 2016; Yin et al, 2017). The method takes a calcium-dependent protein kinase gene VpCDPK13 of a powdery mildew susceptible variety, namely Hebino grape (reference genome) as reference, designs a specific primer, and adopts a gene cloning technology to clone from a Huadong grape strain Baihe-35-1 genome cDNA library to obtain a separated powdery mildew-resistant calcium-dependent protein kinase gene, so that the method has important theoretical value and practical significance for providing gene resources and molecular markers for accurate molecular breeding of powdery mildew resistance of grapes.

Disclosure of Invention

The invention discloses a gene VpCDPK13 of Calcium Dependent Protein Kinase (CDPKs) separated from China wild Vitis vinifera Baihe-35-1, and proves that the gene has the function of powdery mildew resistance. The application also relates to the use of VpCDPK13 for the treatment of powdery mildew in grapes.

In one embodiment, the isolated powdery mildew resistant Portland calcium dependent protein kinase gene VpCDPK13 of the present application has the nucleotide sequence shown as SEQ ID NO. 1.

One aspect of the present patent application relates to an isolated powdery mildew resistant grape calcium dependent protein kinase encoded by the isolated powdery mildew resistant calcium dependent protein kinase gene VpCDPK13 described herein having the amino acid sequence shown in SEQ ID NO. 13.

One aspect of the present patent application relates to the use of an isolated powdery mildew resistant calcium dependent protein kinase gene VpCDPK13 in the creation of powdery mildew resistant transgenic grape varieties.

One aspect of the present patent application relates to a method for producing transgenic grape plants resistant to powdery mildew, characterized in that the isolated grape powdery mildew-resistant grape calcium-dependent protein kinase gene VpCDPK13 described herein is introduced into a grape plant and stably expressed.

Therefore, one aspect of the present patent application also relates to a method for improving the powdery mildew resistance of grape plants, which is characterized in that the isolated powdery mildew resistance grape calcium dependent protein kinase gene VpCDPK13 described in the present application is introduced into the grape plants.

In one embodiment of the present application, the present patent application provides real-time fluorescent quantitative PCR detection primers for vitis vinifera white river-35-1 calcium dependent protein kinase VpCDPK13 in east china and primers for reference gene VpActin1, the primer sequences are as follows:

the expression conditions of the VpCDPK13 gene after biotic stress (powdery mildew infection), abiotic stress (salt, low temperature and high temperature), exogenous hormone treatment (salicylic acid (SA), abscisic acid (ABA), methyl jasmonate (MeJA) and ethephon (Eth)) on different leaf age leaves of the Vitis vinifera Baihe-35-1 are respectively detected. The results show that the VpCDPK13 gene is not significantly induced to express at the transcriptional level by powdery mildew, high temperature, salt and various hormones.

The invention firstly constructs a 35S:: VpCDPK13-GFP plant expression vector, and PEG-Ca is used for the expression vector ²⁺ Mediated transformation method it was introduced into tobacco mesophyll cell protoplasts to study the subcellular localization of VpCDPK13 protein. The results show that the GFP-labeled VpCDPK13 full-length protein can be co-localized with mCherry-labeled endoplasmic reticulum retention protein AtCML5, Golgi retention protein AtEMP12 and oil body proteins AtCOL3 and At alpha-DOX 1 At the same time, and cannot be co-localized with peroxisome localization leader peptide PTS 1.

The invention discloses an overexpression transgenic seedless white grape strain of a Vitis vinifera Baihe-35-1 calcium dependent protein kinase gene VpCDPK13 in east China. Through PCR detection and Western Blot detection, the exogenous fusion protein can be confirmed to be expressed at a low level in transgenic grape leaves. After the transgenic grape strains and the non-transgenic grape strains of the same age are transplanted to an artificial climate box incubator to grow for half a year, strong pathogenic grape powdery mildew En NAFU1 is inoculated to leaves of the grape strains, and the growth condition of powdery mildew hyphae and the defense response of grapes are detected by adopting methods such as trypan blue staining, diaminobenzidine staining, aniline blue staining and the like at different time points after inoculation. Counting the number of conidia averagely generated by a single colony of the powdery mildew, measuring the contents of ethylene and salicylic acid which are plant hormones related to defense, and measuring the contents of hydrogen peroxide which is a core defense molecule and a clearing factor anthocyanin thereof. The results show that the leaves of the VpCDPK13 transgenic grape line are infected by powdery mildew, can generate higher level of ethylene and salicylic acid compared with the leaves of wild grape, and lead to the accumulation of hydrogen peroxide in large quantity; meanwhile, the accumulation of callose in mesophyll cells below the invaded epidermal cells is promoted, so that hydrogen peroxide is diffused to surrounding epidermal cells and anthocyanin is caused to be accumulated in the epidermal cells in a large amount, and the allergic death of the epidermal cells is promoted; thereby limiting the growth of powdery mildew hypha and the generation of conidium and improving the powdery mildew resistance of grapes.

The beneficial effects of the patent application of the invention are as follows:

(1) the invention discloses a method for obtaining an isolated powdery mildew resistant grape calcium dependent protein kinase gene VpCDPK13 from Vitis vinifera Baihe-35-1 in east China by a cloning technology. This is the isolated anti-powdery mildew calcineurin kinase gene reported for the first time to date. The gene is transferred into the sclerotinia-free white of a susceptible European grape variety, and the response of a VpCDPK13 transgenic grape strain and a control grape strain (wild type sclerotinia-free white) to powdery mildew infection and the growth and reproduction conditions of corresponding powdery mildew are compared, so that the VpCDPK13 transgenic plant shows stronger inhibition capacity on the growth of powdery mildew hypha and the propagation of conidiospores, and the VpCDPK13 gene is a calcium-dependent protein kinase gene for resisting the powdery mildew, and can improve the powdery mildew resistance of grapes.

(2) The powdery mildew resistant VpCDPK13 gene applied by the invention can improve the powdery mildew resistant hereditary character of grapes and improve the powdery mildew resistant disease resistance of grape cultivars.

Drawings

FIG. 1 is a graph of the expression pattern of VpCDPK13 gene in grape leaves of different leaf ages. The growth conditions of leaves with different leaf ages on the same branch are marked on the left side; the right panel is the relative expression level of VpCDPK13 in different leaf age leaves. The analysis used VpActin1 as an internal reference gene. The mean and standard deviation were from three biological replicates and three technical replicates.

Figure 2 is the expression pattern of VpCDPK13 gene under different stress and hormone treatment conditions. The figure indicates the type of duress treatment and the different sampling time points. The analysis used VpActin1 as an internal reference gene. The mean and standard deviation were from three biological replicates and three technical replicates.

Figure 3 is the subcellular localization of VpCDPK13 full-length protein in tobacco mesophyll cell protoplasts. mCherry-labeled AtCML5 belongs to the endoplasmic reticulum resident protein, AtEMP12 is Golgi resident protein, AtCOL3 and At α -DOX1 belong to the oil body proteins, and PTS1 is the peroxisome localization leader peptide.

FIG. 4 is a Western Blot identification of transgenic grape lines overexpressing VpCDPK 13. Anti-GFP shows that murine GFP monoclonal antibody was used to recognize the YFP tag protein during immunoblotting.

FIG. 5 is a powdery mildew resistance assay of transgenic grape lines overexpressing VpCDPK 13. A. Growth of powdery mildew on leaves of Wild Type (WT) and transgenic grape lines after inoculation with powdery mildew EnNAFIU 120 d. B. Histochemical staining to detect the growth of powdery mildew and the defense reaction of grape cells; trypan blue staining identifies the growth of powdery mildew hyphae and conidiospore production on wild type and transgenic grape leaves after inoculation; diaminobenzidine staining is used for identifying the accumulation condition of hydrogen peroxide on wild grape leaves and transgenic grape leaves after inoculation; and (3) identifying the accumulation condition of callose on wild type and transgenic grape leaves after inoculation by aniline blue staining. C. The number of conidia generated on each single colony on the leaf of the wild type grape and the transgenic grape after 20d inoculation is average. D. And (4) after 20d inoculation, ethylene release amount of wild type and transgenic grape leaves is increased. E. And after 20d of inoculation, the content of free salicylic acid in wild type and transgenic grape leaves is reduced. F. And (5) inoculating the wild grape leaves and the transgenic grape leaves after 20d of inoculation with hydrogen peroxide. G. And (5) inoculating the wild grape leaves and the transgenic grape leaves after 20d of inoculation to obtain anthocyanin accumulation. Indicates a significant difference compared to wild type,. indicates a very significant difference compared to wild type (Student's t-test,. P <0.05,. P < 0.01).

Detailed Description

The following patent applications are described in further detail with reference to the following examples and drawings:

example 1: cloning and expression analysis of Vitis vinifera Baihe-35-1 calcium dependent protein kinase gene VpCDPK13

The extraction step is carried out according to the Kit description method by adopting an OMEGA Plant RNA Kit Plant RNA miniprep Kit to extract the total RNA of the leaf of the white river-35-1 grape. First strand cDNA was synthesized by reverse transcription of RNA using the PrimeScript RTreagent Kit with gDNA Eraser (Perfect Real Time) reverse transcription Kit from TaKaRa, according to the Kit instructions. According to the VviCDPK13 gene sequence identified in the black bino grape genome published in NCBI, Vector NTI software is utilized to design a specific primer, an upstream primer: VpCDPK 13-F: 5 'ATGGGTAATACTTGTGTTGGT 3' (SEQ ID NO:2), downstream primer: VpCDPK 13-R: 5 'ATGTTTTAACGCCTCTCTAAACCC 3' (SEQ ID NO:6), using 'Baihe-35-1' leaf cDNA as a template, and using high fidelity enzyme PrimeSTAR HS DNApolymerase of TAKARA company for PCR amplification, wherein the specific amplification system is as follows: 0.5. mu.L HS Taq, 6.0. mu.L 5 XPCR buffer, 3.0. mu.L dNTP, 1.0. mu.L cDNA template, 1.0. mu.L Forward-primer, 1.0. mu.L Reverse-primer, 17.5. mu.L ddH ₂ And (O). The PCR amplification procedure was: pre-denaturation at 98 deg.C for 10s, annealing at 57 deg.C for 10s, and extension at 72 deg.C for 1min and 30s, and performing 34 cycles, and fully extending at 72 deg.C for 10 min. PCR products were detected by electrophoresis in a 1% agarose gel, imaged on an ultraviolet gel imaging system and photographed. After photographing, a single band of interest was cut and recovered with a gel recovery kit of Genstar corporation, and then ligated to the cloning vector pMD19-T to construct pMD19T-VpCDPK13 plasmid, followed by transformation of E.coli competent cells. Selecting white monoclonal cell from transformed competent cell, culturing at 37 deg.C with shaking table at 180rpm/min for 16-18h, and culturing with bacterial solution PThe clone with CR identified as positive is sent to sequencing verification of the department of sequencing of Poplar in Olympic of Beijing, and the nucleotide sequence and the deduced amino acid sequence of the clone are shown in a sequence table. The cloned VpCDPK13 gene sequence is analyzed, the total length of the gene coding sequence is 1710bp, and 569 amino acids are coded. The nucleotide sequence similarity of the VpCDPK13 gene and its homologous gene vvidpk 13(XM _010660615) in the reference genome of the susceptible grape melanopino is 99.5%, there are 9 single nucleotide differences, and it results in non-synonymous mutations at 3 amino acid sites.

The inventor adopts a real-time fluorescent quantitative PCR technology to detect the expression conditions of the VpCDPK13 gene after biotic stress (powdery mildew infection), abiotic stress (salt, low temperature and high temperature), exogenous hormone treatment (salicylic acid (SA), abscisic acid (ABA), methyl jasmonate (MeJA) and ethephon (Eth)) on different leaf age leaves of the east China grape Baihe-35-1. Leaf samples from different leaf ages are shown in FIG. 1, and the specific stress and hormone treatments were as follows:

treating powdery mildew: grape powdery mildew EnNAFI 1 (A) is inoculated on the white river-35-1 healthy leaves of east China grape potted in the greenhouseErysiphe necator NAFIU 1) (Gao et al, 2016) and inoculated leaves were collected at 0, 24, 48, 72, 96, 120, 144 and 168h after treatment, and RNA was extracted after rapid freezing with liquid nitrogen.

Salt stress treatment: the potted Baihe-35-1 grape plant growing in normal environment is irrigated with saline (300mM NaCl solution), and samples are taken 0, 0.5, 2, 4, 8, 12, 24 and 48 hours after irrigation, and RNA is extracted after liquid nitrogen is rapidly frozen.

Low-temperature stress treatment: the potted Baihe-35-1 grape plant growing in the normal environment is put in the environment of 4 ℃ for low-temperature stress treatment, samples are respectively taken for 0 hour, 0.5 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours and 48 hours after the treatment, and RNA is extracted after the liquid nitrogen is rapidly frozen.

High-temperature stress treatment: placing potted Baihe-35-1 grape plants growing in a normal environment in an environment of 42 ℃ for high-temperature stress treatment, respectively sampling 0, 0.5, 2, 4, 8, 12, 24 and 48 hours after the treatment, and quickly freezing by liquid nitrogen to extract RNA.

And (3) treating the phytohormone: leaves of potted Baihe-35-1 grape plants grown in normal environment are respectively and uniformly sprayed with 50 mu M abscisic acid (ABA), 50 mu M Salicylic Acid (SA), 50 mu M methyl jasmonate (MeJA) and 50 mu M ethephon (Eth), and samples are respectively taken at 0, 0.5, 2, 4, 8, 12, 24 and 48h after spraying, and RNA is extracted after liquid nitrogen is rapidly frozen.

The following real-time fluorescent quantitative PCR detection primers are designed according to the VpCDPK13 gene sequence:

RT-qPCR assay was performed on a Bio-Rad IQ5 real-time fluorescent quantitative PCR instrument using TaKaRa real-time fluorescent quantitative PCR kit. The reaction system is as follows: SYBR Premix Ex Taq II 10.5. mu.L, cDNA template 1.0. mu.L, Forward-primer 0.8. mu.L, Reverse-primer 0.8. mu.L, ddH ₂ O7.4. mu.L. PCR amplification procedure: pre-denaturation at 95 ℃ for 3min, 40 cycles (95 ℃ for 30s, 58 ℃ for 30 s). After PCR cycling, the temperature was maintained at 50 ℃ for 1min, and then melting curve analysis was performed at 0.5 ℃ increments every 10 seconds. The relative expression levels of the genes were analyzed using IQ5 software normalized expression method. Each treatment was performed 3 biological replicates and 3 technical replicates, respectively.

The results showed that VpCDPK13 gene was highly expressed in mature leaf and less expressed in young and etiolated leaf (figure 1); powdery mildew infestation did not induce significant up-regulation of its expression (fig. 2); under abiotic stress, the VpCDPK13 gene did not respond significantly at the transcriptional level to NaCl stress treatment, low temperature stress treatment, and high temperature stress treatment; VpCDPK13 responded relatively strongly to Eth and SA treatment upon exogenous hormone treatment, but the upregulated expression was also not high in magnitude, and was primarily post-treatment responses (fig. 2). These results indicate that the VpCDPK13 gene may function specifically in the ethylene or salicylic acid signaling pathway.

Example 2: subcellular localization analysis of Vitis vinifera Baihe-35-1 calcium dependent protein kinase Gene VpCDPK13

Designing gene specific primers with XbaI and KpnI enzyme cutting sites, wherein an upstream primer: VpCDPK 13-GFP-F: 5' TCTGATCAAGAGACATCTAGAATGGGTAATACTTGTGTTGG 3' (SEQ ID NO:7), downstream primer: VpCDPK 13-GFP-R: 5' GCCCTTGCTCACCATGGTACCATGTTTTAACGCCTCTCTAAACCC 3' (SEQ ID NO:8) (restriction sites in underlined font), and the coding sequence of VpCDPK13 was amplified using pMD19T-VpCDPK13 plasmid as template, using

The PCR one-step directional cloning kit (seamless cloning) is connected to the plant overexpression vector pBI221 containing the GFP label through homologous recombination reaction under a proper molar ratio to construct a fusion overexpression vector 35S:: VpCDPK 13-GFP. The 35S plasmid VpCDPK13-GFP and 35S plasmid AtCML5-mCherry, 35S plasmid AtEMP12-mCherry, 35S plasmid AtCLO3-mCherry, 35S plasmid Ata-DOX1-mCherry and 35S plasmid PTS 1-mChery are mixed uniformly in equal amount, and PEG-Ca is used for mixing uniformly ²⁺ The mediated transformation method was poured into tobacco mesophyll cell protoplasts (Zhao et al, 2016), and observed whether GFP-labeled VpCPDK13 co-localized with mCherry-labeled organelle resident protein using OLYMPUS BX63 orthofluorescent microscope. The results indicate that VpCDPK13 can localize to endoplasmic reticulum, golgi and oil bodies simultaneously, but not to peroxisomes (figure 3).

Example 3: obtaining and identifying transgenic anucleate white strain over-expressed by Vitis vinifera Baihe-35-1 calcium dependent protein kinase VpCDPK13

Designing a gene specific primer with a BamHI enzyme cutting site, wherein an upstream primer: VpCDPK 13-C15-F: 5' TCTGATCAAGAGACAGGATCCATGGGTAATACTTGTGTTGG 3' (SEQ ID NO:9), reverse primer: VpCDPK 13-C15-R: 5' GCCCTTGCTCACCATGGATCCATGTTTTAACGCCTCTCTAAACCC 3' (SEQ ID NO:10) (enzyme cutting sites are underlined), and the coding sequence of VpCDPK13 was amplified using pMD19T-VpCDPK13 plasmid as template, using

The PCR one-step directional cloning kit (seamless cloning) is connected by homologous recombination reaction under the proper molar ratioTo YFP-tag-containing plant overexpression vector C15 (Wang et al, 2007), a fusion overexpression vector 35S:: VpCDPK13-YFP was constructed. The 35S-VpCDPK 13-YFP plasmid is transferred into agrobacterium-infected cells GV3101 by an electrotransformation method, and a C15 empty vector is used as a control. After activation, the transformed competent cells were spread evenly on LBA solid medium plates with kanamycin (50mg/L), gentamicin (25mg/L) and rifampicin (25 mg/L). And (4) carrying out colony PCR detection after the plate grows out the monoclonals, and preserving the bacterial liquid after the monoclonals detected as positive are propagated.

Taking out the bacterial liquid of VpCDPK13-YFP/GV3101 which is preserved in a refrigerator of-80' C and is 35S:, unfreezing the bacterial liquid in a refrigerator of 4 ℃, sucking 200 mu L of the bacterial liquid, inoculating the bacterial liquid in an LBA liquid culture medium added with antibiotics, culturing at the constant temperature of 28 ℃ for 18-20h at 180rpm/min, and activating the agrobacterium. And sucking 100 mu L of the activated bacterial liquid, inoculating the bacterial liquid into 20ml of LBA liquid culture medium containing antibiotics, and carrying out amplification culture at 180rpm/min and 28 ℃ for 14-16 h. Pouring into a sterile centrifuge tube in a clean bench, centrifuging at 5500rpm/min for 10min, removing precipitate, removing supernatant, adding equal volume of 1/2MS solution containing 100 μ M acetosyringone, resuspending, culturing at 180rpm/min at 28 deg.C for 1-2h, measuring concentration with an ultraviolet-visible spectrophotometer, and diluting to OD 600 of 0.5-0.8.

Pouring the bacterial liquid into a sterile culture bottle, putting the transformation acceptor material (the seedless white grape embryonic callus) into the culture bottle filled with the infection liquid, and soaking for 20min, and slightly shaking the culture bottle during the soaking process to ensure that the bacterial liquid is fully contacted with the callus. Placing the infected embryonic callus on sterile filter paper to suck off redundant agrobacterium. Then placed on two layers of filter paper in a sterile glass dish (moistened with 1/2MS liquid medium supplemented with 100. mu.M AS) and incubated for 48h at 26 ℃ in the absence of light. The embryogenic callus after co-culture is washed with sterile water for 3 times, then sterilized with MS solution of 200mg/L of cefradine (Cef) and carbenicillin (Carb) for 10min, and finally washed with sterile water for 3 times. The embryogenic callus after being sterilized is placed on a sterile filter paper to absorb excess sterile water, then the proembryogenic mass is inoculated on a KCC medium (KBN +200mg/L Carb +200mg/L Cef) to be cultured for 3 weeks for delayed screening, and then is transferred to an X3CC medium (X3+200mg/L Carb +200mg/L Cef) for delayed screening for one week. After one month of delayed selection, embryogenic calli were transferred to resistant selection medium containing different concentrations of Basta, cultured in dark at 26 ℃ and subcultured every 4-6 weeks, with the concentration of Basta in the medium required for subculture gradually increasing from 5mg/L to 20mg/L until resistant somatic embryos germinated. After the resistant somatic embryos germinate, the cotyledon embryos are inoculated on a GM culture medium (MS +15g/L of sucrose +1g/L of activated carbon +3g/L of plant gel), cultured under light until the cotyledon turns green, and then inoculated on a rooting culture medium (MS +1mg/L of IBA +30g/L of sucrose +7g/L of agar) to form seedlings. After the resistant plant roots grow to the culture bottle mouth, transplanting the resistant plant roots to an artificial climate chamber for hardening seedlings for 1-2 months, and then transplanting the resistant plant roots to a greenhouse.

VpCDPK13-YFP transgenic plant is tested for the resistance screening of the seedling, the traditional CTAB method is used for extracting the genome DNA of the resistance plant, about 0.1g of leaves are taken, a grinding rod is used for grinding the leaves in a 1.5mL centrifuge tube, 650 mu L of CTAB buffer solution is added, a 65 ℃ oven is placed for 30min, the leaves are taken out and placed at room temperature, equal volume of chloroform isoamyl alcohol is added, the mixture is mixed evenly and centrifuged at 12000rpm/min for 10min, about 400 mu L of supernatant is taken, precooled absolute ethyl alcohol with double volume is added, 30min of precipitation at-20 ℃, 10min of centrifugation at 12000rpm/min is poured out, and the mixture is naturally dried. Dissolve in 20. mu.L of distilled water. The nucleotide sequences on the reporter gene YFP and the promoter 35S are used for designing universal primers (upstream YFP:5'CAGGGTCAGCTTGCCGTAG 3' (SEQ ID NO:11) and downstream 35SA:5'TCCTTCGCAAGACCCTTCCTCTAT 3' (SEQ ID NO:12)) to detect the PCR level of the resistant plants. Among the 89 Basta resistant transgenic lines, 10 lines can amplify the target bands.

In order to examine whether the foreign fusion gene in the PCR positive strain can be normally translated into protein, Western Blot detection was performed on the positive strain. And (3) putting 100mg of fresh leaves into liquid nitrogen for sample grinding, and preserving the ground sample in the liquid nitrogen. Adding a protein extracting solution (pH 8.01M Tris-HCl: 10% SDS: 50% glycerol: B-mercaptoethanol ═ 2:4:4:1) until the sample just submerges, fully mixing, then carrying out boiling water bath for 5min, after the sample returns to the room temperature, centrifuging at 13000rpm for 5min, and taking the supernatant, namely the extracting solution containing the protein. Preparing 8% SDS-PAGE separation gel, solidifying the separation gel, preparing 4% concentrated gel, solidifying the concentrated gel, mixing the sample buffer solution and the protein extracting solution, moving the mixture into a sample inlet hole, performing vertical gel electrophoresis, performing electrophoresis at 70V for about 30min in the migration process in the concentrated gel, then performing electrophoresis at 90V for 3 hours, and stopping electrophoresis when the blue line is about 0.5cm away from the bottom. And adding the prepared membrane transferring buffer solution into the glass groove. PVDF membrane of appropriate size is cut and activated by soaking in a small amount of methanol for half a minute. The excess of SDS-PAGE gel was cleaved. Fixing the glue and the membrane according to the sequence of the blackboard, the sponge, the filter paper, the glue, the membrane, the filter paper, the sponge and the white board, and placing the fixed splint into a membrane conversion buffer solution. And (5) transferring the membrane for 1h at 100V, and placing the electrophoresis tank under ice bath conditions. After the membrane transfer was completed, the PVDF membrane was removed, the side in contact with the gel was facing upward, and washed with TBST buffer on a decolorizing shaker for 10 min. 15mL of TBST membrane sealing solution dissolved with 0.5% skimmed milk powder is prepared, shaken one hour in advance, and the PVDF membrane is placed in the sealing solution to be incubated for 90 min. The murine GFP monoclonal antibody diluted 2000-fold was added and incubated overnight at 4 ℃. Wash 5 times with 20ml TBST for 10min each time. HRP-labeled goat anti-mouse polyclonal antibody diluted 5000 times was added, incubated at room temperature for hours, and then washed 3 times with TBS for 5min each. The PVDF membrane was taken out, developed using a developer from Millipore, subjected to chemiluminescence imaging using a BIO-RAD gel imaging system, and recorded by photography. Finally, the PVDF membrane was incubated in ponceau staining solution, total protein was stained, and then photographed and recorded with a canon single lens reflex camera. The results showed that VpCDPK13-YFP fusion protein could be expressed normally but in lower amounts in most transgenic lines (figure 4).

Example 4: identification of powdery mildew resistance of Vitis vinifera Baihe-35-1 calcium-dependent protein kinase gene VpCDPK13 overexpression transgenic anucleate white strain

After 35S, transplanting a VpCDPK13-YFP transgenic grape strain and a non-transgenic grape strain of the same age into an artificial climate box incubator to grow for half a year, inoculating strong pathogenic grape powdery mildew En NAFI 1 to leaves of the grape strain. A small piece of leaf was cut at 10 days after inoculation, and trypan blue staining, diaminobenzidine staining, and aniline blue staining were performed. First, powdery mildew, which had formed white velvet-like hyphae and larger single colonies on wild type seedless white strain leaves after inoculation of powdery mildew for 20d, but only sporadic colonies were produced on the VpCDPK13-YFP transgenic grape strain leaves, but white hyphae were not evident and large red-brown necrotic patches were found all below the colonies (fig. 5A). After observing the growth condition of hyphae by trypan blue staining and counting the quantity of conidia, the hyphae growth on the transgenic grape strain line is inhibited, the hyphae density is lower and the conidia yield is reduced compared with powdery mildew on wild grape leaves (figures 5B and 5C). After measuring the free salicylic acid and ethylene of the leaf tissue in 20D inoculation by ultra performance liquid chromatography and gas chromatography respectively, the transgenic line leaves were found to have more ethylene accumulation and higher level salicylic acid synthesis (fig. 5D and 5E). Meanwhile, after measuring the content of hydrogen peroxide and the content of anthocyanin by spectrophotometry, higher levels of hydrogen peroxide and anthocyanin are accumulated in the leaves of the transgenic lines, which indicates that the leaves of the transgenic grapes are in a high oxidative stress state (FIGS. 5F and 5G). The results of aniline blue staining indicated that more callose was accumulated in the leaves of the transgenic lines, and that these callose were accumulated mainly in mesophyll cells that were punctured around epidermal cells (FIG. 5B). Therefore, we speculate that overexpression of VpCDPK13-YFP promotes the synthesis of ethylene and salicylic acid under erysiphe necator-induced conditions, activating downstream signals leading to hydrogen peroxide accumulation and callose accumulation; the callose accumulated in mesophyll cells around the punctured epidermal cells prevents the diffusion of hydrogen peroxide from the punctured epidermal cells to the lower mesophyll cells, so that the hydrogen peroxide mainly diffuses to the surrounding epidermal cells, and the accumulation of a large amount of anthocyanin in the epidermal cells and the death of allergic cells are promoted. The large-area necrosis of grape epidermal cells limits the nutrition supply of grape powdery mildew, inhibits the hypha growth and the conidium generation of the grape powdery mildew, and improves the powdery mildew resistance of grapes to a certain extent.

Reference to the literature

Amrine KCH,Blanco-Ulate B,Riaz S,Pap D,Jones L,Figueroa-Balderas R,Walker MA,Cantu D(2015)Comparative transcriptomics of Central Asian Vitis vinifera accessions reveals distinct defense strategies against powdery.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.Mol Plant Pathol 13(1):1-16.doi:10.1111/j.1364-3703.2011.00728.x

Gao YR,HanYT,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 species using detached leaves.Plant Physiol Biochem 98:12-24.doi:10.1016/j.plaphy.2015.11.003

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Sequence listing

<110> northwest agriculture and forestry science and technology university

<120> powdery mildew-resistant grape calcium-dependent protein kinase gene VpCDPK13 and application thereof

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1710

<212> DNA

<213> grape in the east China (vitas pseudooculata)

<400> 1

atgggtaata cttgtgttgg tccaagcatc tccaagaatg gtttcttcca atctgtgtcg 60

gctgcaatgt ggcggagccg tgcaccggaa ggctcggctt cttacacgaa tggagaaact 120

atggatgagg cacaggctac aactaaagaa cctggatcac cattgccagt ccaaaacaaa 180

cccccagagc aaatgacaat ccccaaggag gagcagccta aaaaacccaa gaagccccat 240

caaattaaga gggtgtcgag tgcagggctt aggatagaat ctgtgttgca aacaaaaacc 300

ggaaacttta aggaattttt tattctgggg aggaagcttg gacaagggca atttgggact 360

acatttcttt gtgtgcagaa agctaccagg aaagagtatg cgtgtaaatc aattgcgaaa 420

aggaaattgt taacagatga ggacgtggag gatgtcagaa gggaaattca gataatgcac 480

cacctggcag ggcatccaaa tgtcatatct atcgaggggg cttatgagga tgctgtggca 540

gttcatgttg tcatggaact atgcaaaggt ggggagctat ttgataggat tattcagcgc 600

ggccattaca ctgaaagaaa ggcagctgag cttactagga ctatagttgg ggttgtggag 660

gcttgtcatt ctcttggggt catgcatcga gaccttaagc ctgagaactt tcttttagtc 720

aatgaggagg aggattcact tctcaaaaca attgactttg gattatcagt tttcttcaag 780

ccaggggaaa aatttactga tgtggttggc agcccatact atgtcgcacc agaagttctg 840

agaaagcgtt atggtccaga agcagatgtt tggagtgctg gggtgatcct atacatttta 900

ttgagtggag tgcctccctt ttgggccgaa accgagcaag gtatatttga acaggtcttg 960

catggtgatc ttgacttttc atcagaccct tggccgagta tctcagaaag tgcaaaagat 1020

ttagtaagga gaatgcttgt tcgagaccct agacggcggc tgactgcaca tgaagttttg 1080

tgtcaccctt gggttcaggt tgatggtgta gctcctgaca agcctcttga ttcggcagtt 1140

ttaagtcgct tgaaacaatt ttcagcaatg aacaagctca agaagatggc tcttattgtc 1200

attgcagaga acctatcaga agaagaaata gctggcttaa aagaaatgtt caagatgata 1260

gatacagaca acagtggcca aatcactttt gaagaactca aggctggatt aaaaagagtt 1320

ggtgctaatc ttaaagagtc tgaaatttat gatttaatgc atgcagctga tgttgataac 1380

aatggaacca ttgattatgg ggagttcata gctgccacac tacatctaaa caaagttgag 1440

agagaagatc atttatttgc agctttttcc tactttgata aggatggaag tggctacata 1500

accccagatg agcttcaaca agcctgtgaa gagtttggct tagaggatgt ccgcctggaa 1560

gaaatgatcc gagaagttga tcaggacaat gatggacgca tagattacaa tgagtttgtg 1620

gccatgatgc aaaagggaaa tccaggcatt gggaagaagg gcctgcaaac cagtttcagt 1680

atggggttta gagaggcgtt aaaacattag 1710

<210> 2

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgggtaata cttgtgttgg tcc 23

<210> 3

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

taagccctgc actcgacacc ctc 23

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gtgctggatt ctggtgatgg t 21

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tcccgttcag cagtagtggt g 21

<210> 6

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgttttaac gcctctctaa accc 24

<210> 7

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tctgatcaag agacatctag aatgggtaat acttgtgttg g 41

<210> 8

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gcccttgctc accatggtac catgttttaa cgcctctcta aaccc 45

<210> 9

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tctgatcaag agacaggatc catgggtaat acttgtgttg g 41

<210> 10

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gcccttgctc accatggatc catgttttaa cgcctctcta aaccc 45

<210> 11

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cagggtcagc ttgccgtag 19

<210> 12

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tccttcgcaa gacccttcct ctat 24

<210> 13

<211> 569

<212> PRT

<213> Vitis vinifera (vitas pseudoticulata)

<400> 13

Met Gly Asn Thr Cys Val Gly Pro Ser Ile Ser Lys Asn Gly Phe Phe

1 5 10 15

Gln Ser Val Ser Ala Ala Met Trp Arg Ser Arg Ala Pro Glu Gly Ser

20 25 30

Ala Ser Tyr Thr Asn Gly Glu Thr Met Asp Glu Ala Gln Ala Thr Thr

35 40 45

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

50 55 60

Met Thr Ile Pro Lys Glu Glu Gln Pro Lys Lys Pro Lys Lys Pro His

65 70 75 80

Gln Ile Lys Arg Val Ser Ser Ala Gly Leu Arg Ile Glu Ser Val Leu

85 90 95

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

100 105 110

Leu Gly Gln Gly Gln Phe Gly Thr Thr Phe Leu Cys Val Gln Lys Ala

115 120 125

Thr Arg Lys Glu Tyr Ala Cys Lys Ser Ile Ala Lys Arg Lys Leu Leu

130 135 140

Thr Asp Glu Asp Val Glu Asp Val Arg Arg Glu Ile Gln Ile Met His

145 150 155 160

His Leu Ala Gly His Pro Asn Val Ile Ser Ile Glu Gly Ala Tyr Glu

165 170 175

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

180 185 190

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

195 200 205

Ala Glu Leu Thr Arg Thr Ile Val Gly Val Val Glu Ala Cys His Ser

210 215 220

Leu Gly Val Met His Arg Asp Leu Lys Pro Glu Asn Phe Leu Leu Val

225 230 235 240

Asn Glu Glu Glu Asp Ser Leu Leu Lys Thr Ile Asp Phe Gly Leu Ser

245 250 255

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

260 265 270

Tyr Tyr Val Ala Pro Glu Val Leu Arg Lys Arg Tyr Gly Pro Glu Ala

275 280 285

Asp Val Trp Ser Ala Gly Val Ile Leu Tyr Ile Leu Leu Ser Gly Val

290 295 300

Pro Pro Phe Trp Ala Glu Thr Glu Gln Gly Ile Phe Glu Gln Val Leu

305 310 315 320

His Gly Asp Leu Asp Phe Ser Ser Asp Pro Trp Pro Ser Ile Ser Glu

325 330 335

Ser Ala Lys Asp Leu Val Arg Arg Met Leu Val Arg Asp Pro Arg Arg

340 345 350

Arg Leu Thr Ala His Glu Val Leu Cys His Pro Trp Val Gln Val Asp

355 360 365

Gly Val Ala Pro Asp Lys Pro Leu Asp Ser Ala Val Leu Ser Arg Leu

370 375 380

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

385 390 395 400

Ile Ala Glu Asn Leu Ser Glu Glu Glu Ile Ala Gly Leu Lys Glu Met

405 410 415

Phe Lys Met Ile Asp Thr Asp Asn Ser Gly Gln Ile Thr Phe Glu Glu

420 425 430

Leu Lys Ala Gly Leu Lys Arg Val Gly Ala Asn Leu Lys Glu Ser Glu

435 440 445

Ile Tyr Asp Leu Met His Ala Ala Asp Val Asp Asn Asn Gly Thr Ile

450 455 460

Asp Tyr Gly Glu Phe Ile Ala Ala Thr Leu His Leu Asn Lys Val Glu

465 470 475 480

Arg Glu Asp His Leu Phe Ala Ala Phe Ser Tyr Phe Asp Lys Asp Gly

485 490 495

Ser Gly Tyr Ile Thr Pro Asp Glu Leu Gln Gln Ala Cys Glu Glu Phe

500 505 510

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

515 520 525

Asp Asn Asp Gly Arg Ile Asp Tyr Asn Glu Phe Val Ala Met Met Gln

530 535 540

Lys Gly Asn Pro Gly Ile Gly Lys Lys Gly Leu Gln Thr Ser Phe Ser

545 550 555 560

Met Gly Phe Arg Glu Ala Leu Lys His

565

Claims (3)

1. Isolated powdery mildew resistant gene of grape calcium dependent protein kinase as shown in SEQ ID NO. 1VpCDPK13The application in the production of powdery mildew resistant grape varieties.
2. 2. A method for producing a powdery mildew resistant grape plant, characterized in that an isolated powdery mildew resistant grape calcium dependent protein kinase gene represented by SEQ ID NO. 1 is usedVpCDPK13Introduced into grape plants and stably expressed.
3. 3. A method for improving powdery mildew resistance of grape plants is characterized in that the separated powdery mildew resistant grape calcium dependent protein kinase gene shown in SEQ ID NO. 1VpCDPK13Is introduced intoThe grape plant.

Images