Application of PmchoC gene in prevention and treatment of soft rot of kiwi fruit

Abstract

The invention discloses application of a PmchoC gene in prevention and treatment of kiwi fruit soft rot, compared with a wild type, after the PmchoC gene of pestalotiopsis microspora is knocked out, the integrity of a cell wall is obviously reduced, the sporulation quantity and the germination rate are obviously reduced, and the diameter of a scab is obviously reduced after the pestalotiopsis microspora is inoculated to kiwi fruits; the gene is proved to be capable of directly influencing the pathogenicity of pestalotiopsis microspora; by spraying the dsRNA of the PmchoC gene, the diameter of the scab of the kiwi fruit is reduced by 55.26%, the diameter of the scab of the leaf is reduced by 67.88%, and the PmchoC-dsRNA relieves the disease symptom of the soft rot of the kiwi fruit by inhibiting the expression of the PmchoC gene in pestalotiopsis microspora. According to the method, a spraying-induced gene silencing technology is applied to prevention and treatment of the soft rot of the kiwi fruit for the first time, and the problems that traditional disease-resistant breeding is long in period, resistance is prone to loss, and the disease outbreak risk is large are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of a PmchoC gene in preventing and controlling kiwifruit soft rot. Background Art

[0002] Kiwifruit is rich in vitamins and minerals, has a unique flavor, and is deeply loved by consumers. As the origin of Actinidia plants and the birthplace of kiwifruit cultivars, China has become the country with the largest kiwifruit cultivation area and the highest annual output. However, in recent years, the primary fungal disease that infects before and after harvest, fruit soft rot, has continued to break out, resulting in a serious decline in fruit quality and healthy fruit rate, directly affecting the domestic sales and export of kiwifruit, causing significant economic losses to farmers and sales manufacturers. The Chinese kiwifruit industry is facing a severe test of fruit soft rot.

[0003] Based on the current literature, Botryosphaeria dothidea, Diaporthe, Pestalotiopsis microspora, and Alternaria alternata are considered to be the main pathogens of kiwifruit soft rot in my country. There are obvious differences in the pathogens of different varieties in different regions; the differences in strain types may be related to the temperature and humidity environment, the source of pathogens, and the infection ability of different cultivation areas. Among them, Pestalotiopsis microspora was first reported as the main pathogen of kiwifruit soft rot in China, and the detection rate in the field has increased year by year. Pestalotiopsis microspora is a fungus of the genus Pestalotiopsis. In addition to infecting kiwifruit, it can also infect other economic crops, causing diseases such as berry tea leaf spot, oil palm leaf spot, green plum wilt, guava dry disease, red leaf photinia leaf spot, citrus fruit spot, and black tiger wheel spot. However, the infection mechanism and prevention of this pathogen have not been studied in depth. Therefore, exploring the important pathogenic genes of this pathogen and clarifying its pathogenic mechanism are of great scientific significance for the targeted prevention and control of kiwifruit soft rot and other diseases caused by this pathogen.

[0004] Spray-induced gene silencing (SIGS) is a control technology based on the important theory of small RNA shuttle transport mechanism and gradually developed based on cross-border RNAi technology. SIGS technology has the advantages of high targeting, no residue, rich targets and flexible design. The time required to develop dsRNA corresponding to SIGS is shorter than that required to cultivate transgenic plants. It can respond to new invasive fungal diseases relatively quickly and will not cause environmental pollution and crop residues like chemical pesticides. It has greatly broadened the application of RNAi technology in plant diseases.

[0005] Phosphatidylcholine (PC) is the most abundant type of phospholipid in the membrane system of eukaryotic organisms. It plays an important role in maintaining the integrity and function of membrane structure and is involved in various life activities such as cell growth and differentiation, material metabolism, signal recognition and transduction, cell autophagy, and cell cycle. The fungicides widely used in production, such as pyraclostrobin and isopyruvate, are believed to have a bactericidal effect by inhibiting the activity of phosphatidylethanolamine methyltransferase in the phosphatidylethanolamine (PE) methylation pathway. At present, there are relatively few green control measures for kiwifruit soft rot. Screening out key pathogenic gene targets can not only clarify the pathogenic mechanism of soft rot pathogens, but also help develop efficient and green disease prevention and control technologies. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a target gene for controlling the pathogenic bacteria of kiwifruit soft rot, Pseudomonas microsporus, namely, the phosphatidylethanolamine methylation gene PmchoC, which is involved in regulating the ROS metabolism, chitin metabolism, glucan metabolism and cell wall integrity of Pseudomonas microsporus. The PmchoC gene is used as a target and a corresponding SIGS spraying system is constructed. The method can effectively control the soft rot of kiwifruit.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The phosphatidylethanolamine methylation gene PmchoC of Pseudomonas microsporus has a nucleotide sequence as shown in SEQ ID NO.1, and an amino acid sequence encoded by it as shown in SEQ ID NO.2.

[0009] A kiwifruit soft rot control agent includes an inhibitor of the phosphatidylethanolamine methylation gene PmchoC of Pseudomonas microsporus, the nucleotide sequence of the gene PmchoC is shown in SEQ ID NO. 1. Preferably, the gene PmchoC inhibitor includes a dsRNA that targets and silences the PmchoC gene, and the dsRNA consists of a nucleotide sequence as shown in SEQ ID NO. 5 and a reverse complementary nucleotide.

[0010] The application of the above-mentioned control agent in preventing and controlling plants infected with Microsporum polytrichomonas.

[0011] A method for inhibiting kiwifruit soft rot: spraying kiwifruit plants with dsRNA that targets and silences the phosphatidylethanolamine methylation gene PmchoC of Pseudomonas microsporus, wherein the nucleotide sequence of the gene PmchoC is shown in SEQ ID NO. 1. Preferably, the dsRNA consists of nucleotides as shown in SEQ ID NO. 5 and reverse complementary nucleotides thereof.

[0012] The PmchoC gene of Pseudomonas microsporus was used as a target gene, which is involved in regulating ROS metabolism, chitin metabolism, glucan metabolism and cell wall integrity. Its function was verified by the following methods:

[0013] ①Construct a PmchoC gene knockout vector, and then obtain the PmchoC gene knockout strain through Agrobacterium transformation. The morphology, hyphae, spore production ability and germ tube length of the wild type and mutant of Pseudomonas microsporus were observed. It was found that knocking out the PmchoC gene affected the growth of Pseudomonas microsporus, the conidia morphology was abnormal, and the spore germination rate and germ tube length were decreased.

[0014] ② The expression levels of genes related to chitin metabolism and glucan metabolism were determined under the stress of Pseudomonas microsporus with the PmchoC gene knocked out, and the integrity of the cell wall of the strain was detected. It was found that the PmchoC gene played an important role in Pseudomonas microsporus's resistance to the stress of high-concentration ions and oxidative conditions, and was related to the cell wall integrity of Pseudomonas microsporus.

[0015] ③ Five days after inoculation of the PmchoC complement mutant, the diameter of the diseased spot reached 20.06 mm, while the diameter of the diseased spot of kiwifruit inoculated with the PmchoC gene knockout mutant was only 8.98 mm five days after inoculation.

[0016] Therefore, the PmchoC gene is involved in regulating the cell wall growth of Pseudomonas microsporus and plays an important role in the reproductive development of the pathogen. The PmchoC gene can be used as a new target for the prevention and control of kiwifruit soft rot.

[0017] Furthermore, the corresponding SIGS spraying system was constructed with the PmchoC gene as the target and its dsRNA as the material, and it was confirmed that this method can effectively prevent and control kiwifruit soft rot. After spraying Pmcho-dsRNA, the kiwifruit fruits and leaves were inoculated with Pseudomonas microsporus. It was found that the expression level of PmchoC gene in leaves decreased by 52.67%, the diameter of leaf lesions decreased by 67.88%, and the biomass of pathogens in leaves decreased by 80.90%; the expression level of PmchoC gene in fruits decreased by 89.72%, the diameter of lesions decreased by 55.26%, and the biomass of pathogens in fruits decreased by 68.71%. The above results show that PmchoC-dsRNA can alleviate the symptoms of kiwifruit soft rot by inhibiting the expression of PmchoC gene in Pseudomonas microsporus.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0019] The present invention discloses for the first time at home and abroad the sequence of the phosphatidylethanolamine methylation gene PmchoC of the endogenous synthesis pathway of phosphatidylcholine in Pseudomonas microsporus - the PE synthesis pathway. In addition, there is no report on the function of this gene in regulating the reproductive development and pathogenicity of Pseudomonas microsporus. The PmchoC gene of Pseudomonas microsporus was knocked out and complemented to verify the gene function. It was found that compared with the wild type, the cell wall integrity was significantly reduced after the gene was knocked out, and the spore production and germination rate were significantly reduced. In addition, the diameter of the lesions of Pseudomonas microsporus inoculated on kiwifruit after the PmchoC gene was knocked out was significantly reduced, confirming that the gene can directly affect the pathogenicity of Pseudomonas microsporus; by spraying the dsRNA of the gene, the diameter of the fruit lesions decreased by 55.26%, and the diameter of the leaf lesions decreased by 67.88%, indicating that PmchoC-dsRNA can inhibit the expression of the PmchoC gene in Pseudomonas microsporus, thereby alleviating the symptoms of kiwifruit soft rot. Spraying the dsRNA of the PmchoC gene is helpful for the green and sustainable prevention and control of kiwifruit soft rot, providing an ideal target for the research of disease-resistant molecular breeding and comprehensive prevention and control measures, which is of great significance for the sustainable development of the kiwifruit industry. In addition, the present invention applies spray-induced gene silencing technology to the prevention and treatment of kiwifruit soft rot for the first time, breaking the problems of long cycle, easy loss of resistance and high risk of disease outbreak in traditional disease-resistant breeding. At the same time, it can also effectively solve the damage of chemical pesticides to plants and the environment and the problem of pesticide residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1The construction method of the PmchoC knockout mutant ΔPmchoC and the PmchoC complement mutant C-ΔPmchoC of Pseudomonas microsporus (a, b) and the mutant PCR verification (c). b is the electrophoresis of PCR verification of PmchoC knockout and revertant mutants using HYG-F / HYG-R as primers, and c is the electrophoresis of PCR verification of PmchoC knockout and revertant mutants using HYG-F / Down arm-R as primers.

[0021] Figure 2 The morphological observation, hyphae observation and spore production ability of the wild type and mutants of Pseudomonas microsporus. a. Colony morphology of mutants and wild types (PDA, 5d), b. Hyphae morphology of mutants and wild types under electron microscope, c. Germ tube morphology of spore germination of mutants and wild types, d. Germ tube length of spore germination of wild types and mutants (24h), e. Spore germination rate of wild types and mutants.

[0022] Figure 3 Results of the PmchoC gene function verification experiment. a. Lesion morphology of wild type and mutants of Pseudomonas microsporus inoculated on kiwifruit, b. Difference in lesion size of wild type and mutants inoculated on kiwifruit, c. Fluorescence observation of mutants and wild type under 488nm excitation light after PI staining, d. Colony morphology of strains under different stresses (6d), e. Colony diameter of strains under different stresses (6d), f. Expression of genes related to chitin synthesis, g. Expression of genes related to chitin metabolism, h. Expression of genes related to glucan synthesis, i. Colony morphology in stress experiment, j. Statistical results of colony size in stress experiment.

[0023] Figure 4 The dsRNA was absorbed by Pseudomonas microsporus and kiwifruit tissues. a. Electrophoresis results of in vitro synthesized PmchoC-dsRNA and fluorescein-labeled EGFP-dsRNA, b. Electrophoresis detection of PmchoC-dsRNA expressed based on L4440 vector, c. Colony morphology of Pseudomonas microsporidia after spraying PmchoC-dsRNA, d. Expression level of PmchoC gene of Pseudomonas microsporidia after spraying PmchoC-dsRNA, e. Fluorescence distribution of fruit 6h, 12h, 18h and 24h after spraying EGFP-dsRNA on the fruit stalk, f. Fluorescence distribution of leaf petiole 24h after spraying fluorescently labeled EGFP-dsRNA, g. Laser confocal observation of fruit stalk 24h after spraying fluorescently labeled EGFP-dsRNA, h. Laser confocal observation of petiole and leaf mesophyll cells 24h after spraying EGFP-dsRNA at the distal end of the leaf petiole.

[0024] Figure 5Spraying PmchoC-dsRNA can reduce the infection of Pseudomonas microsporidia on kiwifruit leaves and fruits. a. The size of the lesions on the leaves 3 days after inoculation with Pseudomonas microsporidia, b. Schematic diagram of spraying PmchoC-dsRNA on the leaves and inoculating with Pseudomonas microsporidia, c. The statistical results of the lesion diameter on the leaves 3 days after inoculation with Pseudomonas microsporidia, d. The expression of PmchoC in the leaf tissue after infection with Pseudomonas microsporidia, e. The biomass of the pathogen in the leaves 6 days after inoculation with Pseudomonas microsporidia, f. The fruit was inoculated with Pseudomonas microsporidia after spraying PmchoC-dsRNA and water, g. The biomass of the pathogen in the fruit 6 days after inoculation, h. The expression of PmchoC in the fruit tissue after infection with Pseudomonas microsporidia, i. The size of the fruit lesions 6 days after inoculation with Pseudomonas microsporidia. DETAILED DESCRIPTION

[0025] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples can be purchased unless otherwise specified.

[0026] Example 1: Functional verification of the phosphatidylethanolamine methylation gene PmchoC in Pseudomonas microsporus

[0027] The invention discloses the function of the PmchoC gene in kiwifruit soft rot fungus Pseudomonas microsporus, wherein the nucleotide sequence of the PmchoC gene is shown as SEQ ID NO.1, and the amino acid sequence of PmchoC is shown as SEQ ID NO.2.

[0028] 1. Obtaining PmchoC gene knockout and complement mutants

[0029] (1) Construction of PmchoC gene knockout vector

[0030] According to the principle of homologous recombination, the target gene fragment is replaced by the resistance gene fragment to achieve the knockout of the target gene PmchoC. The construction method of the PmchoC gene knockout vector is as follows:

[0031] ① Design primers tRNA-F / R containing the target gene (Table 1), use the intermediate vector pHLW-sgRNA-tRNA of sgRNA (construct the gRNA scaffold and tRNA fragment into the PylsgRNA-ATU3bLacZ plasmid, the sequence of gRNA scaffold is shown in SEQ ID NO.3, and the tRNA sequence is shown in SEQ ID NO.4) as a template for PCR, and recover the obtained PCR product by gel and combine it with the vector STU-Cas9 (the LbCpf1 endonuclease fragment and SV40 poly (A) are connected to the POE-3XFLAG-DN vector by In-Flsion Cloning to obtain the C2-pCpf1-TDT-SV4DN vector, and the gRNA scaffold is connected to the SV40 poly (A), and the obtained fragment is connected to the C2-pCpf1-TDT-SV4DN vector by In-Flsion Cloning. The STU-INTER vector was obtained by cloning and ligation. The STU-INTER vector was double-digested with SPeI and BamHI and ligated with the Cas9 protein gene sequence to obtain the STU-Cas9 vector. After restriction digestion and ligation with BsaI and T4 ligase, the competent Escherichia coli cells were transformed to construct the sgRNA / Cas9 binary expression vector containing the target gene.

[0032] ② After the vector GFP-Cas9 was cut with EcoRI, the up arm+HYG+down arm fragment containing the hygromycin resistance gene HYG and the upper and lower arms of the target gene of 1 kb was connected and sent to the company for sequencing verification. The constructed vector was named GFP-Cas9-updown.

[0033] ③ Design primers HH-F and HH-R, use the sgRNA / Cas9 binary expression vector containing the target gene obtained in step ① as a template to amplify sgRNA by PCR, use the endonuclease SpeI to cut the vector GFP-Cas9-updown plasmid, then recover the PCR product and the enzyme-cut product by gel, connect them with the homologous recombinase, and send them to the company for sequencing verification. The obtained plasmid is the gene knockout vector containing the target gene.

[0034] (2) Construction of PmchoC gene restoration vector

[0035] ① Design primers tRNA-F / R containing the target gene, perform PCR using the intermediate vector pHLW-sgRNA-tRNA of sgRNA as a template, recover the obtained PCR product by gel, and perform cyclic restriction digestion and ligation with the vector STU-Cas9 using BsaI and T4 ligase to construct an sgRNA / Cas9 binary expression vector containing the target gene.

[0036] ② After synonymous mutation of the knockout sgRNA target site of the PmchoC gene, PmchoC-M was obtained. The vector GFP-Cas9 was cut with EcoRI and connected to PmchoC-M and 1kb up arm+PmchoC-M+down arm fragments on the upper and lower arms. The fragments were sent to the company for sequencing verification. The constructed vector was named GFP-Cas9-updownm.

[0037] ③ Design primers HH-F and HH-R, use the sgRNA / Cas9 binary expression vector containing the target gene obtained in step ① as a template to amplify sgRNA by PCR, use the endonuclease SpeI to digest the GFP-Cas9-updownm plasmid obtained in the previous step, then recover the PCR product and the enzyme digestion product by gel, connect them with the homologous recombinase, and send them to the company for sequencing verification. The obtained plasmid is the gene recovery vector containing the target gene.

[0038] (3) Genetic transformation of Pseudomonas microsporus

[0039] The knockout vector and the restoration vector containing the target gene were used as transformation vectors. Agrobacterium was used to transform Pseudomonas microsporus. The candidate transformants were selected to extract DNA. The primers HYG-F / HYG-R for amplifying the hygromycin B resistance gene were used for PCR and electrophoresis detection. After being sent for testing, the pure PmchoC gene knockout mutant ΔPmchoC and the complement mutant C-ΔPmchoC ( Figure 1 a, 1b and 1c).

[0040] The results showed that knocking out the PmchoC gene led to changes in colony morphology, an increase in aerial hyphae, and abnormal hyphae morphology ( Figure 2 a, 2b), compared with the wild type (WT) and C-ΔPmchoC strains, the conidia of ΔPmchoC on PDA were abnormal in morphology, and the spore germination rate and germ tube length were decreased ( Figure 2 c, 2d and 2e).

[0041] (4) Functional verification of the PmchoC gene

[0042] The detection method of the effect of PmchoC gene on the pathogenicity of Pseudomonas microsporus is as follows: Take the kiwi fruit of the 'Donghong' variety as the material, take the activated transformant and the wild-type strain's bacterial cake (diameter = 0.8 cm), and cover the fruit's needle-punctured wound with the mycelium surface. Place the inoculated kiwi fruit in a moisturizing box at 25°C for 6 days. Take pictures and measure the diameter of the lesions. Set up 3 replicates for each group.

[0043] The detection method of the effect of PmchoC gene on the cell wall function of Pseudomonas microsporus is as follows: the hyphae are stained with propidium iodide (PI). As with the climbing slide method, 50 μg / mL of PI staining solution is added to the slide and stained in the dark for 10 minutes, then the excess staining solution is washed off with distilled water, and the fluorescence intensity is observed under a fluorescence microscope. The activated transformant and the wild-type strain cakes are inoculated on Congo red (CR) and fluorescent brightener 28c (CFW), respectively, and cultured in the dark at 25°C for 6 days. The colony diameter is measured every 24 hours, and the colony growth diameter is determined by the cross method.

[0044] The detection method of the effect of PmchoC gene on the stress resistance of Pseudomonas microsporus and the metabolism of glucan and chitin is as follows: (A) 5mM H2O2 or 7mM H2O2; (B) 0.7M KCl or 1M NaCl were added to the PDA medium respectively. The bacterial cakes (diameter = 0.8cm) of the activated mutant and wild-type strains were inoculated on the above-mentioned medium with stress conditions, and the photos were observed and taken after 6 days, and the colony growth diameter was determined by the cross method.

[0045] Real-time fluorescence quantitative PCR: The total RNA of wild-type and mutant Pseudomonas microsporus was extracted using the Fast Pure Universal Plant Total RNA Isolation Kit. cDNA was synthesized using One-Stepg DNA Removal and cDNA Synthesis Super Mix. After reverse transcription, the sample was diluted 2 times with RNase-free H2O for subsequent experiments. Primers were designed to detect chitin synthesis-related genes (ChsA, ChsB, Chs2, ChsD, ChsF, ChsG1, ChsG2), chitin decomposition-related genes (Chi1, Chi2, CTS3, Chi3) and glucanase-related genes (Ags3, Pmd3, FksP, Exg1) of Pseudomonas microsporus (see Table 1 for details). The internal reference gene was Actin. The fluorescence quantitative PCR kit Green Premix Pro Taq HS qPCR Kit was used for detection. The real-time fluorescence quantitative PCR instrument model was ABIStepOnePlusTM Real-Time PCR System with Tower, and the dye used was ROX.

[0046] The results showed that compared with the lesions formed by wild-type Pseudomonas microsporus inoculated on kiwifruit, the diameter of the lesions formed by ΔPmchoC was reduced by 45.97% ( Figure 3a and 3b), while the reverse mutant had no difference with the wild type. After PI staining, ΔPmchoC had stronger fluorescence under excitation light than the wild type, and all hyphae glowed under excitation light ( Figure 3 c), while ΔPmchoC was more sensitive in PDA medium supplemented with Congo red (CR) and fluorescent whitening agent 28c (CFW) ( Figure 3 d); After adding CFW, the colony diameter of ΔPmchoC was only 59.75% of that of the wild type ( Figure 3 e); after adding CR, the diameter of ΔPmchoC colonies was only 71.10% of that of the wild type (3e). This indicates that the PmchoC gene is related to cell wall integrity. The mutant's increased sensitivity to CFW and CR is speculated to be related to the content of chitin and glucan. The expression of genes related to chitin synthesis or decomposition and glucanase was further quantitatively analyzed. The results showed that among the chitin synthesis-related enzymes, except for the CHSG2 gene, which was upregulated by 1.36 times compared with the wild type, all other genes were downregulated, especially the ChsA gene (chitin synthase A), which was downregulated by 50.75% ( Figure 3 f). All chitin decomposition-related enzyme genes were up-regulated, among which the CHI1 (chitinase) gene was significantly up-regulated by 1.94 times ( Figure 3 g). At the same time, quantitative analysis of glucan synthase-related genes was performed, and all genes except PMD3 were down-regulated, among which the expression of the extracellular β-(1,3)-glucan synthase EXG1 gene was down-regulated by 68.28% ( Figure 3 h). ΔPmchoC showed sensitivity under oxidative stress, while the reverse mutant was similar to the wild type ( Figure 3 i and 3j). This indicates that the PmchoC gene plays an important role in the resistance of Pseudomonas microsporus to the stress of high concentration ions and oxidative conditions. The above data show that the PmchoC gene is necessary for Pseudomonas microsporus to exert full virulence, and knocking out the PmchoC gene can significantly reduce its pathogenicity in causing kiwifruit soft rot.

[0047] Table 1 Primer sequences

[0048]

[0049]

[0050]

[0051] Example 2: In vitro synthesis of dsRNA of the phosphatidylethanolamine methylation gene PmchoC and construction of the Escherichia coli L4440 expression system

[0052] 1. In vitro synthesis of PmchoC-dsRNA:

[0053] (1) The isolated and purified Pseudomonas microsporus was inoculated on a potato culture medium with cellophane. After 5 days, the mycelium was collected and the total RNA was extracted by the CTAB method. The first chain of cDNA was synthesized under the action of reverse transcriptase.

[0054] (2) Synthesis of fluorescent EGFP-dsRNA: First, amplify the template with T7 promoter, and use primers EGFP-dsT7F / EGFP-dsR and EGFP-F / EGFP-T7dsR to amplify the fragments with T7 promoter at the 5' end of the sense strand and the fragments with T7 promoter at the 5' end of the antisense strand as templates. Under the action of T7 RNA polymerase and fluorescent RNA labeling mixture, transcribe fluorescently labeled EGFP-dsRNA.

[0055] (3) Synthesis of PmchoC-dsRNA: First, the template with T7 promoter was amplified, and the fragments with T7 promoter at the 5' end of the sense strand and the 5' end of the antisense strand were amplified using primers PmchoC-dsT7F / PmchoC-dsR and PmchoC-dsF / PmchoC-dsT7R, respectively. PmchoC-dsRNA was transcribed in vitro using the Novagen T7 RNAi Transcription Kit, and electrophoresis showed that the amount of transcription products was high, about 500 ng / μL. The synthesized dsRNA was treated with DnaseI / RNaseT1 and purified to remove residual DNA template, single-stranded RNA and excess fluorescein d-NTP. Finally, the purified dsRNA was further purified using the magnetic bead method.

[0056] 2. Construction of the E. coli L4440 expression system of PmchoC-dsRNA. The specific method is as follows:

[0057] (1) Use primers PmchoC4440-F / PmchoC4440-R to amplify the PmchoC gene so that the target gene carries the homology arm of the L4440 vector.

[0058] The amplification system is as follows:

[0059]

[0060] The reaction conditions are:

[0061]

[0062] The product was detected by agarose gel electrophoresis, and the concentration was adjusted to 200-400 ng / mL and stored at -80°C for future use.

[0063] (2) Linearize the L4440 vector with the help of SamI enzyme.

[0064]

[0065] (3) Under the action of homologous recombinase, the target gene fragment with homologous arms is connected to the L4440 vector by homologous recombination. The amplification system is as follows:

[0066]

[0067] The reaction conditions are: 37°C, 60 min; cool to 4°C or immediately cool on ice.

[0068] (4) Transformation of recombinant products: Thaw DH5α competent cells on ice, take 10μL of recombinant products and add them to 100μL of competent cells, gently tap the tube wall to mix (do not oscillate to mix), and place on ice for 30 minutes; heat shock in a 42℃ water bath for 45 seconds, and immediately place on ice to cool for 2-3 minutes. Add 900μL LB medium and shake at 37℃ for 1 hour. Preheat the LB solid medium plate with Amp resistance in a 37℃ incubator. Centrifuge at 5000rpm (2400×g) for 5 minutes and discard 900μL of supernatant. Resuspend the bacteria with the remaining medium and gently spread it on the plate with the correct resistance using a sterile spreader. Incubate inverted in a 37℃ incubator for 12-16 hours.

[0069] (5) Colony PCR verification: the successfully verified plasmids were stored at -80°C for future use.

[0070] (6) HT115 (DE3) competent cell transformation: HT115 (DE3) super competent cells can be used for IPTG induction to express and produce double-stranded RNA (dsRNA). The transformation method is the same as above, and the bacterial solution that has been successfully sequenced is stored at -80°C for later use.

[0071] (7) The successfully verified bacterial solution was cultured in LB liquid medium for 5 h, and a final concentration of 1 mmoL / L PTG solution was added and continued to be shaken and cultured for 5 h. Total RNA was extracted and treated with RNase. 1.2% agarose gel electrophoresis was performed to verify whether PmchoC-dsRNA was correctly expressed. The dsRNA was adjusted to 50 ng / μL and set aside.

[0072] Example 3: Application and verification of phosphatidylethanolamine methylation gene PmchoC-dsRNA

[0073] Application of dsRNA to plants, leaves and fruits: The concentration of fluorescein-labeled EGFP-dsRNA was adjusted to 50 ng / μL and dripped on the petiole of intact leaves, the fruit stalk and the surface of plant stems. After 12 hours, it was observed with a laser confocal microscope. After dripping, the leaves and fruits were observed for fluorescence using a handheld fluorescence instrument. After spraying PmchoC-dsRNA on the leaves, the leaf veins were punctured or not punctured and inoculated with Pseudomonas microsporus. The fruits were punctured at the equator and inoculated with Pseudomonas microsporus.

[0074] The results showed that Pseudomonas microsporus could effectively absorb PmchoC-dsRNA, and the expression of PmchoC gene was significantly downregulated after absorption ( Figure 4 c and 4d). At the same time, the plants can also absorb EGFP-dsRNA well and penetrate into the entire fruit within 48h ( Figure 4 e and 4g), it can penetrate into the whole leaf within 24 hours, and is found in the conducting tissue, intercellular spaces and cells, indicating that EGFP-dsRNA can be self-transmitted in the plant ( Figure 4 f and 4h). After spraying Pmcho-dsRNA, the kiwifruit fruits and leaves were inoculated with Pseudomonas microsporus: the diameter of the leaf lesions was found to be reduced by 67.88% ( Figure 5 a and Figure 5 c), the expression level of PmchoC gene in leaves decreased by 55.26% ( Figure 5 d), the biomass of leaf pathogens decreased by 80.9% ( Figure 5 e); The diameter of the fruit lesion area decreased by 55.26% ( Figure 5 f and Figure 5 i), the biomass of pathogens in the fruit decreased by 68.71% ( Figure 5 g), the expression level of PmchoC gene in the fruit decreased by 89.60% ( Figure 5 h). The above results indicate that PmchoC-dsRNA can alleviate the symptoms of kiwifruit soft rot by inhibiting the expression of PmchoC gene in Pseudomonas microsporus.

[0075] The present invention has found that phospholipids are essential substances for fungal growth and pathogenicity, and the PmchoC gene is related to phospholipid synthesis and is significantly induced during the infection of Pseudomonas microsporus. Furthermore, the present invention constructs a PmchoC gene knockout mutant ΔPmchoC and a complemented mutant C-△PmchoC to determine the pathogenicity of the PmchoC gene. The study found that compared with the wild-type kiwifruit soft rot fungus, the virulence of the ΔPmchoC strain was significantly impaired, and the fungal biomass of the ΔPmchoC strain after infecting kiwifruit leaves and fruits was significantly less than that of the wild type. The above results show that the PmchoC gene is a key pathogenic gene of Pseudomonas microsporus and is necessary for the strain to exert its full virulence. Furthermore, using the PmchoC gene as a target and its dsRNA as a material, a corresponding SIGS spraying system was constructed, and it was confirmed that this method can effectively prevent and control kiwifruit soft rot.

[0076] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A kiwifruit soft rot control agent, characterized in that: Pseudomonas microsporus phosphatidylethanolamine methylation gene PcqI The inhibitor of PcqI The nucleotide sequence is shown in SEQ ID NO.

1.

2. The kiwifruit soft rot control agent according to claim 1, characterized in that The gene PcqI Inhibitors include targeted silencing PcqI dsRNA of the gene.

3. The kiwifruit soft rot control agent according to claim 2, characterized in that The dsRNA consists of nucleotides with a sequence as shown in SEQ ID NO. 5 and nucleotides that are reverse complementary thereto.

4. Use of the control agent according to any one of claims 1 to 4 in controlling plants infected with Pseudomonas microsporus.

5. A method for preventing and controlling plant soft rot, characterized in that: Targeted silencing of phosphatidylethanolamine methylation genes in Pseudomonas microsporus by spraying PcqI dsRNA, the gene PcqI The nucleotide sequence is shown in SEQ ID NO.

1.

6. The method according to claim 5, characterized in that The dsRNA consists of nucleotides with a sequence as shown in SEQ ID NO.5 and nucleotides that are reverse complementary thereto.

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