Application of a tomato Corynespora cassiicola effector protein in pest trapping, prevention and control

Abstract

The present invention provides an application of a tomato Corynespora cassiicola effector protein in pest trapping and control, belonging to the technical field of genetic engineering. The nucleotide sequence of the tomato Corynespora cassiicola effector protein is shown as SEQ ID NO.1 in the sequence listing, and the amino acid sequence is shown as SEQ ID NO.6 in the sequence listing. The tomato Corynespora cassiicola is Corynespora cassiicola Corynespora cassiicola , after treating tobacco seeds and plants with the effector protein Cca0022 solution of the present invention, it has an attracting effect on Bemisia tabaci, and can be combined with chemical control. For the trapped Bemisia tabaci, targeted spraying can be carried out to reduce the usage amount of chemical agents, and at the same time achieve the effect of controlling Bemisia tabaci on tobacco plants.

Description

Technical Field

[0001] The present invention relates to the application of an effector protein of Corynespora cassiicola in the trapping and control of pests, belonging to the field of genetic engineering technology. Background Art

[0002] The whitefly ( Bemisiatabaci ) is also known as the sweet potato whitefly, cotton whitefly, etc. It belongs to the genus Bemisia of the family Aleyrodidae in the order Hemiptera. It was first discovered and named on tobacco in Greece in 1889. It can damage more than 600 kinds of plants and is a highly polyphagous worldwide pest. It is called the "super pest" because of its wide host range, strong environmental adaptability, and strong resistance to chemical insecticides. In agricultural production, chemical agents are mainly used to control the whitefly. Through detection, it is found that the whitefly has developed varying degrees of resistance to insecticides such as organophosphates, nicotine-based, and pyrethroid insecticides, and the control difficulty is gradually increasing. Moreover, most growers use pesticides irregularly, with behaviors such as untimely application, high application concentration, inaccurate application, mixing of multiple pesticides, and frequent application, resulting in frequent occurrences of the phenomenon of "difficult to control pests and ineffective pesticides". The long-term, large-scale, and unreasonable use of chemical pesticides has made problems such as pest resistance and pesticide residues increasingly serious, becoming a bottleneck restricting the further development of the protected vegetable industry. How to develop new control paths has become the research focus of scientific researchers. Protein-based biological materials are a hot spot for the innovative research and application of green pest control due to their multiple advantages such as high safety and mass production.

[0003] There is no report in the prior art on using effector proteins to trap the whitefly. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention provides the application of an effector protein of Corynespora cassiicola in the trapping and control of pests, achieving the following invention objectives: The screened effector protein of Corynespora cassiicola has an obvious trapping effect on the whitefly.

[0005] To solve the above technical problems, the present invention adopts the following technical solutions:

[0006] The application of an effector protein of Corynespora cassiicola in the trapping and control of pests, wherein the nucleotide sequence of the effector protein of Corynespora cassiicola is as shown in SEQ ID NO.1 in the sequence listing, and the amino acid sequence is as shown in SEQ ID NO.6 in the sequence listing.

[0007] The pest is the whitefly.

[0008] The Corynespora cassiicola is Corynespora polyspora.

[0009] The application of the effector protein of Corynespora cassiicola in the trapping and control of the tobacco whitefly.

[0010] Sixteen candidate effector proteins were screened from the reference genome of the Cladosporium fulvum strain, and then five candidate effector proteins that were up-regulated at both 24 h and 48 h after induction by tomato leaves were screened. Two effector proteins with less impact on the normal growth of Nicotiana benthamiana leaves were further screened, and finally effector protein Cca0022 with an attracting effect on Bemisia tabaci was screened out.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] After the tobacco seeds and plants are treated with the effector protein Cca0022 solution of the present invention, it has an attracting effect on Bemisia tabaci, and can be combined with chemical control. Targeted spraying can be carried out on the attracted Bemisia tabaci to reduce the usage amount of chemical agents, and at the same time achieve the effect of controlling Bemisia tabaci on tobacco plants.

[0013] After the tobacco plants are sprayed with the effector protein Cca0022 solution of the present invention for 24 h and then Bemisia tabaci is released, the attracting rates for Bemisia tabaci are 75.14% (when Bemisia tabaci is released for 6 h), 81.58% (when Bemisia tabaci is released for 12 h), and 87.06% (when Bemisia tabaci is released for 24 h), respectively. Description of the Drawings

[0014] Figure 1 It is a heat map of the expression levels of candidate effector proteins in Cladosporium fulvum Cca105;

[0015] Figure 2 It is a diagram of the growth status of tobacco leaves transiently expressing different effector proteins;

[0016] Figure 3 It is a bar chart of the selection rate of Bemisia tabaci for tobacco plants treated with the effector protein Cca0022 solution;

[0017] Figure 4 It is a bar chart of the selection rate of Bemisia tabaci for tobacco plants treated with the effector protein Cca5208 solution. Detailed Embodiments

[0018] The tomato variety for testing is Jinpeng No. 1, which is a tomato variety susceptible to Corynespora cassiicola leaf spot;

[0019] The tested Corynespora cassiicola pathogen is Cladosporium fulvum Cca105, which is preserved in the Green Prevention and Control Laboratory of Facility Vegetable Diseases and Pests, Weifang University of Science and Technology.

[0020] Example 1 Pathogenicity Test of Cladosporium fulvum Cca105

[0021] After activating and culturing Corynespora cassiicola Cca105 on PDA medium, scrape the mycelium and inoculate it into PDA liquid medium. Culture it in a shaking incubator at 25 °C and 160 rpm for 5 days to obtain mycelial pellets. For Jinpeng No. 1 tomatoes with 6 true leaves, take the second and third true leaves from the bottom. Inoculate mycelial pellets of Corynespora cassiicola Cca105 with a diameter of 2 mm on the back of the leaves. Set 4 inoculation points on each true leaf, and inoculate one mycelial pellet at each inoculation point. Inoculate 10 tomato leaves each time, and repeat the experiment 3 times. 6 days after inoculation, observe the disease incidence of tomato leaves. Light brown to dark brown lesions appear on the tomato leaves, and the green color fades around the lesions, showing a yellow halo. Count and calculate the percentage of the number of lesions to the total number of inoculation points. The appearance of lesions at the inoculation points on the small leaves is considered as disease incidence, and the disease incidence is 83.3%.

[0022] Example 2 Analysis and Prediction of Candidate Effector Proteins

[0023] Using the genomic sequencing information of Corynespora cassiicola strains released by the National Center for Biotechnology Information (NCBI) in the United States (https: / / www.ncbi.nlm.nih.gov / nuccore / NSJI00000000) as the reference genome, with the version number NSJI00000000.1; adopt bioinformatics methods to screen candidate effector proteins.

[0024] According to the criteria of having a signal peptide, a size not exceeding 300 AA, a transmembrane structure ≤ 1, no anchor site, subcellular localization outside the cell, the number of cysteines in the protein ≥ 6 for unknown function proteins, and an effector protein prediction score ≥ 0.8, a total of 16 candidate effector proteins were screened from the reference genome of Corynespora cassiicola strains (see Table 1), and they were named Cca9079, Cca9097, Cca9101, Cca0022, Cca0909, Cca1956, Cca3101, Cca5047, Cca4034, Cca6409, Cca6434, Cca2944, Cca3771, Cca5208, Cca9104, and Cca0129 respectively.

[0025] Table 1 Candidate Effector Proteins Screened from the Genome of Corynespora cassiicola Strains

[0026]

[0027] Example 3 Screening of Candidate Effector Proteins with Upregulated Expression after Induction by Tomatoes

[0028] Use fluorescence quantitative PCR to detect the expression levels of 16 candidate effector proteins of Corynespora cassiicola on tomatoes.

[0029] After activating and culturing the Cca105 strain of Corynespora cassiicola on a PDA solid plate, scrape about 0.1 g of mycelium and inoculate it into 100 mL of liquid PDA medium. Incubate it in an oscillating incubator at 25 °C and 160 rpm for 5 days to obtain mycelial pellets with a diameter of 2-3 mm.

[0030] Inoculate the mycelial pellets with a diameter of 2-3 mm onto the 2nd to 4th true leaves of tomato at the 5-6 leaf stage. Evenly spread the mycelial pellets on the tomato leaves so that each mycelial pellet comes into contact with the leaf. At 24 h and 48 h after inoculation, sample together with the leaves and mycelial pellets as test samples, and use the uninoculated mycelial pellets (before inoculation) as control samples.

[0031] Use the TransZol total RNA extraction kit from Beijing Quanshijin Biotechnology Co., Ltd. to extract the total RNA of the samples, perform reverse transcription to obtain cDNA, use the obtained cDNA as a template, perform fluorescence quantitative PCR detection, and calculate the relative gene expression levels of the effector proteins in Corynespora cassiicola Cca105 after 24 h and 48 h of induction by tomato leaves. The heat map results of the relative gene expression levels are shown in Figure 1 . Among these 16 candidate effector proteins, 5 effector proteins were up-regulated at both 24 h and 48 h after inoculation, namely Cca0022, Cca2944, Cca3101, Cca5208, and Cca9101.

[0032] Example 4 Screening candidate effector proteins with less impact on the normal growth of Nicotiana benthamiana leaves

[0033] Using Nicotiana benthamiana as the experimental material, conduct an Agrobacterium-mediated transient expression assay in tobacco to observe the effect of effector proteins on plant cell growth.

[0034] The specific method is as follows:

[0035] Extract the total RNA of Corynespora cassiicola Cca105 and perform reverse transcription to obtain cDNA. Respectively use the cDNA as a template, and through PCR amplification, clone the target fragments of the coding genes of the effector proteins Cca0022, Cca2944, Cca3101, Cca5208, and Cca9101;

[0036] The nucleotide sequence of Cca0022 is shown as SEQ ID NO.1 in the sequence listing;

[0037] The nucleotide sequence of Cca2944 is shown as SEQ ID NO.2 in the sequence listing;

[0038] The nucleotide sequence of Cca3101 is shown as SEQ ID NO.3 in the sequence listing;

[0039] The nucleotide sequence of Cca5208 is shown as SEQ ID NO.4 in the sequence listing;

[0040] The nucleotide sequence of Cca9101 is shown as SEQ ID NO.5 in the sequence listing;

[0041] The amino acid sequence of Cca0022 is shown as SEQ ID NO.6 in the sequence listing;

[0042] The amino acid sequence of Cca2944 is shown as SEQ ID NO.7 in the sequence listing;

[0043] The amino acid sequence of Cca3101 is shown as SEQ ID NO.8 in the sequence listing;

[0044] The amino acid sequence of Cca5208 is shown as SEQ ID NO.9 in the sequence listing;

[0045] The amino acid sequence of Cca9101 is shown as SEQ ID NO.10 in the sequence listing.

[0046] The target fragments of the coding genes of the above effector proteins Cca0022, Cca2944, Cca3101, Cca5208 and Cca9101 were ligated to the transient expression vector pYBA1133, transformed into competent Escherichia coli DH5α cells, spread on LA selective medium containing kanamycin (50 ng / mL), and cultured overnight at 37°C in an inverted position. Positive transformants were selected and verified by sequencing. After correct sequencing verification, positive recombinant Escherichia coli was obtained.

[0047] 100 μL of the bacterial liquid of positive recombinant Escherichia coli was inoculated into 20 mL of liquid LB medium and cultured overnight at 37°C with shaking at 220 rpm. The recombinant plasmid was extracted using a plasmid miniprep kit. The recombinant plasmid was transformed into competent Agrobacterium tumefaciens GV3101 cells and cultured with shaking at 37°C and 220 rpm for 48 h. The cells were collected by centrifugation (12000 rpm × 5 min), and resuspended with MMA solution (10 mM MgCl 2 , 10 mM MES, 0.15 mM AS) and adjusted to a concentration of OD600 = 1.0 to obtain a bacterial suspension. When Nicotiana benthamiana grew to the 4-5 leaf stage, the bacterial suspension was injected into the interior of the second and third true leaves counted from the bottom of Nicotiana benthamiana using a syringe without a needle. One injection point was set for each leaf, and the injection volume for each injection point was 200 μL. Transient expression of INF (infestans host-specific elicitor) protein was used as a positive control. After injection, it was cultured in the dark for 12 h and then normally cultured for 6 d. The changes at the injection point of Nicotiana benthamiana leaves were observed and photographed for recording.

[0048] The results showed that the tobacco leaves transiently expressing Cca2944, Cca3101, and Cca9101 exhibited varying degrees of local chlorosis, malformation, or cell necrosis, which were similar to the state of tobacco leaves transiently expressing INF protein (positive control), while the tobacco leaves transiently expressing effector proteins Cca0022 and Cca5208 did not show obvious changes ( Figure 2 ), indicating that the expression of effector proteins Cca0022 and Cca5208 in tobacco cells did not affect the normal growth of tobacco cells.

[0049] Example 5 Obtaining Recombinant Effector Proteins

[0050] By using the method of homologous recombination, the nucleotide sequences of effector proteins Cca0022 and Cca5208 were respectively constructed onto the prokaryotic expression vector pET-28a(+). The resulting recombinant plasmids were transformed into competent cells of Escherichia coli (DH5α), spread on LA plates containing kanamycin (50 ng / mL), and cultured overnight at 37°C in an inverted position. Positive transformants were selected and sent to a sequencing company for sequencing verification.

[0051] 100 μL of the positive recombinant Escherichia coli bacterial solution that had been verified correctly by sequencing was inoculated into 20 mL of liquid LB medium, and cultured overnight at 37°C with shaking at 220 rpm. The recombinant plasmid was extracted using a plasmid miniprep kit. The recombinant plasmid was transformed into competent BL21(DE3) cells for prokaryotic expression. After spreading on plates, it was cultured overnight at 37°C in an inverted position. Single colonies of positive transformants were picked and inoculated into 20 mL of LB medium, cultured overnight at 37°C and 220 rpm to prepare bacterial strains. The bacterial strains were inoculated into LB medium at a volume ratio of 1:100 and cultured at 220 rpm for 16 h. IPTG (final concentration 0.5 mM) was added to the fermentation broth to induce the expression of the fusion protein.

[0052] For the fermentation broth that had completed prokaryotic expression, the cells were collected by centrifugation (12000 rpm × 5 min). After lysing the cells, the target proteins were collected and purified; the concentrations of effector proteins Cca0022 and Cca5208 were measured using a BCA kit (Nanjing Jiancheng) and were 0.19 mg / mL and 0.24 mg / mL respectively; SDS PAGE gel electrophoresis combined with Western blot was used to detect the two prokaryotic expression proteins Cca0022 and Cca5208, and bands with sizes of approximately 10 KDa and 15 KDa (including the tag protein) were detected respectively, which were consistent with the expected protein sizes.

[0053] Example 6 Trapping Test of Effector Protein Cca0022 of Corynespora cassiicola on Bemisia tabaci

[0054] The effector protein Cca0022 solution obtained in Example 5 was diluted to 0.06 μmol / L and 0.12 μmol / L respectively with phosphate buffer solution.

[0055] A cubic insect rearing cage was made of 100-mesh insect-proof net and aluminum alloy frame, with a specification of 60.0 cm × 60.0 cm × 60.0 cm. A 100-mesh insect-proof net was used to make an insect release port (Φ = 2.5 cm) in the center of the top for releasing Bemisia tabaci, which was tied with a rope when not in use.

[0056] The experiment set up treatment groups and control groups.

[0057] Treatment group: Treated with effector protein Cca0022 solution; Control group: Treated with phosphate buffer solution.

[0058] The specific experimental process was as follows:

[0059] (1) Seed soaking and germination acceleration

[0060] The mature and plump tobacco seeds (NC89) were soaked in the effector protein Cca0022 solution with a concentration of 0.06 μmol / L for 8 h, and the temperature was maintained at 25 °C. Then the seeds were taken out and placed in a petri dish filled with moist filter paper, and the filter paper was wetted with the above protein solution of 0.06 μmol / L. Finally, the petri dish was placed in an incubator at 25 °C for germination acceleration under constant temperature and humidity conditions.

[0061] In the control group, the seeds were soaked with phosphate buffer solution, and the filter paper was wetted with phosphate buffer solution.

[0062] (2) Transplanting

[0063] After germination, the tobacco seeds were sown in a 75-hole seedling tray filled with seedling substrate and cultured in an intelligent greenhouse at 25 - 27 °C. When the plants grew to the three-leaf stage, the tobacco plants were transplanted into flower pots and cultured in an intelligent greenhouse at 25 - 27 °C.

[0064] (3) Spraying effector protein

[0065] 28 days after transplanting, a solution of effector protein Cca0022 with a concentration of 0.12 μmol / L was sprayed on the tobacco leaves, and 2 mL of the protein solution was sprayed on each plant.

[0066] The control group was sprayed with phosphate buffer solution.

[0067] (4) Releasing adult Bemisia tabaci

[0068] Twenty-four hours after spraying the effector protein solution or phosphate buffer on tobacco plants, 50 newly emerged adult Bemisia tabaci were released at the insect release port for both the treatment group and the control group. The number of B. tabaci landing on the tobacco plant leaves was checked 6 h, 12 h, and 24 h after releasing the adult B. tabaci. A total of 10 replicates were set up, with 3 tobacco plants in each replicate.

[0069] The formula for calculating the selection rate of B. tabaci for tobacco plants is: (the number of B. tabaci landing on the tobacco plant leaves / the total number of insects) × 100%;

[0070] The formula for calculating the trapping rate of the effector protein in the treatment group for B. tabaci is: (((the number of B. tabaci landing on the tobacco plant leaves in the treatment group - the number of B. tabaci landing on the tobacco plant leaves in the control group) / the number of B. tabaci landing on the tobacco plant leaves in the treatment group)) × 100%.

[0071] The results of the selection rate are as Figure 3 shown. B. tabaci preferred to feed on the tobacco plants in the treatment group. At 6 h, 12 h, and 24 h after releasing the adult B. tabaci, the selection rates of B. tabaci for the tobacco plants in the treatment group were 46.75%, 46%, and 50.75% respectively, which were significantly higher than the selection rates of the control group, 11.5%, 8.5%, and 6.5% ( P=0.000 ).

[0072] The trapping rates of the effector protein Cca0022 solution for B. tabaci were 75.14% (6 h), 81.58% (12 h), and 87.06% (24 h) respectively; it shows that the effector protein Cca0022 solution has the effect of trapping B. tabaci, and it can be combined with chemical control. Targeted spraying can be carried out on the trapped B. tabaci to reduce the usage amount of chemical agents, and at the same time achieve the effect of controlling B. tabaci on tobacco plants.

[0073] Example 7 Trapping test of the effector protein Cca5208 of Stemphylium lycopersici on Bemisia tabaci

[0074] The experiment was set up with a treatment group of the effector protein Cca5208 solution and a control group (treated with phosphate buffer).

[0075] The specific experimental process of the treatment group with the effector protein Cca5208 solution was the same as that of the treatment group with the effector protein Cca0022 solution in Example 6, and the only difference was that the effector protein Cca5208 solution was used to replace the effector protein Cca0022 solution.

[0076] The results are as Figure 4As shown, the selection rates of Bemisia tabaci for tobacco plants treated with the effector protein Cca5208 solution at 6 h, 12 h, and 24 h were 24.75%, 26.5%, and 24.75% respectively, while the selection rates of Bemisia tabaci for tobacco plants in the control group at 6 h, 12 h, and 24 h were 28.75%, 30.5%, and 27.5% respectively, and the differences were not significant ( P>0.05 ), indicating that the effector protein Cca5208 solution has no obvious trapping effect on Bemisia tabaci.

Claims

1. An application of an effector protein of tomato coryneformis target spot pathogen in pest trapping and prevention, characterized in that: The nucleotide sequence of the tomato coryneformis target spot pathogen effector protein is shown in SEQ ID NO.1 in the sequence list, and the amino acid sequence is shown in SEQ ID NO.6 in the sequence list; The pest is Bemisia tabaci.

2. The use of a tomato Corynespora target spot pathogen effector protein in pest trapping and prevention according to claim 1, characterized in that: The tomato coryneformis target spot pathogen is multi-homogeneous coryneformis.

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