Application of tomato corynespora leaf spot pathogen effect protein in pest trapping, prevention and control

Abstract

The invention provides an application of a tomato corynespora leaf spot pathogen effect protein in pest trapping, prevention and control, and belongs to the technical field of genetic engineering. The nucleotide sequence of the tomato corynespora parvata effect protein is as shown in SEQ ID NO.1 in a sequence table, and the amino acid sequence of the tomato corynespora parvata effect protein is as shown in SEQ ID NO.6 in the sequence table. After tobacco seeds and plants are treated by a solution of the effect protein Cca0022, the effect protein Cca0022 has a trapping effect on bemisia tabaci and can be combined with chemical prevention and control, fixed-point pesticide spraying is carried out on the trapped bemisia tabaci, the use amount of chemical agents is reduced, and meanwhile the effect of preventing and controlling the bemisia tabaci of the tobacco plants is achieved.

Description

Technical Field

[0001] The invention relates to application of an effector protein of tomato coryneformis target spot pathogen in pest trapping and prevention, and belongs to the technical field of genetic engineering. Background Art

[0002] Bemisia tabaci Bemisiatabaci ) is also known as sweet potato whitefly, cotton whitefly, etc. It belongs to the genus Aleyrodidae of the Hemiptera order. It was first discovered and named on tobacco in Greece in 1889. It can harm more than 600 species of plants and is a highly omnivorous global pest. It is called a "super pest" because of its wide host range, strong adaptability to the environment, and strong resistance to chemical pesticides. In agricultural production, chemical agents are mainly used to prevent and control whiteflies. Through testing, it is found that whiteflies have developed varying degrees of resistance to pesticides such as organophosphorus, nicotine, and insecticide polyesters, and the difficulty of prevention and control is gradually increasing. In addition, most growers use pesticides in an irregular manner, and there are behaviors such as untimely application of pesticides, high concentration of pesticides, inaccurate use of pesticides, mixing of multiple pesticides, and frequent application of pesticides, which leads to the frequent occurrence of "insects are difficult to prevent and drugs are useless". The long-term, large-scale and irrational use of chemical pesticides has led to increasingly serious problems such as pest resistance and pesticide residues, which has become a bottleneck restricting the further development of the facility vegetable industry. How to open up new prevention and control paths has become the research focus of scientific researchers. Protein-based biomaterials are a hot spot for innovative research and development and application of green pest control because of their multiple advantages such as high safety and mass production.

[0003] There is no report in the prior art on using effector proteins to attract whiteflies. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides an application of a tomato Corynespora target spot pathogen effector protein in pest attraction and control, to achieve the following invention objectives: the screened tomato Corynespora target spot pathogen effector protein has an obvious attraction effect on Bemisia tabaci.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions: An application of a tomato Corynespora target spot pathogen effector protein in pest trapping and control, wherein the nucleotide sequence of the tomato Corynespora 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.

[0006] The pest is Bemisia tabaci.

[0007] The tomato coryneformis target spot pathogen is multi-homogeneous coryneformis.

[0008] The application of the tomato Coryneformis target spot pathogen effector protein in the trapping and prevention of tobacco whiteflies.

[0009] The present invention screened out 16 candidate effector proteins from the reference genome of the polyhomocybin strain, and then screened 5 candidate effector proteins whose expression was upregulated at both 24h and 48h after induction in tomato leaves, and further screened out 2 effector proteins that had little effect on the normal growth of Nicotiana benthamiana leaves, and finally screened out the effector protein Cca0022 that had an attracting effect on Bemisia tabaci.

[0010] Compared with the prior art, the present invention achieves the following beneficial effects: After tobacco seeds and plants are treated with the effector protein Cca0022 solution of the present invention, it has an attracting effect on whiteflies, and can be combined with chemical control to perform targeted spraying on the attracted whiteflies, thereby reducing the use of chemical agents and achieving the effect of controlling whiteflies on tobacco plants.

[0011] After the effector protein Cca0022 solution of the present invention was sprayed on tobacco plants for 24 hours, Bemisia tabaci was released, and the trapping rates of Bemisia tabaci were 75.14% (6 hours after the release of Bemisia tabaci), 81.58% (12 hours after the release of Bemisia tabaci), and 87.06% (24 hours after the release of Bemisia tabaci). BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a heat map of the expression of candidate effector proteins in Coryneformis polytomata Cca105; Figure 2 This is a diagram of the growth status of tobacco leaves transiently expressing different effector proteins; Figure 3 It is a bar graph of the selection rate of tobacco whiteflies on tobacco plants treated with effector protein Cca0022 solution; Figure 4 The bar graph shows the selection rate of tobacco whitefly on tobacco plants treated with effector protein Cca5208 solution. DETAILED DESCRIPTION

[0013] The tomato variety used in the test was Jinpeng No. 1, a tomato variety susceptible to Corynosporium target spot disease; The target pathogen of tomato Coryneformis spore was Coryneformis multifiliis Cca105, which was maintained by the Green Control Laboratory of Vegetable Pests and Diseases of Weifang University of Science and Technology.

[0014] Example 1 Pathogenicity test of polymyxin Cca105 After activating and culturing the multi-main coryneform spore Cca105 on PDA medium, the mycelium was scraped and inoculated into PDA liquid medium, and cultured in a shaking incubator at 25°C and 160rpm for 5 days to obtain mycelial balls. For the Jinpeng No. 1 tomato with 6 true leaves, the second and third true leaves from the bottom were taken, and mycelial balls of multi-main coryneform spore Cca105 with a diameter of 2mm were inoculated on the back of the leaves. Four inoculation points were set for each true leaf, and one mycelial ball was inoculated at each inoculation point. Ten tomato leaves were inoculated each time, and the experiment was repeated 3 times. Six days after inoculation, the disease incidence of tomato leaves was observed. The leaves of tomatoes produced light brown to dark brown spots, and the green around the spots faded, showing a yellow halo. The percentage of the number of spots to the total number of inoculation points was counted and calculated. The occurrence of spots at the inoculation points on the leaflets was considered to be the disease, and the incidence rate was 83.3%.

[0015] Example 2 Analysis and prediction of candidate effector proteins The genome sequencing information of the multi-homocybin strain published by the National Center for Biotechnology Information (NCBI) of the United States (https: / / www.ncbi.nlm.nih.gov / nuccore / NSJI00000000) was used as the reference genome, with the version number NSJI00000000.1; bioinformatics methods were used to screen candidate effector proteins.

[0016] According to the criteria of having a signal peptide, a size not exceeding 300AA, a transmembrane structure ≤1, no anchor site, subcellular localization outside the cell, an unknown functional protein with ≥6 cysteine ​​residues in the protein, and an effector protein prediction score ≥0.8, a total of 16 candidate effector proteins were screened from the reference genome of the multi-main coryneform strain (see Table 1), and they were named Cca9079, Cca9097, Cca9101, Cca0022, Cca0909, Cca1956, Cca3101, Cca5047, Cca4034, Cca6409, Cca6434, Cca2944, Cca3771, Cca5208, Cca9104 and Cca0129.

[0017] Table 1 Candidate effector proteins screened from the genomes of multiple main Coryneformis strains

[0018] Example 3 Screening of candidate effector proteins whose expression is upregulated after tomato induction Fluorescence quantitative PCR was used to detect the expression levels of 16 candidate effector proteins of tomato Coryneformis target spot pathogen.

[0019] After activating and culturing the Cca105 strain of Polymyces sporei on a PDA solid plate, about 0.1 g of mycelium was scraped and inoculated into 100 mL of liquid PDA culture medium, and cultured in a shaking incubator at 25°C and 160 rpm for 5 days to obtain mycelium balls with a diameter of 2-3 mm.

[0020] Inoculate mycelial pellets with a diameter of 2-3 mm on the 2nd to 4th true leaves of tomatoes in the 5-6 leaf stage. Spread the mycelial pellets evenly on the tomato leaves so that each mycelial pellet touches the leaves. Collect samples together with the leaves and mycelial pellets 24h and 48h after inoculation as test samples, and use the uninoculated mycelial pellets (before inoculation) as the control sample.

[0021] The total RNA of the sample was extracted using the TransZol total RNA extraction kit from Beijing Quanshijin Biotechnology Co., Ltd., and reverse transcription was performed to obtain cDNA. The obtained cDNA was used as a template for fluorescence quantitative PCR detection, and the relative expression levels of the effector proteins in the multi-primary coryneformis Cca105 after 24h and 48h of induction in tomato leaves were calculated. The heat map results of the relative expression levels of genes are shown in Figure 1 Among these 16 candidate effector proteins, 5 effector proteins were upregulated at both 24h and 48h after inoculation, namely Cca0022, Cca2944, Cca3101, Cca5208 and Cca9101.

[0022] Example 4 Screening for candidate effector proteins that have little effect on the normal growth of Nicotiana benthamiana leaves Using Nicotiana benthamiana as the experimental material, an Agrobacterium-mediated tobacco transient expression experiment was carried out to observe the effects of effector proteins on plant cell growth.

[0023] The specific method is as follows: Total RNA of Cca105 of Polymyxin was extracted and reverse transcribed to obtain cDNA. Using cDNA as template, PCR amplification was performed to clone the target fragments of the coding genes of effector proteins Cca0022, Cca2944, Cca3101, Cca5208 and Cca9101; The nucleotide sequence of Cca0022 is shown in SEQ ID NO.1 in the sequence list; The nucleotide sequence of Cca2944 is shown in SEQ ID NO.2 in the sequence list; The nucleotide sequence of Cca3101 is shown in SEQ ID NO.3 in the sequence list; The nucleotide sequence of Cca5208 is shown in SEQ ID NO.4 in the sequence list; The nucleotide sequence of Cca9101 is shown in SEQ ID NO.5 in the sequence list; The amino acid sequence of Cca0022 is shown in SEQ ID NO.6 in the sequence listing; The amino acid sequence of Cca2944 is shown in SEQ ID NO.7 in the sequence listing; The amino acid sequence of Cca3101 is shown in SEQ ID NO.8 in the sequence listing; The amino acid sequence of Cca5208 is shown in SEQ ID NO.9 in the sequence listing; The amino acid sequence of Cca9101 is shown in SEQ ID NO.10 in the sequence listing.

[0024] The target fragments of the genes encoding the above-mentioned effector proteins Cca0022, Cca2944, Cca3101, Cca5208 and Cca9101 were connected to the transient expression vector pYBA1133, transformed into Escherichia coli DH5α competent cells, spread on LA selective medium containing kanamycin (50 ng / mL), and cultured inverted at 37°C overnight. The positive transformants were selected and sequenced to verify that they were correct. After sequencing verification, positive recombinant Escherichia coli were obtained.

[0025] Take 100 μL of the positive recombinant Escherichia coli and inoculate it into 20 mL of liquid LB medium, shake and culture it at 37°C and 220 rpm overnight, and extract the recombinant plasmid using a plasmid extraction kit. Transform the recombinant plasmid into Agrobacterium GV3101 competent cells, shake and culture it at 37°C and 220 rpm for 48 hours, collect the bacteria by centrifugation (12000 rpm×5 min), resuspend the bacteria with MMA solution (10 mM MgCl2, 10 mM MES, 0.15 mM AS) and adjust the concentration to OD600=1.0 to obtain a bacterial suspension. When N. benthamiana grows to the 4-5 leaf stage, use a syringe with the needle removed to inject the bacterial suspension into the second and third true leaves of N. benthamiana from the bottom, set one injection point for each leaf, and the injection volume for each injection point is 200 μL. Transiently expressed INF (infestans host-specific elicitor) protein is used as a positive control. After injection, the leaves were cultured in dark for 12 hours and then cultured normally for 6 days. The changes of the injection points on the Nicotiana benthamiana leaves were observed and photographed for record.

[0026] The results showed that tobacco leaves transiently expressing Cca2944, Cca3101, and Cca9101 showed different degrees of localized yellowing, deformity, or cell necrosis, which was similar to the state of tobacco leaves transiently expressing INF protein (positive control), while tobacco leaves transiently expressing effector proteins Cca0022 and Cca5208 showed no obvious changes ( Figure 2), indicating that the expression of effector proteins Cca0022 and Cca5208 in tobacco cells does not affect the normal growth of tobacco cells.

[0027] Example 5 Obtaining recombinant effector protein The nucleotide sequences of effector proteins Cca0022 and Cca5208 were constructed into the prokaryotic expression vector pET-28a(+) by homologous recombination. The obtained recombinant plasmids were transformed into competent Escherichia coli (DH5α) cells, spread on LA plates containing kanamycin (50 ng / mL), and cultured inverted at 37°C overnight. The positive transformants were selected and sent to a sequencing company for sequencing verification.

[0028] 100 μL of the positive recombinant E. coli culture verified by sequencing was inoculated into 20 mL of liquid LB medium, and cultured overnight at 37°C and 220 rpm. The recombinant plasmid was extracted using a plasmid extraction kit. The recombinant plasmid was transformed into BL21 (DE3) competent cells for prokaryotic expression, and cultured overnight at 37°C after plating. A single colony of the positive transformant was picked and inoculated into 20 mL of LB medium, and cultured overnight at 37°C and 220 rpm to prepare the strain. The strain was inoculated into LB medium at a volume ratio of 1:100, and cultured at 220 rpm for 16 h. IPTG (final concentration of 0.5 mM) was added to the fermentation broth to induce the expression of the fusion protein.

[0029] After the prokaryotic expression of the fermentation broth was completed, the bacteria were collected by centrifugation (12000rpm×5min), and after the cells were lysed, the target protein was collected and purified; the concentrations of the effector proteins Cca0022 and Cca5208 were determined using a BCA kit (Nanjing Jiancheng), which were 0.19mg / mL and 0.24mg / mL, respectively; SDS PAGE gel electrophoresis combined with Western blot was used to detect the two prokaryotic expression proteins Cca0022 and Cca5208, and bands of approximately 10 KDa and 15 KDa (including tagged proteins) were detected, respectively, which were consistent with the expected protein size.

[0030] Example 6 Trial on the attraction of the tomato Coryneformis target spot pathogen Cca0022 to Bemisia tabaci The effector protein Cca0022 solution obtained in Example 5 was diluted to 0.06 μmol / L and 0.12 μmol / L respectively using phosphate buffer; A square insect cage with a size of 60.0 cm × 60.0 cm × 60.0 cm was made of 100-mesh insect-proof net and an aluminum alloy frame. An insect release port (Φ=2.5 cm) was made of 100-mesh insect-proof net in the center of the top to release whiteflies. It was tied with a rope when not in use.

[0031] The experiment was set up with treatment group and control group.

[0032] The treatment group was treated with effector protein Cca0022 solution, and the control group was treated with phosphate buffer solution.

[0033] The specific experimental process is: (1) Seed soaking and germination Soak mature and plump tobacco seeds (NC89) in a 0.06 μmol / L solution of the effector protein Cca0022 for 8 hours at 25°C. Then take out the seeds and place them in a culture dish filled with moist filter paper, which is moistened with the above protein solution at 0.06 μmol / L. Finally, place the culture dish in a 25°C constant temperature box and germinate under constant temperature and moisture conditions.

[0034] In the control group, seeds were soaked with phosphate buffer and filter paper was moistened with phosphate buffer.

[0035] (2) Colonization After germination, sow the tobacco seeds in a 75-hole seedling tray filled with seedling medium and culture them in an intelligent greenhouse at 25-27℃. When the plants grow to the 3-leaf stage, plant them in pots. After planting, culture the tobacco plants in an intelligent greenhouse at 25-27℃.

[0036] (3) Spraying effector protein 28 days after planting, a solution of the effect protein Cca0022 with a concentration of 0.12 μmol / L was sprayed on the tobacco leaves, with 2 mL of the protein solution sprayed on each plant.

[0037] The control group was sprayed with phosphate buffer.

[0038] (4) Release of adult whiteflies 24 hours after the tobacco plants were sprayed with the effect protein solution or phosphate buffer, 50 newly emerged whiteflies were released from the insect release port for the treatment group and the control group, and the number of whiteflies landing on the tobacco plant leaves was checked 6 hours, 12 hours and 24 hours after the release of the whiteflies. A total of 10 replicates were set, with 3 tobacco plants in each replicate.

[0039] The formula for calculating the selection rate of tobacco whiteflies on tobacco plants is: (number of whiteflies landing on tobacco plant leaves / total number of insects) × 100%; The calculation formula for the attraction rate of the effector protein in the treatment group for whiteflies is: ((the number of whiteflies landing on the leaves of tobacco plants in the treatment group - the number of whiteflies landing on the leaves of tobacco plants in the control group) / the number of whiteflies landing on the leaves of tobacco plants in the treatment group)) × 100%.

[0040] The selection rate results are as follows Figure 3As shown in the figure, whiteflies were more inclined to feed on the tobacco plants in the treatment group. At 6h, 12h and 24h after the release of whitefly adults, the selection rates of whiteflies on the tobacco plants in the treatment group were 46.75%, 46% and 50.75%, respectively, which were significantly higher than the selection rates of 11.5%, 8.5% and 6.5% in the control group ( P=0.000 ).

[0041] The attraction rates of the effector protein Cca0022 solution on whiteflies were 75.14% (6h), 81.58% (12h) and 87.06% (24h), respectively; this indicates that the effector protein Cca0022 solution has the function of attracting whiteflies and can be combined with chemical control to carry out targeted spraying on the attracted whiteflies, thereby reducing the use of chemical agents and achieving the effect of controlling whiteflies on tobacco plants.

[0042] Example 7 Trial on the attraction of tomato gray leaf spot pathogenic fungus effector protein Cca5208 to Bemisia tabaci The experiment was set up as an effector protein Cca5208 solution-treated group and a control group (phosphate buffer-treated).

[0043] The specific experimental process of the effector protein Cca5208 solution treatment group was the same as that of the effector protein Cca0022 solution treatment group in Example 6, with the only difference being that the effector protein Cca5208 solution was used to replace the effector protein Cca0022 solution.

[0044] The results are as follows Figure 4 As shown, the selection rates of tobacco plants treated with the effector protein Cca5208 solution by whiteflies at 6h, 12h and 24h were 24.75%, 26.5% and 24.75%, respectively, while the selection rates of tobacco plants in the control group at 6h, 12h and 24h were 28.75%, 30.5% and 27.5%, respectively, with no significant difference ( P>0.05 ), indicating that the effector protein Cca5208 solution has no obvious attracting effect on whiteflies.

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.

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 pest is Bemisia tabaci.

3. 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.

4. The use of a tomato Corynespora target spot pathogen effector protein in pest trapping and prevention according to claim 1, characterized in that: The application of the tomato Coryneformis target spot pathogen effector protein in the trapping and prevention of tobacco whiteflies.

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