Application of RNAi in improving the control of Chilo suppressalis

Abstract

The present invention relates to the application of silencing caspase5 gene in improving the effect of controlling insects. Silencing caspase5 gene in target insects can improve the control effect of Cry9A protein on the target insects.

Description

Technical Field

[0001] The invention relates to the field of biological control, and in particular to the application of silencing caspase5 gene in improving the effect of controlling insects. Background Art

[0002] Insecticidal proteins expressed by Bacillus thuringiensis (Bt) can effectively control target pests and reduce the use of chemical pesticides. However, the effect of using only insecticidal proteins to control target pests is limited. Summary of the invention

[0003] One aspect of the present invention provides the use of silencing caspase5 gene expression in target insects to improve the control effect on the target insects.

[0004] In a specific embodiment, the efficiency of silencing the expression of caspase5 gene in the target insect is above 60%.

[0005] In one embodiment, the target insect is a Lepidoptera insect.

[0006] In a specific embodiment, the target insect is an insect of the family Pyralidae.

[0007] In a specific embodiment, the target insect is Chilo suppressalis Walker.

[0008] In a specific embodiment, silencing the expression of caspase5 gene in the target insect is used to improve the control effect of Cry protein on the target insect.

[0009] In a specific embodiment, the Cry protein is Cry9Aa protein.

[0010] In a specific embodiment, the Cry protein is Cry9Aa3 protein, for example, its amino acid sequence is shown in Genbank No. ADE60735.1.

[0011] In a specific embodiment, the Cry protein is a Cry9Aa655 protein with an amino acid sequence such as SEQ ID No.11.

[0012] In a specific embodiment, the sequence of the positive strand in the dsRNA for silencing the expression of the caspase5 gene in the target insect is shown in Genbank No. GQ249293.1, or in SEQ ID No. 3. When used as RNA, t is replaced by u.

[0013] The second aspect of the present invention provides a combined drug, which includes dsRNA and Cry protein for silencing the expression of caspase5 gene in target insects. The dsRNA and Cry protein can be administered sequentially or simultaneously, preferably sequentially; preferably, the Cry protein is administered 48 hours after the dsRNA is administered.

[0014] In a specific embodiment, the sequence of the sense strand in the dsRNA is shown in Genbank No. GQ249293.1, or in SEQ ID No. 3. When used as RNA, t is replaced by u.

[0015] In a specific embodiment, the Cry protein is Cry9Aa protein.

[0016] In a specific embodiment, the Cry protein is Cry9Aa3 protein, for example, its amino acid sequence is shown in Genbank No. ADE60735.1.

[0017] In a specific embodiment, the Cry protein is a Cry9Aa655 protein with an amino acid sequence such as SEQ ID No.11.

[0018] Beneficial effects of the present invention:

[0019] The present invention discovered for the first time that silencing the expression of the Cscaspase5 gene of the Chilo suppressalis can significantly enhance the activity of the Cry9A protein against the Chilo suppressalis, which lays a foundation for the development of a new generation of biological insecticides and transgenic plants and enriches the gene resource library of insecticide engineering microorganisms in my country. DETAILED DESCRIPTION

[0020] The above contents of the present invention are further described in detail below in the form of preferred implementation cases, but they do not constitute a limitation to the present invention.

[0021] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased through commercial channels.

[0022] Example 1

[0023] 1. Cloning of Cscapase5 gene of Chilo suppressalis and preparation of dsCscapase5

[0024] According to the genome of the lepidopteran pest Chilo suppressalis Walker (NCBI Genomeassemly GCA_004000445.1), a primer pair for the full length of the caspase5 gene (Cscaspase5, Genbank No. GQ249293.1) of Chilo suppressalis Walker was designed, wherein the upstream primer was Cscaspase5F (SEQ ID No. 1) and the downstream primer was Cscaspase5R (SEQ ID No. 2).

[0025] Total RNA from the third instar larvae of Chilo suppressalis was extracted using the RNeasy Mini Kit (74106, QIAGEN), and reverse transcription was performed using a reverse transcription kit (R223-01, Nanjing Novozymes Biotechnology Co., Ltd.) to obtain cDNA. PCR was then performed using the cDNA as a template and Cscaspase5F and Cscaspase5R as primers. The PCR product was cloned into the pEASYBluntZero plasmid (Beijing Quanshijin Biotechnology Co., Ltd.) to obtain the pEASYBluntZero-Cscaspase5 positive plasmid containing the full-length Cscaspase5 gene.

[0026] On the SnapDragon-dsRNA Design website ( https: / / www.flyrnai.org / cgi-bin / RNAi_ find_primers.pl ) designed a dsRNA fragment for RNA interference (dsCscapase5, the base sequence of its positive strand is shown in SEQ ID No. 3). Then, T7 RiboMAX TM The Express RNAi System kit (P1700, Promega) used pEASYBluntZero-Cscaspase5 as a template and dsCscaspase5F (SEQ ID No. 4) and dsCscaspase5R (SEQ ID No. 5) as primers to synthesize the dsRNA fragment of Cscaspase5, which was finally dissolved in enzyme-free water to obtain a dsCscapase5 aqueous solution.

[0027] Comparative Example 1

[0028] Preparation of negative control dsGFP

[0029] The gfp DNA with the sequence shown as SEQ ID No.6 was synthesized by Shanghai Bioengineering Co., Ltd.

[0030] Utilizing T7 RiboMAX TMThe Express RNAi System kit (P1700, Promega) uses gfp DNA as a template and gfpF (SEQ ID No. 7) and gfpR (SEQ ID No. 8) as primers to synthesize a dsRNA fragment of gfp (dsgfp, the base sequence of its positive chain is shown in SEQ ID No. 6), which is finally dissolved in enzyme-free water to obtain a dsgfp aqueous solution.

[0031] Example 2

[0032] Preparation of Cry9Aa655 protein

[0033] 1. Protein Extraction

[0034] Using cry9Aa655-F (SEQ ID No. 9) and cry9Aa655-R (SEQ ID No. 10) as primers and the synthetic cry9Aa3 gene (Genbank No. GQ249293.1, its amino acid Genbank No. ADE60735.1) as a template, the DNA sequence of amino acids 1-655 (SEQ ID No. 11) of the cry9Aa3 gene was cloned and connected to the pEB expression vector to obtain pEB-cry9Aa655, and transformed into Escherichia coli BL21 (DE3) to obtain BL21 (DE3) / pEB-cry9Aa655. At the same time, pEB was transformed into Escherichia coli BL21 (DE3) without a vector to obtain BL21 (DE3) / pEB.

[0035] Escherichia coli BL21 (DE3) / pEB-cry9Aa655 was inoculated into 5 mL of LB liquid medium containing 1‰ ampicillin, and cultured at 37°C 220r / min for 8h to obtain an activated culture solution; 1% of the activated culture solution was inoculated into 300 mL of LB liquid medium containing 1‰ ampicillin, and cultured at 37°C 220r / min until OD 600=0.8; add IPTG to a final concentration of 0.5mmol / L, and then induce at 18℃ 150r / min for 14h to obtain an induced culture solution; centrifuge the induced culture at 12000g for 8min to collect the bacteria, and suspend the collected bacteria in binding buffer (50mM imidazole, 20mM Tris-HCl, 500mMNaCl, pH 8.0), wherein the ratio of binding buffer to bacteria is 7:100 based on the volume ratio of binding buffer to induction culture solution; ultrasonically disrupt the cells for 5min (ultrasonic power: 70%; ultrasonic: 3s, pause: 5s); centrifuge at 13000g for 15min, collect the supernatant, and analyze the supernatant by SDS-PAGE to confirm that the target protein Cry9Aa655 is successfully expressed (the BL21(DE3) / pEB supernatant obtained under the same conditions is used as a negative control).

[0036] 2. Affinity Purification

[0037] Add 20 mL of supernatant obtained in the previous step to 2 mL of nickel (Ni) affinity column material into the purification column; elute impurities with 5 column volumes of binding buffer; elute the target protein Cry9Aa655 with 10 mL of eluent II and collect; desalt and purify the collected Cry9Aa655 protein using a desalting column (HiPrep 26 / 10, Cytiva) in an AKTA-Avant protein purification system (Cytiva), wherein the buffer is 20 mmol / L Tris-HCl (pH 8.0); the flow rate is 8 mL / min, UV280 nm is detected, and the desalted protein solution is collected at the protein peak.

[0038] Example 3

[0039] Determination of biological activity against Chilo suppressalis

[0040] 1. Preparation of dsRNA-SPc Complex

[0041] The dsCscapase5 aqueous solution prepared in Example 1 was diluted with enzyme-free water to 5 concentrations; the dsCscapase5 aqueous solution at the 5 concentrations was mixed with the nanomaterial SPc at a mass ratio of dsCscapase5 to SPc of 1:1 to obtain a dsCscapase5-SPc solution; 1% of the total volume of the final solution (i.e., dsCscapase5-SPc complex) of fatty alcohol ether sodium sulfate was added to the dsCscapase5-SPc solution, mixed evenly, and incubated at room temperature for 15 minutes to obtain a dsCscapase5-SPc complex. Among them, the preparation of the nanomaterial SPc is prepared according to A facile-synthesized star polycation constructed as a highly efficient gene vector in pest management (Li, J., Qian, J., Xu, Y., Yan, S., Shen, J., and Yin, M., ACS Sustainable Chem. Eng. 7, 6316-6322, 2019.).

[0042] The dsGFP-SPc complex was prepared in the same manner as a negative control.

[0043] 2. Determination of silencing efficiency of dsCscaspase5

[0044] Artificial feed formula for Chilo suppressalis: 40.0g soybean meal, 30.0g yeast powder, 15.0g casein, 12.0g sucrose, 145.0g fresh wild rice stem, 18.0g agar, 4.5g sodium ascorbate (Vc), 0.25g cholesterol, 0.40g choline chloride, 0.15g Wilkinson salt, 0.020g vitamin B complex, 1.50g sorbic acid, 1.50g methyl parahydroxybenzoate, 0.6ml 40% formaldehyde, 731.08ml water. The formula of vitamin B complex is as follows: nicotinamide 0.0060g, thiamine hydrochloride (VB1) 0.0016g, riboflavin (VB2) 0.0030g, pyridoxine hydrochloride (VB6) 0.0016g, lysine inositol (VB12) 0.00004g, folic acid 0.0016g, calcium pantothenate 0.0060g, biotin 0.00016g. The preparation process thereof is shown in Example 1 of CN200910080336.2.

[0045] Use ultrapure water to dilute the dsCscapase5-SPc complex and dsgfp-SPc complex to 333 μL, respectively, and then add them to 1 g of artificial feed and mix them evenly, so that the concentrations of dsCscapase5 in the artificial feed are 6.25 μg / g, 12.5 μg / g, 25 μg / g, 50 μg / g and 100 μg / g, respectively, and the concentration of dsgfp in the artificial feed is 100 μg / g. The evenly mixed artificial feed is divided into finger-shaped tubes, each of which is inoculated with 15 newly hatched larvae of Chilo suppressalis, and each treatment group is repeated 3 times. After feeding for 48 hours, RNA of the larvae of Chilo suppressalis was extracted by the same operation as in Example 1, and reverse transcribed to obtain cDNA, and the obtained cDNA was used as a template, Q-Cscaspase5-F (SEQ ID No. 12) and Q-Cscaspase5-R (SEQ ID No. 13) were used as primers to perform qRCR with a qRCR kit (Q131-02, Nanjing Novozyme Biotechnology Co., Ltd.) to detect the RNA interference efficiency. The preparation system of qRCR is shown in Table 1.

[0046] Table 1

[0047]

[0048] QuantStudio 6Flex System (ABI) was used for detection, and the amplification conditions were: 95°C pre-denaturation for 5 min, 95°C for 10 s, 60°C for 30 s, 40 cycles, and melting curve analysis was performed. According to the 2-△△Ct method, the relative expression of the Cscaspase5 gene after dsCscaspase5 treatment was calculated according to the Ct value compared with the dsgfp control group, and the silencing efficiency (or interference efficiency) was calculated based on the relative expression. The results are shown in Table 2. Wherein, silencing efficiency = (1-relative expression) × 100%.

[0049] Table 2

[0050]

[0051] Note: Different lowercase letters after the data in the same column indicate significant differences at the P<0.001 level.

[0052] The results in Table 2 show that when the concentration of dsCscaspase5 was greater than 25 μg / g, the silencing efficiency reached more than 60%. Considering the interference efficiency and the use cost, 25 μg / g dsRNA was finally selected as the concentration for subsequent experiments.

[0053] 3. Bioactivity Assay of dsCscaspase5 and Cry9A Combined Use

[0054] 1) dsCscaspase5-Cry9A treatment: Chilo suppressalis larvae interfered with 25 μg / g dsCscaspase5 for 48 h (same operation as in Section 2 above) were transferred into finger-shaped tubes containing artificial diet with a Cry9Aa655 protein content of 200 μg / g, with 32 larvae in each tube, and repeated 3 times. The finger-shaped tubes were placed in an incubator with a temperature of 27±2°C, a humidity of 70±5%, and a photoperiod of 14:10 h, and the mortality rate was counted when the larvae were fed with the artificial diet containing Cry9Aa655 protein for 7 days.

[0055] 2) dsgfp-Cry9A treatment: Chilo suppressalis larvae interfered with 25 μg / g dsgfp for 48 h (same operation as in Section 2 above) were transferred into finger-shaped tubes filled with artificial diet containing 200 μg / g Cry9Aa655 protein, with 32 larvae in each tube, and repeated 3 times. The finger-shaped tubes were placed in an incubator at a temperature of 27±2°C, a humidity of 70±5%, and a photoperiod of 14:10 h, and the mortality rate was counted after feeding with artificial diet for 7 days.

[0056] 3) dsCscaspase5 treatment: Chilo suppressalis larvae interfered with 25 μg / g dsCscaspase5 for 48 h (same operation as in Section 2 above) were transferred into finger-shaped tubes filled with artificial diet, with 32 larvae in each tube, and repeated 3 times. The finger-shaped tubes were placed in an incubator at a temperature of 27±2°C, a humidity of 70±5%, and a 14:10 h photoperiod, and the mortality rate was counted after feeding with artificial diet for 7 days.

[0057] 4) dsgfp treatment: The fall armyworm larvae that had been interfered with 25 μg / g dsgfp for 48 h (the same operation as in Section 2 above) were transferred into finger-shaped tubes filled with artificial diet, with 32 larvae in each tube, and repeated 3 times. The finger-shaped tubes were placed in an incubator at a temperature of 27±2°C, a humidity of 70±5%, and a 14:10 h photoperiod, and the mortality rate was counted after feeding with artificial diet for 7 days.

[0058] The results are shown in Table 3.

[0059] The results in Table 3 show that the mortality rate of larvae treated with dsCscaspase5-Cry9A was 48.23%, significantly higher than 15.38% of larvae treated with dsgfp-Cry9A; in addition, there was no significant difference in the mortality rate of larvae of the striped stem borer treated with dsCscaspase5 and the negative control dsgfp. The above results show that although silencing the Cscaspase5 gene alone cannot kill the striped stem borer, that is, silencing the Cscaspase5 gene has no insecticidal activity against the striped stem borer; however, silencing the Cscaspase5 gene of the striped stem borer can significantly improve the insecticidal effect of the Cry9Aa655 protein against the striped stem borer.

[0060] Table 3

[0061]

[0062] Note: Different lowercase letters after the data in the same column indicate significant differences at the P<0.001 level.

Claims

1. Use of dsRNA for silencing the expression of caspase5 gene in target insects to improve the control effect of Cry9Aa protein on the target insects, wherein the target insects are at least one of the Pyralidae insects.

2. The use according to claim 1, characterized in that: The efficiency of silencing the expression of caspase5 gene in the target insect is above 60%.

3. The use according to claim 1, characterized in that: The target insect is Chilo suppressalis Walker.

4. The use according to claim 1, characterized in that: The amino acid sequence of the Cry9Aa protein is shown in Genbank No. ADE60735.

1.

5. The use according to claim 1, characterized in that: The Cry9Aa protein is the Cry9Aa655 protein with an amino acid sequence such as SEQ ID No.

11.

6. The use according to claim 1, characterized in that: The sequence of the sense strand in the dsRNA for silencing the expression of the caspase5 gene in the target insect is shown in Genbank No. GQ249293.1, or in SEQ ID No.

3.

7. A combined drug comprising dsRNA and Cry9Aa protein for silencing the expression of caspase5 gene in target insects, wherein the target insects are the target insects in the use according to any one of claims 1 to 6.

8. The combined drug according to claim 7, characterized in that: The sequence of the sense strand in the dsRNA is shown in Genbank No. GQ249293.1, or in SEQ ID No.

3.

9. The combined drug according to claim 7, characterized in that: The amino acid sequence of the Cry9Aa protein is shown in Genbank No. ADE60735.

1.

10. The combined drug according to claim 7, characterized in that: The Cry9Aa protein is the Cry9Aa655 protein with an amino acid sequence such as SEQ ID No.11.

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