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Bemisia tabaci lethal gene and application thereof, RNA interference agent and preparation method and application of interference agent

A technology of RNA interference and whitefly, applied in the field of genetic engineering, can solve the problems of less whitefly and scarcity of applications, and achieve the effect of reducing viral diseases

Active Publication Date: 2021-12-28
金华市暖田农业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are many control measures for whitefly, the application of RNAi (RNA interference) technology is still scarce, especially there are too few targets for whitefly. The target is imminent

Method used

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  • Bemisia tabaci lethal gene and application thereof, RNA interference agent and preparation method and application of interference agent
  • Bemisia tabaci lethal gene and application thereof, RNA interference agent and preparation method and application of interference agent
  • Bemisia tabaci lethal gene and application thereof, RNA interference agent and preparation method and application of interference agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] A whitefly lethal gene, specifically the coding gene Aspartateaminotransferase of aspartate aminotransferase, its gene sequence is shown in SEQ ID NO.1, specifically:

[0037]ATGACCTCTTCGGTTTTCTCCTCCATTGAGCCTGGAATACTGAACGAAATATGCGCATTGCACGAGGAATTTTGCAGCGATCCTTACAGGAAGAAGGTGGATCTTGTTAAAGGAGCTTATCCGACGGACGAAGGAAAACCATGGGTTTTGCCTGTGGTCAGGAAAACTGAAATCATTCTGGCAAACGATGAAAATACCTTTCATGAGTCTGAACATTTTCTGAGCTCCAGCGAGTTTACTGATGCAGCCACTTCCTCACTACTGGGCGAAAAATCCATCGCCGTTCAAGACAAGAGGAAGAGTTTGGGACGCTCACACTTTTACTTATCTGACCCCCATTGGAACGCCTACGAAGTCATAATCATGCAAGCAGGTTTCGAGAAAAGCTCTCGTTACCGATATTGGAATGCGGAGAAACAATGCATTGACATGGATGGACTTTTGGAGGATTTGTCAAAGGCAGAGCCAACTTCGGTCGTTTTACTCCAAGCGTGTGCGCATAACCCGACTGGGTGTGATCCTACCCCCGAACAGTGGGCAAAAATAGCTTCTCTCATGAAGAAAAATAGATTGTTCCCATTCTTCGACATAGCTTATCAAGGTTTGGCGTCTGGTGATATGGACGAAGATGCAGAGGCTGTTCGCTATTTCGTTGACCAGGGTTTCGAGTTACTGTGCGCGCAATCTTTTTCTAAGAACTTTGGTCTTTACGGTGAACGAGTAGGAAGCCTCACTATTGTCTCAAATTGTGCCCATACAATGCCTCATATAGTATCCAAGATGATTAACATCGCTGAGGGCAATTATCTGGT...

Embodiment 2

[0063] A kind of RNA interfering agent of the present invention, dsRNA is synthesized by the Aspartate aminotransferase gene fragment of embodiment 1, and the DNA sequence of RNA interfering agent is as shown in SEQ ID NO.4, is that the target DNA fragment of embodiment 1 is synthesized kit with PROMEGAdsRNA Carrying out the synthesis of dsRNA to obtain the dsRNA containing the Bemisia tabaci lethal gene fragment Aspartateaminotransferase, specifically:

[0064] CGAGUUUACUGAUGCAGCCACUUCCUCACUACUGGGCGAAAAAUCCAUCGCCGUUCAAGACAAGAGGAAGAGUUUGGGACGCUCACACUUUUACUUAUCUGACCCCCAUUGGAACGCCUACGAAGUCAUAAUCAUGCAAGCAGGUUUCGAGAAAAGCUCUCGUUACCGAUAUUGGAAUGCGGAGAAACAAUGCAUUGACAUGGAUGGACUUUUGGAGGAUUUGUCAAAGGCAGAGCCAACUUCGGUCGUUUUACUCCAAGCGUGUGCGCAUAACCCGACUGGGUGUGAUCCUACCCCCGAACAGUGGGCAAAAAUAGCUUCUCUCAUGAAGAAAAAUAGAUUGUUCCCAUUCUUCGACAUAGCUUAUCAAGGUUUGGCGUCUGGUGAUAUGGACGAAGAUGCAGAGGCUGUUCGCUAU。

[0065] Its preparation method comprises the following steps:

[0066] (1) Collect 50-60 Bemisia tabaci...

Embodiment 3

[0086] A kind of RNA interfering agent of embodiment 2 is applied in reducing whitefly gene expression, and its application method comprises the following steps:

[0087] (1) Prepare 15% sucrose solution (water as solvent), fully dissolve, filter with 0.22 μm bacterial filter, and then mix with different volumes of AST dsRNA to obtain 400 ng / μL feeding nutrient solution.

[0088] (2) Add the feeding nutrient solution to the sealing film, and then cover the droplet with the sealing film to remove air bubbles as much as possible to form a pouch containing the feeding nutrient solution. Bemisia tabaci were placed in a glass tube with the pouch containing the feeding nutrient solution facing the light source.

[0089] (3) Put a black cotton plug on it, wrap it with a light-shielding sleeve that can cover the glass tube, and place it in an incubator. The required culture conditions are a light ratio of 14:10 and a humidity of 80%.

[0090] (4) After feeding for 48 h, calculate the...

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Abstract

The invention discloses a bemisia tabaci lethal gene and application thereof, an RNA interference agent and a preparation method and application of the RNA interference agent. The bemisia tabaci lethal gene is a coding gene of asparaginic acid transferase. The bemisia tabaci lethal gene is related to the gene expression quantity and activity of asparaginic acid transferase, can catalyze the transamination reaction, transfers amino from asparaginic acid to alpha-ketoglutaric acid, and can be applied to killing bemisia tabaci by destroying the gene. The RNA interference agent is dsRNA synthesized according to a bemisia tabaci lethal gene, and the preparation method of the RNA interference agent comprises the following steps: synthesizing a cDNA first chain, and carrying out RT-PCR amplification; and synthesizing the dsRNA. The RNA interference agent provided by the invention can reduce the relative gene expression quantity of AST, thereby reducing the ability of bemisia tabaci to metabolize absorbed amino acids, affecting the digestion and absorption of bemisia tabaci to amino acids, and affecting the growth and development of bemisia tabaci, thereby effectively controlling the population growth of bemisia tabaci, and further reducing the propagation of tomato chlorotic virus diseases.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a bemisia tabaci lethal gene and its application, an RNA interference agent and its preparation method and application. Background technique [0002] In recent years, the harm of tomato chlorosis virus has not been significantly reduced, but the control of whitefly is still difficult. Physical prevention and control mainly includes strengthening field management, cleaning up weeds and poisonous seedlings in the field in time, setting up insect-proof nets to prevent the invasion of whitefly, hanging sticky boards to trap and kill whitefly, etc. Chemical control mainly involves spraying insecticides such as 5% buprofezin (Petidyl) wettable powder, 10% imidacloprid wettable powder, and 25% thiamethoxam water (Actay) dispersant. Although there are many control measures for whitefly, the application of RNAi (RNA interference) technology is still scarce, especially there a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/113A01N57/16A01P7/04
CPCC12N9/1096C12N15/1137A01N57/16C12Y206/01001C12N2310/14C12N2320/30
Inventor 史晓斌卢丁伊慧刘勇张德咏张战泓张卓谭新球郑立敏高阳彭静
Owner 金华市暖田农业有限公司
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