Application of nilaparvata lugens NCS2 gene or encoded protein as target spot in preparation of medicine for preventing and treating nilaparvata lugens

A brown planthopper, gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of aggravating environmental pollution and stimulating the reproduction of pests, and achieve the effects of inhibiting reproduction, inhibiting individual development, inhibiting growth and reproduction.

Pending Publication Date: 2022-07-29
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The application of insecticides not only kills the natural enemies of brown planthoppers, but also stimulates the reproduction of pests, causing the population to become rampant. At the same time, it also aggravates environmental pollution and brings risks to the quality and safety of agricultural products.

Method used

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  • Application of nilaparvata lugens NCS2 gene or encoded protein as target spot in preparation of medicine for preventing and treating nilaparvata lugens
  • Application of nilaparvata lugens NCS2 gene or encoded protein as target spot in preparation of medicine for preventing and treating nilaparvata lugens
  • Application of nilaparvata lugens NCS2 gene or encoded protein as target spot in preparation of medicine for preventing and treating nilaparvata lugens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Rice pest brown planthopper NCS2 gene.

[0027] Step 1: Extraction of total RNA from N. lugens and cloning of NCS2 gene

[0028] The adult N. lugens (100 mg) was placed in a grinding tube, an appropriate amount of silica grinding beads and 1 mL of Trizol reagent were added, and the tissue grinder was shaken and ground three times at 60 Hz for 30 seconds each time, and stood on ice for 3 minutes. The sample was centrifuged at 12,000 rpm and 4°C for 10 minutes, and 800 μL of the supernatant was transferred to a new 1.5 mL RNase free EP tube; 500 μL of chloroform was added, vigorously shaken for 30 seconds, and allowed to stand at room temperature for 3 minutes. Centrifuge at 12000rpm, 4°C for 10 minutes, pipette the supernatant into a new 1.5mL RNase freeEP tube, add an equal volume of isoacetone, gently invert and mix for 30 seconds, and let stand at room temperature for 20 minutes. Centrifuge at 12,000 rpm, 4°C for 20 minutes, a precipitate can be seen at the bottom of...

Embodiment 2

[0044] 1. Synthesis of dsRNA

[0045] The following specific primers containing the T7 promoter were used:

[0046] Forward:

[0047]

[0048] Reverse:

[0049]

[0050] The underlined sequence is the T7 promoter sequence.

[0051] dsGFP primer sequences are as follows

[0052] dsGFP-F:

[0053]

[0054] dsGFP-R:

[0055]

[0056] Using the recombinant plasmid pMD-NCS2 extracted in Example 1 as a template, a high-concentration DNA fragment of about 550 bp was amplified by PCR with the above-mentioned primers (the nucleotide sequence is shown in SEQ ID NO. 4), and this was used as a template. Synthesize dsNCS2 (the nucleotide sequence of the sense strand is shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is the reverse complement of the sequence shown in SEQ ID NO.3), and the template synthesized by dsGFP is a well-known sequence .

[0057] Prepare the double-stranded RNA synthesis reaction system:

[0058] ATP solution, UTP solutio...

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Abstract

The invention discloses application of a brown planthopper NCS2 gene or an encoded protein as a target spot in preparation of a drug for preventing and treating brown planthopper, and relates to the technical field of plant genetic engineering. According to the application disclosed by the invention, the effect of the rice pest nilaparvata lugens NCS2 gene in prevention and treatment of nilaparvata lugens is found, and the nilaparvata lugens has an efficient RNAi effect, so that the expression of the nilaparvata lugens NCS2 gene in a pest body can be interfered by utilizing the advantage of the nilaparvata lugens NCS2 gene. DsRNA of the NCS2 gene is obtained through an RNAi technology, and after the NCS2 gene is silenced in a microinjection mode, the transcriptional level of the NCS2 gene is reduced, individual development and ovary development of the brown planthopper can be effectively inhibited, the egg laying amount can be reduced, and the ability of inhibiting growth, development and reproduction of pests is achieved, so that reproduction of the brown planthopper is inhibited, and prevention and treatment of the brown planthopper are achieved. When NCS2 gene function research is carried out, it is found that an obvious lethal effect is generated when an RNAi technology is used for interfering expression of the gene in 2-5-age nymphs of brown planthopper.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to the application of the NCS2 gene or the encoded protein of N. lugens as a target in preparing a drug for controlling N. lugens. Background technique [0002] Intracellular calcium ion is an important second messenger in eukaryotic cells. Calcium ion-binding proteins mediate intracellular calcium signaling. Most calcium-binding proteins contain one or more pairs of EF-hand domains. The EF-hand domain has a high affinity for calcium ions. The EF-hand domain is usually 29 amino acids and has a helix-loop-helix structure (Tsujimoto T, Jeromin A, Saitoh N, Roder JC, Takahashi T. 2002. Neuronal calciumsensor 1 and activity-dependent facilitation of P / Q- type calcium currents atpresynaptic nerve terminals. Science, 295(5563):2276-2279.). Members of the neural calcium receptor (NCS) family belong to calcium-binding proteins containing EF-hand domains. NCS genes are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/435C12N15/113C12N15/89A01N63/60A01P7/04
CPCC07K14/43563C12N15/113C12N15/89A01N63/60C12N2310/141
Inventor 鲍艳原张璐
Owner ZHEJIANG UNIV
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