Insect cytochrome P450 gene and application thereof

A technology of insect cells and pigments, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve problems such as pesticide agricultural production and ecological environment threats

Inactive Publication Date: 2015-06-03
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The control of locust plagues still mainly adopts chemical control methods, and the large-scale appl...

Method used

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  • Insect cytochrome P450 gene and application thereof
  • Insect cytochrome P450 gene and application thereof
  • Insect cytochrome P450 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: Acquisition of full-length cDNA sequence of migratory locust cytochrome P450 gene

[0016] Based on the migratory locust transcriptome database, bioinformatics methods were used to search the cytochrome P450 genes of migratory locusts, and the partial sequences of related genes were obtained after sequence analysis. The full-length cDNA sequence of the migratory locust cytochrome P450 gene (LmCYP4G102) was obtained by rapid cloning PCR technology at the end of the cDNA. The nucleotide sequence length was 1686bp, encoding 561 amino acids.

Embodiment 2

[0017] Example 2: Acquisition of cytochrome P450 gene fragment and dsRNA of migratory locust

[0018] 1) Obtaining the cytochrome P450 gene fragment of migratory locust

[0019] According to the sequence of the characteristic region of the cytochrome P4504G family, primer premier 5.0 software was used to design specific primers. The sequence of the upstream primer was SEQ ID NO: 4, and the sequence of the downstream primer was SEQ ID NO: 5. All primers were provided by Sangon Bioengineering (Shanghai ) Co., Ltd. Synthesis. Using the plasmid containing SEQ ID NO: 1 as a template, the cytochrome P450 gene fragment of migratory locust was amplified by PCR, and used SV Gel and PCR Clean-Up System (Promega) kit was used to purify the obtained cytochrome P450 gene fragment of migratory locust.

[0020] 2) Synthesis of dsRNA from migratory locust cytochrome P450 gene fragment

[0021] Using the migratory locust cytochrome P450 gene fragment SEQ ID NO: 3 obtained in step 1) as a t...

Embodiment 3

[0022] Example 3: dsRNA synthesis of cytochrome P450 gene fragments to kill migratory locust experiments

[0023] 1. Injection of dsRNA synthesized by migratory locust cytochrome P450 gene CYP4G102 fragment

[0024] The nymphs of migratory locust with uniform size and health status on the second day of the second instar were selected, and a control group injected with dsGFP and an experimental group injected with dsRNA (dsCYP4G102) of SEQ ID NO: 3 were set up. The dsRNA synthesized in vitro was injected into the body cavity of nymphs with a microinjector, the injection volume of dsRNA was 4 μg / head, 60 nymphs in each group, and 3 biological replicates were set up. After the injection, the nymphs were placed in a sarong and raised in an artificial climate box (light:dark time=14h:10h, temperature 30±2°C, humidity 60%), and fed with fresh wheat seedlings.

[0025] 2. Detection of silencing efficiency of migratory locust cytochrome P450 gene

[0026] The nymphs 24 hours after d...

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Abstract

The invention provides an insect cytochrome P450 gene and the application thereof, and particularly relates to the insect cytochrome P450 gene, gene fragments, dsRNA and the application of the dsRNA to killing pests. Firstly, the cytochrome P450 gene of a migratory locust is cloned and sequenced to obtain a CYP4G102 gene, an overall nucleotide sequence of the CYP4G102 gene is SEQ ID NO:1, and an amino acid sequence of the CYP4G102 gene is SEQ ID NO:2; secondly, the gene fragments of which the sequence is SEQ ID NO:3 are obtained and used for synthesizing the dsRNA. After the synthesized dsRNA is injected into the body cavity of the migratory locust, the insect dies due to excessive in-vivo water loss caused by the fact that the water retention capacity of the body wall is reduced after ecdysis, and the death rate reaches 100 percent. The insect cytochrome P450 gene provides a molecular target or pest control based on RNA interference and can be used for the pest control.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an insect cytochrome P450 gene, a gene fragment and its dsRNA and the application of the dsRNA in killing pests. Background technique [0002] In our country, the sustainable development of the agricultural industry plays a pivotal role in the national development strategy, and the integrated control of agricultural pests is an important task for agricultural plant protection. Migratory locusts are migratory and explosive, and locust plagues are major natural disasters in the agricultural history of our country. The control of locust plagues still mainly adopts chemical control methods, and the large-scale application of pesticides has caused serious threats to agricultural production and the ecological environment. Under the guidance of the development concept of green plant protection, exploring new environmentally friendly pest control methods has become the development direction...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/10C12N15/113A01N57/16A01P7/04
Inventor 余志涛张学尧刘耀明张建珍马恩波
Owner SHANXI UNIV
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