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High-lethality gene vATPase B and application thereof to resisting henosepilachna vigintioctopunctata

A technology of gene and target gene, applied in the application field of highly lethal gene vATPaseB and its anti-ladybug, can solve the problems of no insecticidal activity, no research on the function of the ladybug gene, and achieve effectiveness and sensitivity Good performance, friendly environment and easy operation

Active Publication Date: 2019-12-20
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the use of RNAi technology can specifically inhibit the expression of genes, this technology has been widely used in targeted interference with pest genes to achieve the purpose of pest control. However, there is no research on the gene function of the solanum beetle at home and abroad. Reported target genes without insecticidal activity

Method used

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  • High-lethality gene vATPase B and application thereof to resisting henosepilachna vigintioctopunctata
  • High-lethality gene vATPase B and application thereof to resisting henosepilachna vigintioctopunctata
  • High-lethality gene vATPase B and application thereof to resisting henosepilachna vigintioctopunctata

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 The acquisition of gene vATPase B kit synthetic dsRNA

[0033] We constructed its transcriptome library based on the genome of the ladybug Solanum solani, and then based on the constructed transcriptome library, researched and screened the genes related to the growth and development of the ladybug Solanum, and screened the vATPase B gene fragment, such as SEQID NO .1 shown. The dsRNA is then synthesized.

[0034]1. Extraction of total RNA and synthesis of first-strand cDNA from ladybug Solanum solani.

[0035] Get 10 2nd instar larvae of Ladybug solani and place in 2ml centrifuge tube, utilize TRIzol method to extract the total RNA of Ladybug solani, utilize reverse transcription kit (PrimeScript TM RT reagent Kit with gDNA Eraser, TAKARA) followed the steps in the instructions for reverse transcription to synthesize the first strand of cDNA.

[0036] 2. Primer design

[0037] The gene sequence of vATPase B was obtained from the transcriptome library of L...

Embodiment 2

[0049] Example 2 Obtaining of Gene vATPase B Bacterial Liquid Expression dsRNA

[0050] 1. Construction of dsvATPase B and L4440 expression vectors

[0051] Two restriction sites were selected on the sequence of L4440, namely BamHI (GGATCC) and SacI (GAGCTC). According to the sequence information of L4440 (this sequence information has been published), the primer P1 of dsvATPase B and the primer P2 of dsGFP were respectively added with two homologous arms related to the restriction site, and the dsvATPase B construction expression vector was designed. Primer P3, and primer P4 related to constructing expression vector of dsGFP (Table 1). With the cDNA in Example 2 as a template, the reaction system and amplification program of PCR amplification are as shown in Example 2, and the dsvATPase B and dsGFP target fragments of the construction vector are obtained, and the DNA purification recovery kit (Universal DNA Purification Kit, TIANGEN) recovered the two PCR products obtained ...

Embodiment 3

[0054] Example 3 Application of dsRNA in Inhibiting the Growth and Development of Ladybug Solanum

[0055] 1. Preparation of host plants and artificial incubators for ladybird solanum

[0056] The eggplant variety fed with eggplant twenty-eight star ladybird is Wansheng Marshal round eggplant seedlings, and the artificial incubator is a 90mm petri dish with filter paper and humidified cotton balls.

[0057] 2. The application of dsvATPase B synthesized by the kit in inhibiting the growth and development of the ladybug Solanum solani

[0058] Feeding group of lady beetle dsvATPase B: 10 1st instar larvae of lady beetle solani were placed in a petri dish with filter paper and humidified cotton balls. Soak circular eggplant leaf discs with a diameter of 12 mm in the dsvATPase B solutions synthesized by the kit at a concentration of 50 ng / μL, 100 ng / μL, 250 ng / μL and 500 ng / μL for 1 min, air-dry for 1 h, and then feed the larvae, replacing them every 24 h Leaf disks, after two d...

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Abstract

The invention discloses a high-lethality gene vATPase B and an application thereof to resisting ladybugs. Through research, a gene vATPase B is obtained from henosepilachna vigintioctopunctata, and when the expression of the gene is silenced, the henosepilachna vigintioctopunctata can be efficiently prevented and treated, so that a target gene dsRNA namely dsvATPase B having high lethal capacity on the henosepilachna vigintioctopunctata is designed, and the target gene dsRNA has high lethality effects on the henosepilachna vigintioctopunctata, is good in sensitivity, high in parasite destroying efficiency, good in preventing and treating effects and convenient to use and operate, also has many advantages of being environmentally-friendly and the like, and has good application prospects inpreventing and treating the henosepilachna vigintioctopunctata.

Description

technical field [0001] The invention belongs to the technical field of pest control. More specifically, it relates to a highly lethal gene vATPase B and its application against ladybugs. Background technique [0002] Henosepilachnavigintioctopunctata (Fabricius) belongs to the family Coleoptera and is an important agricultural pest with a wide range of host plants. It mainly damages eggplant, potato and tomato and other solanaceous vegetables. Both the larvae and adults feed on the leaves. They like to cluster on the back of the leaves and feed on the lower epidermis and mesophyll. The damaged leaves usually form irregular transparent spots or perforations. In severe cases, the plants will wilt or even die. Ladybug solani has a wide range of distribution in my country, especially in the south of the Yangtze River. In recent years, due to climate warming, trade development, and the expansion of vegetable cultivation area in protected areas, its food is constantly available ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/113A01N57/16A01P7/04
CPCA01N57/16C12N9/14C12N15/1137C12N2310/14C12Y306/01003
Inventor 潘慧鹏吕晶郭威杨春晓郭木娟陈诗敏邱宝利刘卓琦
Owner SOUTH CHINA AGRI UNIV
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