Henosepilachna vigintioctopunctata high lethal gene and application thereof to controlling henosepilachna vigintioctopunctata

A 28 star lady beetle, gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problem of no insecticidal activity, no research on the gene function of the 28 star lady beetle, etc., to achieve effective The effect of good performance and sensitivity, environmental friendliness and convenient operation

Active Publication Date: 2019-11-19
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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  • Henosepilachna vigintioctopunctata high lethal gene and application thereof to controlling henosepilachna vigintioctopunctata
  • Henosepilachna vigintioctopunctata high lethal gene and application thereof to controlling henosepilachna vigintioctopunctata
  • Henosepilachna vigintioctopunctata high lethal gene and application thereof to controlling henosepilachna vigintioctopunctata

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Obtaining of synthetic dsRNA of embodiment 1 gene RPL13 kit

[0034]We constructed the 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, screened the RPL13 gene, and based on the RACE (rapid -amplification of cDNA ends) technology to amplify the full-length CDS sequence of the RPL13 gene, and the kit used by RACE is from TAKARA company ( RACE 5’ / 3’Kit cat#634858), the experimental steps are detailed in the kit instructions. The primers for 5'RACE and 3'RACE were designed as P7 and P8 in Primer Table 1. The kits used for the PCR reaction and cloning involved in the RACE process are as described above, and the CDS sequence of the RPL13 gene was obtained from the sequencing results, as shown in SEQ ID NO.1. The dsRNA is subsequently synthesized based on the sequence information.

[0035] 1. Extra...

Embodiment 2

[0050] Obtaining of embodiment 2 gene RPL13 bacterium expressing dsRNA

[0051] 1. Construction of dsRPL13 and L4440 expression vectors

[0052] 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 made public), add the homology arms related to the two restriction sites to the primer P1 of dsRPL13 and the primer P2 of dsGFP respectively, and design the primer P3 related to the construction of expression vector of dsRPL13 , Primer P4 (Table 1) related to construction of expression vector for dsGFP. Using 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 target fragments of dsRPL13 and dsGFP of the construction vector are obtained, and the DNA purification recovery kit (Universal DNA Purification Kit) is used , TIANGEN) recovered the two PCR products obtained...

Embodiment 3

[0055] Example 3 Application of dsRNA in Inhibiting the Growth and Development of Ladybug Solanum

[0056] 1. Preparation of host plants and artificial incubators for ladybird solanum

[0057] 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.

[0058] 2. The application of artificially synthesized dsRPL13 in inhibiting the growth and development of Ladybug solani

[0059] RPL13 dsRNA feeding group (dsRPL13): 10 1st instar larvae of Ladybug solani were placed in a petri dish with filter paper and humidified cotton balls. Soak round eggplant leaves with a diameter of 12 mm in dsRPL13 solutions with a concentration of 10 ng / μL, 50 ng / μL, 100 ng / μL, 250 ng / μL and 500 ng / μL for 1 min, air-dry them at room temperature for 1 h, and then feed the larvae, and replace the leaf discs every 24 h , after two days of continuous feed...

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Abstract

The invention discloses a henosepilachna vigintioctopunctata high lethal gene and application thereof to controlling henosepilachna vigintioctopunctata. The henosepilachna vigintioctopunctata gene RPL13 is screened out, the high lethal effect can be generated on the henosepilachna vigintioctopunctata by silencing the expression of the gene as the target gene, and therefore the technology capable of efficiently controlling the henosepilachna vigintioctopunctata is developed, that is, the henosepilachna vigintioctopunctata is directly fed with the target gene dsRNA with high lethal capacity, andthe control aim is realized through the lethal effect of the target gene dsRNA on the henosepilachna vigintioctopunctata. The method is convenient to operate, high in effectiveness and sensitivity and high in insect killing efficiency, has the advantages of being friendly to the environment and the like and has quite broad application prospects.

Description

technical field [0001] The invention belongs to the technical field of pest control. More specifically, it relates to a high-lethal gene for ladybug solani and its application in preventing and controlling ladybug. Background technique [0002] Henosepilachnavigintioctopunctata (Fabricius) belongs to the family Coleoptera and mainly damages eggplant, potato and other Solanaceae vegetables. It has a wide range of host plants and is an important agricultural pest. 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 co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/113C07K14/435A01N57/16A01P7/04
CPCA01N57/16C07K14/43563C12N15/113
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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