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dsRNA (double-stranded ribonucleic acid) of sitobion avenae symbiotic bacteria gene and application thereof in reducing sitobion avenae survival rate

A technology of aphids and DNA molecules, applied in the direction of DNA / RNA fragments, applications, recombinant DNA technology, etc., can solve the problem of few research reports on aphids

Inactive Publication Date: 2015-01-07
NANYANG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are few reports on the indirect control of aphids by silencing symbiont-related genes through RNAi technology

Method used

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  • dsRNA (double-stranded ribonucleic acid) of sitobion avenae symbiotic bacteria gene and application thereof in reducing sitobion avenae survival rate
  • dsRNA (double-stranded ribonucleic acid) of sitobion avenae symbiotic bacteria gene and application thereof in reducing sitobion avenae survival rate
  • dsRNA (double-stranded ribonucleic acid) of sitobion avenae symbiotic bacteria gene and application thereof in reducing sitobion avenae survival rate

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Experimental program
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Effect test

Embodiment 1

[0042] Example 1. Obtaining of dsRNA of Pseudomonas putida29698 gene in aphid endosymbiotic bacteria

[0043] 1. Extraction of total RNA and synthesis of cDNA

[0044] About 20 tube aphids were taken respectively, and total RNA was extracted according to the instructions of the Trizol kit of Beijing Quanshijin Company, purified with RNA Cleaning Kit, and the first strand of cDNA was synthesized by reverse transcription. The operation steps refer to the instructions of the kit.

[0045] 2. Primer design and gene cloning

[0046] The gene 29698 related to the aphid symbiotic bacteria was obtained according to the intestinal transcriptome sequencing of Aphid aphidalis, and the primers P1-F and P1-R were designed by Primer Primer5.0 software and synthesized by Beijing Huada Gene Company. The green fluorescent protein gene (GFP) fragment was amplified from the 16318hGFP plasmid preserved in the laboratory of the School of Life Science and Technology of Nanyang Normal University, a...

Embodiment 2、29698

[0065] Application of embodiment 2, 29698dsRNA in inhibiting the growth of aphids

[0066] 1. Preparation of artificial diet for aphids and preparation of feeder

[0067] References for the preparation of artificial feed and feeder (Li Caixia, Gao Lifeng, Gao Lingling, Li Runzhi. Research on feeding aphids in pure artificial nutrient solution. Journal of Shanxi Agricultural University, 1997, 17(3): 225-228. Li C X , Gao L F, Gao L L, Li R Z. Study on the rearing of aphids on an artificially solidic diets. Journal of Shanxi Agricultural University, 1997,17(3):225-228.(in Chinese)), with 0.2μm Filter the artificial feed with a bacterial filter, dispense it into 2.0mL sterilized centrifuge tubes and store in a -20°C refrigerator to avoid repeated freezing and thawing.

[0068] 2. Application of dsRNA (29698dsRNA) in inhibiting the growth of aphids

[0069] Aphid feeding methods refer to the following documents: Jiao Min, Liu Shusheng. The technology of using artificial feed to ...

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Abstract

The invention discloses a dsRNA (Double-stranded Ribonucleic Acid) of a sitobion avenae symbiotic bacteria gene and application thereof in reducing the sitobion avenae survival rate. The dsRNA provided by the invention is a double-stranded RNA consisting of the nucleotide shown as a sequence 1 in a sequence list and the nucleotide shown as a reverse complementary sequence. According to the dsRNA provided by the invention, the Pseudomonas putida 29698 gene of the symbiotic bacteria in a sitobion avenae body is silenced by an in-vitro dsRNA feeding method through the utilization of a RNAi (RNA interfere) technology, lethal effect is produced on the sitobion avenae, and the dead rate of the sitobion avenae is gradually increased with the prolongation of the feeding time. The real-time fluorescent quantitative polymerase chain reaction study of the Pseudomonas putida 29698 gene of the symbiotic bacteria in the fed avenae body shows that the expression of the 29698 gene is obviously inhibited and that a conservative sequence of the Pseudomonas putida 29698 gene of the symbiotic bacteria in the avenae body can be applied in a study improving the wheat avenae resistance through a plant mediating RNAi technology.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to dsRNA of aphid symbiotic bacteria gene and its application in reducing the survival rate of aphids. Background technique [0002] Aphids are one of the main pests that endanger crop production. In recent years, factors such as global warming and changes in farming systems have significantly enhanced the reproductive capacity and adaptability of aphids, and their harm has become increasingly serious. According to statistics, from 2010 to 2011, the aphid-damaged areas of major crops such as wheat, corn, cotton, rape, and soybean in my country accounted for 62.5%, 14%, 90%, 32%, and 23% of the planting area in that year, respectively. Taking wheat aphids as an example, wheat aphids gather as nymphs on wheat leaves, stems, ears, etc., and suck juice, causing irregular shrinkage, curling, and chlorosis of leaves, and in severe cases, plant death. The honeydew secreted by the wheat aphid ad...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/11C12N15/63A01N57/16A01P7/04
Inventor 喻修道夏兰琴孙永伟陈志勤刘宗才贾殿勇段鹏飞
Owner NANYANG NORMAL UNIV
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