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A kind of dsRNA of aphid symbiont gene and its application in reducing the survival rate of aphid
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A technology of survival rate and wheat long tube aphid, applied in the field of dsRNA and its application in reducing the survival rate of aphids, can solve the problems of few research reports on aphids
Inactive Publication Date: 2016-08-03
NANYANG NORMAL UNIV
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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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Embodiment 1
[0042] Example 1. Obtaining of dsRNA of Pseudomonasputida29698 gene in aphid endosymbiotic bacteria
[0043] 1. Extraction of total RNA and synthesis of cDNA
[0044] About 20 tube aphids were taken, and total RNA was extracted according to the instructions of the Trizol kit of Beijing Quanshijin Company, purified with RNACleaningKit, 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 PrimerPri-mer5.0 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, and primers P2-F ...
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 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. LiCX, GaoLF, GaoLL, LiRZ.Study ontherearingofaphidsonaartificiallyholidicdiets.JournalofShanxiAgriculturalUniversity, 1997,17(3):225-228.(inChinese)) carry out, filter the artificial feed with 0.2μm bacteria filter, divide into 2.0mL sterile centrifuge tubes and store in Store in a refrigerator at -20°C, 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 raise aphids. Entomologica...
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Abstract
The invention discloses a dsRNA of aphid symbiotic bacteria gene and its application in inhibiting the growth of aphids. The dsRNA provided by the present invention is a double-stranded RNA composed of the nucleotides shown in Sequence 1 in the sequence listing and the nucleotides shown in its reverse complementary sequence. The dsRNA provided by the present invention adopts the method of feeding dsRNA in vitro, and uses RNAi technology to silence the gene of Pseudomonas putida 29698, the endosymbiotic bacterium Pseudomonas putida 29698. The mortality rate of the long pipe aphid was gradually increased. The real-time fluorescent quantitative PCR study of the symbiotic bacterium Pseudomonas putida 29698 gene in aphids after feeding showed that the expression of the 29698 gene was significantly inhibited. It indicated that the conserved sequence of the aphid endosymbiont Pseudomonas putida 29698 gene can be applied to the study of improving wheat aphid resistance through plant-mediated 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...
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