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dsRNA of aphid gene and application of dsRNA to aphid survival rate reduction

A kind of survival rate and aphid technology, applied in the field of bioengineering, can solve the problems of lack of aphid resistance gene and difficult breeding of aphid resistance

Inactive Publication Date: 2016-01-20
NANYANG NORMAL UNIV
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Problems solved by technology

[0004] In view of the huge loss caused by aphids to agricultural production, and the lack of effective aphid-resistant genes in the existing germplasm resources of crops, conventional aphid-resistant breeding is difficult to be effective; excavate and identify new aphid-resistant genes or aphid dsRNA, and use transgenic technology to breed aphid-resistant new species. Germplasm is of great significance for ensuring food security and environmental security in our country

Method used

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  • dsRNA of aphid gene and application of dsRNA to aphid survival rate reduction

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Embodiment 1

[0037] The acquisition of the dsRNA of embodiment 1 aphid gene

[0038] (1) Extraction of total RNA and synthesis of cDNA

[0039] 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.

[0040] (2) Primer design and gene cloning

[0041]Primers P1-F and P1-R were designed using PrimerPrimer5.0 software and synthesized by Beijing Huada Gene Company. GFP gene ( GFP ) fragment was amplified from the 16318hGFP plasmid preserved in the laboratory of the School of Life Science and Technology, Nanyang Normal University, and primers P2-F and P2-R were designed using PrimerPrimer5.0 software.

[0042] P1-F: 5'- TAATACGACTCACTATAGGG CAACAGTCAGCGGAAGTAC-3' (the underlined sequence is the 1st to 19th positions of SEQ ID No: 2);

[0043] P1-R...

Embodiment 2

[0060] Example 2 SaUP Application of dsRNA in inhibiting the growth of aphids

[0061] (1) Preparation of artificial feed for aphids and preparation of feeders

[0062] References for preparation of artificial feed and feeder (Li Caixia, Gao Lifeng, Gao Lingling, Li Runzhi. A study on feeding aphids in pure artificial nutrient solution. Journal of Shanxi Agricultural University, 1997, 17(3): 225-228.) , filter the artificial feed with a 0.2μm bacterial filter, dispense it into 2.0mL sterilized centrifuge tubes and store in a -20°C refrigerator to avoid repeated freezing and thawing.

[0063] (2) dsRNA ( SaUP dsRNA) in inhibiting the growth of aphids

[0064] Aphid feeding methods refer to the following documents: Jiao Min, Liu Shusheng. The technology of using artificial feed to raise aphids. Entomological Journal of East China, 2004,13(2):102-109.

[0065] Aphid aphid SaUP dsRNA feeding group: put 20 3rd-instar nymphs of A. sativa in each aphid feeder, and add 0 and 1...

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Abstract

The invention relates to a dsRNA of an aphid gene and application of the dsRNA to aphid survival rate reduction. The dsRNA of the aphid gene is particularly a double-chain RNA formed by nucleotide with the sequence shown by SEQ ID No:1 and nucleotide with a reverse complement sequence. According to the application, the dsRNA is transferred into aphids, so that the goals of preventing and controlling the aphids, reducing the aphid survival rate or inhibiting the aphid SaUP gene expression are realized. An in-vitro dsRNA feeding method is adopted, an RNAi technology silent English grain aphid SaUP gene is used, and the lethal effect of the English grain aphids is caused; the fed aphid SaUP gene is subjected to real-time fluorogenic quantitative PCR (polymerase chain reaction). Study proves that the expression of the SaUP genes is obviously inhibited. The result shows that the sequence of the aphid SaUP gene can be applied to the study of improving the wheat aphid resistance by a plant-mediated RNAi technology.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to dsRNA of aphid 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 ap...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/11A01N57/16A01P7/04
Inventor 喻修道陈志勤贾殿勇郑远段建平
Owner NANYANG NORMAL UNIV
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