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Application of two dsRNAs (double-stranded ribonucleic acids) and combination thereof in controlling aphid damage

An aphid and DNA molecule technology, applied in DNA/RNA fragmentation, application, recombinant DNA technology, etc., can solve the problems of lack of aphid-resistant germplasm resources in wheat, unclear resistance mechanism, and ineffective conventional breeding.

Active Publication Date: 2014-07-09
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There is a lack of aphid-resistant germplasm resources in wheat, and the resistance mechanism is still unclear. Conventional breeding is difficult to work, and it causes huge economic losses to agricultural production every year.

Method used

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  • Application of two dsRNAs (double-stranded ribonucleic acids) and combination thereof in controlling aphid damage
  • Application of two dsRNAs (double-stranded ribonucleic acids) and combination thereof in controlling aphid damage
  • Application of two dsRNAs (double-stranded ribonucleic acids) and combination thereof in controlling aphid damage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1. Acquisition of 23028dsRNA and coo2dsRNA related genes of Aphid aphidis

[0049] 1. Extraction of total RNA and synthesis of cDNA

[0050] 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.

[0051] 2. Primer design and gene cloning

[0052] According to the results of the transcriptome sequencing of the plant aphid in our laboratory, the growth and development-related gene 23028 (sequence 1) and the sequence of coo2 (sequence 7), the salivary secretion protein of green peach aphid, were compared with DNAman5.0, and the conserved sequence was selected , using Primer Primer5.0 software to design primers P1 and P2 (Table 1), synthesized by Beijing Huada Gene Company. The green fluorescent protein gene ...

Embodiment 2

[0069] Embodiment 2, the application of dsRNA in inhibiting the growth of aphids

[0070] 1. Preparation of artificial diet for aphids and preparation of feeder

[0071] References for the preparation of artificial feed and feeder (Li Caixia, Gao Lifeng, Gao Lingling, Li Runzhi. Research on raising aphids in pure artificial nutrient solution. Journal of Shanxi Agricultural University, 1997, 17(3): 225-228. Li C X ,Gao LF ,Gao LL,Li RZ. 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 The artificial feed was filtered with a bacterial filter, distributed into 2.0mL sterilized centrifuge tubes and stored in a -20°C refrigerator to avoid repeated freezing and thawing.

[0072] 2. Application of dsRNA (23028dsRNA, coo2dsRNA) in inhibiting the growth of aphids

[0073] Aphid feeding methods refer to the following documents: Jiao Min, Liu Shusheng. The technology of raising aphids ...

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Abstract

The invention discloses application of two dsRNAs (double-stranded ribonucleic acids) and combination thereof in controlling aphid damage. The dsRNA provided by the invention is disclosed as 1), 2) or 3): 1) dsRNA composed of nucleotide disclosed as Sequence 4 in the sequence table and nucleotide disclosed as reverse complementary sequence thereof; 2) dsRNA composed of nucleotide disclosed as Sequence 5 in the sequence table and nucleotide disclosed as reverse complementary sequence thereof; and 3) composed of the following two dsRNAs: dsRNA disclosed as 1) and dsRNA disclosed as 2). The experiment proves that dsRNAs of grain aphid growth and development related gene 23028 and salivary secretion protein gene coo2 are obtained, and the grain aphid in-vivo growth and development related gene 23028 and salivary secretion protein gene coo2 are silenced by an in-vitro dsRNA feeding method by using an RNAi (ribonucleic acid interference) technique, thereby resulting in hindrance of grain aphid growth and development and generating lethal effect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of two dsRNAs and their combination in controlling aphid damage. Background technique [0002] Wheat aphids (especially wheat aphids) are one of the main pests that harm wheat production in China. According to statistics, the area of ​​wheat aphid damage in China can reach as high as 17 million hectares every year, accounting for 62% of the total wheat planting area, resulting in a 15% reduction in production— 30%, up to 50% in severe cases. In recent years, due to factors such as global warming and changes in farming systems, the reproductive ability and adaptability of aphids have been significantly enhanced, and their harm has become increasingly serious. At present, aphid control is mainly based on spraying pesticides, but a large amount of pesticides are not only harmful to humans and animals, but also cause serious environmental pollution. Breeding aphid-resis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113A01N57/16A01N63/00A01P7/04C12N15/63C12N5/10C12N1/21C12R1/19
Inventor 夏兰琴张珉李雪峰
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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