Carboxylesterase gene dsRNA and applications thereof in prevention and treatment of sitobion avenae

A kind of wheat tube aphid, transgenic technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2015-01-21
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method also has certain limitations. Different insects or different genes of the same insect have different sensitivities to RNAi entering through the oral cavity. The key factor affecting the success rate of feeding dsRNA is the efficiency of dsRNA uptake by insect epithelial cells.

Method used

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  • Carboxylesterase gene dsRNA and applications thereof in prevention and treatment of sitobion avenae
  • Carboxylesterase gene dsRNA and applications thereof in prevention and treatment of sitobion avenae
  • Carboxylesterase gene dsRNA and applications thereof in prevention and treatment of sitobion avenae

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Experimental program
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Effect test

Embodiment 1

[0049] Embodiment 1, obtaining of dsRNA of the aphid aphid carboxylesterase gene

[0050] 1. Extraction and reverse transcription of total RNA

[0051] 1. Total RNA extraction

[0052] 1) Put 50 tube aphids into a 1.5 mL EP centrifuge tube without RNase, add 60 μL Trizol Reagent, and grind to a homogenized state with an electric grinder;

[0053] 2) Take out 1-2 μL of total RNA for 1.5% agarose gel electrophoresis detection, and measure the 260 / 280 absorbance value, 260 / 230 absorbance value and nucleic acid concentration with NanoDrop at the same time, and store the rest at -80°C for later use.

[0054] The result is as figure 1 As shown, it can be seen that the extracted total RNA is relatively complete, the 5S, 18S, and 28S bands of the total RNA are relatively clear, and the RNA is not degraded.

[0055] 2. Reverse transcription

[0056] According to the reverse transcription kit, the operation is as follows:

[0057] (1) Prepare genomic DNA removal system mixture: 5×g...

Embodiment 2

[0099] Embodiment 2, the application of the aphid aphid CarE gene dsRNA in the prevention and control of the aphid aphid

[0100] 1. Analysis of the expression level of CarE gene in different ages

[0101] Before the feeding experiment, the tube aphids of different ages were picked to detect the expression of CarE gene. For each instar (1st instar larvae, 2nd instar larvae, 3rd instar larvae, 4th instar larvae, and adults), about 20 aphids were selected to extract total RNA and then detect gene expression.

[0102] Use Primer5.0 to design fluorescent quantitative primers for different genes, and the fluorescent quantitative primers are as follows:

[0103] Primers used to detect the CarE gene of the aphid:

[0104] CarERTF: 5'-TCCTGGAAACGTGGGCTTGAA-3' (position 300-320 of Sequence 2);

[0105] CarERTR: 5'-AACAGACGCTGATCCTGCACTTT-3' (reverse complement of positions 404-426 of Sequence 2).

[0106] Primers used to detect the internal reference gene RpL7Actin:

[0107] Inter...

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Abstract

The invention discloses a carboxylesterase gene dsRNA and applications thereof in prevention and treatment of sitobion avenae. The dsRNA is a double-chain RNA consisting of nucleotide shown in a sequence 1 in a sequence table and nucleotide shown in a reverse complementary sequence of the sequence 1. Experiments proves that carboxylesterase genes in sitobion avenae are reduced to different degrees after the sitobion avenae is fed with carboxylesterase gene dsRNA, which means that dsRNA taken in by a lancet enters the cells of the sitobion avenae to generate RNAi cascade reaction and the expression of a target gene is interfered. In a morphological view, the growth state of the sitobion avenae is changed, and the ecdysis index of the sitobion avenae fed with the carboxylesterase gene dsRNA is significantly reduced in comparison with a control. Therefore, carboxylesterase gene dsRNA can be used in prevention and control of sitobion avenae. The invention lays a foundation for construction of new germplasm of wheat by using plant-mediated RNAi technology in the future.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a carboxylesterase gene d s RNA and its application in the control of the wheat aphid. Background technique [0002] Wheat is the main food crop in our country. There are many kinds of pests in wheat fields, the main pests are underground pests, wheat midges, wheat aphids, wheat spiders, leaf-eating armyworms, etc. Among them, wheat aphids are the most serious harm to wheat. Wheat aphids belong to the family Aphididae of the order Homoptera, commonly known as putty insects, youhan, honey insects, etc., and are one of the main pests on wheat in my country. Its species mainly include Sitobion avenae, Schizaphis graminum, Rhopalosiphum padi Linnaeus, Acyrthosiphon dirhodum and so on. Aphids elongatus, digemia and aphids are widely distributed in various regions of the world, and all occur in wheat-growing areas in my country. The two-pronged aphid is mainly distributed in Northwest Chi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/63C12N1/21C12N1/15C12N1/19A01N63/02A01P7/04
Inventor 赵章武梁荣奇邓菲
Owner CHINA AGRI UNIV
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