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Method for separation and identification of odorant binding protein (OBP) genes in insect's antenna

A technology of tentacles and genes, applied in the field of pest biological control, can solve problems such as environmental damage, cotton bollworm resistance, pollution, etc., and achieve the effects of improving efficiency, easy operation, and low cost

Inactive Publication Date: 2012-04-04
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because Bt cotton is less effective against mirid gnats, the control of mirid gnats now mainly depends on chemical pesticides, which not only easily leads to the generation of insecticide resistance of Lepidoptera pests such as cotton bollworm, but also causes huge damage and pollution to the environment, so , it is necessary to develop cost-effective, environmentally friendly control methods

Method used

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  • Method for separation and identification of odorant binding protein (OBP) genes in insect's antenna
  • Method for separation and identification of odorant binding protein (OBP) genes in insect's antenna
  • Method for separation and identification of odorant binding protein (OBP) genes in insect's antenna

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Extraction of antennal RNA, synthesis of first-strand cDNA by reverse transcription and synthesis of ds cDNA by LD-PCR

[0051] Get 200 pairs of antennae of Lygus alfalfa that just emerged, utilize the Trizol method to carry out the extraction of RNA, electrophoresis detects RNA quality (see figure 1 ), while the concentration of RNA measured by spectrophotometer was 618.8ng / μl. The first-strand cDNA and ds cDNA were synthesized by SMART technology and LD-PCR method. Finally, 1.5% agarose gel electrophoresis was used to detect the distribution of LD-PCR products. The principle of SMART is as follows figure 2 . The electrophoresis of LD-PCR products is shown in image 3 . The analysis results showed that the size of LD-PCR products ranged from 200bp to 2000bp, and the distribution range was relatively wide, indicating that the products included mRNAs of different genes, and there were several brighter bands, representing genes with relatively high expres...

Embodiment 2

[0052] Example 2: Digestion of ds cDNA by Sfi I and separation of ds cDNA by CHROMA SPIN-400 column

[0053] In order to connect the ds cDNA amplified by LD-PCR with the pDNR-LIB vector (commercially purchased), the ds cDNA was digested with Sfi I endonuclease, and the ds cDNA was separated by size using a CHROMASPIN-400 column , respectively packed in 16 sterile centrifuge tubes, one drop per tube. Finally, the size distribution of ds cDNA fragments detected by 1.5% agarose electrophoresis, such as Figure 4 , and finally according to the electrophoresis results, the ds cDNA and pDNR-LIB vector in tubes 7, 8, 9, and 10 were ligated.

Embodiment 3

[0054] Example 3: Electrotransformation of DH5α Escherichia coli cells with recombinant plasmids

[0055] In order to obtain higher conversion efficiency, electric conversion is used. The electroporation instrument is a product of Bio-Rad Company, and the electric shock parameters are set to 2000V, 200Ω. The electric shock time is 2-3 seconds.

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Abstract

The invention discloses a method for antenna cDNA library construction adopting a SMART technology, and separation and identification of odorant binding protein (OBP) genes in an insect's antenna. The method can realize effective separation and identification of the OBP genes in adelphocoris lineolatus through a bioinformatic method. Through the method, nucleotide sequences of 14 OBP genes of adelphocoris lineolatus are screened out. Because of important effects of the OBP genes on insect olfactory identification, the OBP genes have important effects on insect biological control.

Description

technical field [0001] The present invention relates to the technical field of pest biological control using the olfactory system of insects; further, the present invention relates to the method of using SMART technology to construct antennae cDNA library and combining bioinformatics methods to separate and identify insect odor binding proteins OBPs. Background technique [0002] Miridae, belonging to the Miridae family of Hemiptera, is an important class of pests in cotton production in my country. In 1997, my country began to commercially plant Bt cotton. By 2007, the planting area of ​​Bt cotton had reached 3.8 million hectares, accounting for 69% of the total cotton planting area in the country. The planting of Bt cotton has effectively controlled the harm of major lepidopteran pests such as cotton bollworm, and greatly reduced the use of chemical pesticides. Subsequently, a series of successions have taken place in the status of cotton field pests, and the population of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68G06F19/24
Inventor 谷少华张永军张雪莹吴孔明郭予元
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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