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RNA interference vector of bactrocera dorsalis cytochrome P450 genes and construction method and application thereof

An RNA interference and cytochrome technology, applied in the field of genetic engineering, to achieve the effect of reducing the amount of use and broad market prospects

Active Publication Date: 2014-09-24
CITRUS RES INST SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no one has used the Bactrocera dorsalis cytochrome P450 gene to construct RNA interference vectors to control Bactrocera dorsalis

Method used

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  • RNA interference vector of bactrocera dorsalis cytochrome P450 genes and construction method and application thereof
  • RNA interference vector of bactrocera dorsalis cytochrome P450 genes and construction method and application thereof
  • RNA interference vector of bactrocera dorsalis cytochrome P450 genes and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Cloning and recovery and purification of the cytochrome P450 gene of embodiment 1

[0043] 1. Extraction of Bactrocera dorsalis total RNA and acquisition of full-length cDNA

[0044] Total RNA from the Bacteralis dorsalis source provided by the Plant Protection College of Southwest University was extracted using a total RNA extraction kit. For specific steps, refer to the instructions of the total RNA extraction kit. The extracted total RNA electrophoresis figure 1 shown.

[0045] The extracted total RNA was reverse-transcribed into cDNA using the PrimeScriptTMRTreagentkitwithgDNAEraser reverse transcription kit. For specific steps, refer to the instructions of the reverse transcription kit.

[0046] 2. Cloning, recovery and purification of the target fragment of cytochrome P450 gene

[0047] (1) Search the full-length sequence of the Bacteralis dorsalis cytochrome P450 gene on the NCBI website, compare the conserved sequences, and design the forward and reverse spec...

Embodiment 2

[0055] After the end of the PCR program, take out 5-8 μL of the PCR amplification products, and spot them on 110V1% agarose gel to detect the forward and reverse target fragments by electrophoresis, and stain with ethidium bromide for 10 minutes after 35 minutes. The spectrum in the gel imaging system is photographed and analyzed, such as figure 2 As shown, the size of the amplified forward / reverse target fragment is about 600bp, which is the same as the expected result. Spot the remaining PCR products on 110V1.2% agarose gel for recovery and electrophoresis. After 40 minutes, cut off the gel block containing the target fragment with a blade on a gel cutter, and put it into a sterile 1.5mL centrifuge tube In this method, the target fragment is recovered using the DNA Recovery and Purification Kit. For the operation steps, please refer to the instruction manual of the DNA Recovery Kit. Transformation of embodiment 2 recombinant plasmids

[0056] 1. In vitro ligation of targe...

Embodiment 3

[0073] Example 3 Construction of RNA interference expression vector

[0074] 1. Ligation of the target fragment with the vector PFGC5941

[0075] (1) The plasmid pMD-19-P1F1 and the carrier PFGC5941 of the forward target fragment of the cytochrome P450 gene extracted in Example 2 were subjected to double enzyme digestion with XhoI and NcoI respectively. The enzyme digestion system was: Buffer45μL, XhoI1.5μL, NcoI1.5μL, BSA0.5μL, pMD-19-P1F1 or PFGC594120μL, adjust the total volume of the enzyme digestion system to 50μL with ddH2O, put it into a centrifuge tube, and bathe in a water bath at 37°C for 4 hours.

[0076] (2) Take 3-5 μL of the digested product of pMD-19-P1F1 and carrier PFGC5941 obtained in step 1 for electrophoresis detection, and run the recovery gel to recover the digested product. The electrophoresis of PFGC5941 digested by XhoI and NcoI is as follows: Figure 4 As shown, the electrophoresis of pMD-19-P1F1 digested by XhoI and NcoI is as follows Figure 5 sh...

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Abstract

The invention provides an RNA interference vector of bactrocera dorsalis cytochrome P450 genes and a construction method and application of the RNA interference vector and belongs to the technical field of gene engineering. The method comprises the steps that a vector PFGC5941 is used as a frame, and positive and negative specificity target fragments of the bactrocera dorsalis cytochrome P450 genes are inserted in the two sides of an intron of the vector PFGC5941 through double digestion to form an important region of the RNA interference vector; after the constructed RNA interference expression vector is transferred to a plant, the RNA interference vector is transcribed in a somatic cell of the plant to form a double-stranded RNA hairpin structure under the drive of a strong promoter CaMV35S of the RNA interference vector, so that normal expression and translation of a homologous gene fragment in the body of bactrocera dorsalis which eats the plant are influenced. Consequently, normal growth and development of insects can be interfered, and the purpose that the plant resists the bactrocera dorsalis is achieved; the method plays an important role in controlling the bactrocera dorsalis, and the use amount of chemical pesticides is reduced.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to an RNA interference carrier, in particular to an RNA interference carrier of Bactrocera dorsalis cytochrome P450 gene and its construction method and application. Background technique [0002] Bactroceradorsalis, also known as Bactroceradorsalis orientalis, is a dangerous quarantine pest in the world. It is widely distributed in major citrus producing areas in China. It poses a serious threat to China's citrus industry and causes huge economic losses to citrus every year. It can harm more than 250 kinds of fruits and vegetables. Bacteralis dorsalis has a wide range of hosts and can harm more than 250 kinds of fruits, vegetables and crops such as citrus, mango and carambola. With the change of climate, the adjustment of planting structure and the increase of international trade, its harm area gradually expands, which brings serious economic losses to the fruit and vegeta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/113C12N15/66C12N15/84A01H5/00
Inventor 冉春岳建苏刘浩强丛林李鸿筠
Owner CITRUS RES INST SOUTHWEST UNIV
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