The rna interference vector of Bactrocera dorsalis cytochrome p450 gene and its construction method and application

A technology of RNA interference and cytochrome, which is applied in the field of genetic engineering to achieve broad market prospects and reduce usage

Active Publication Date: 2017-01-11
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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  • The rna interference vector of Bactrocera dorsalis cytochrome p450 gene and its construction method and application
  • The rna interference vector of Bactrocera dorsalis cytochrome p450 gene and its construction method and application
  • The rna interference vector of Bactrocera dorsalis cytochrome p450 gene and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Cloning, recovery and purification of cytochrome P450 gene

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

[0044] The total RNA extraction kit was used to extract the total RNA derived from Bactrocera dorsalis provided by the School of Plant Protection of Southwest University. For specific steps, please refer to the instructions for the total RNA extraction kit. The extracted total RNA electropherogram is as follows figure 1 Shown.

[0045] The extracted total RNA was reverse transcribed into cDNA using PrimeScriptTM RT reagent kit with gDNA Eraser 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 for the full-length sequence of the cytochrome P450 gene of Bactrocera dorsalis on the NCBI website, compare the conservative sequences, and design forward and r...

Embodiment 2

[0056] Example 2 Transformation of recombinant plasmid

[0057] 1. In vitro connection of target fragment and plasmid

[0058] The forward target fragment and the reverse target fragment of the cytochrome P450 gene recovered in Example 1 were respectively connected to the vector pMD-19 in vitro, and the reaction system (10 μL) was as follows:

[0059] pMD-19 Vector 0.5μL

[0060] Insert DNA 4.5μL

[0061] Ligation Solution 5μL

[0062] The above operations are best performed on ice. After adding the drugs, put the mixture into a PCR machine and connect overnight at 16°C to obtain two recombinant plasmids of the forward fragment and the reverse fragment of the cytochrome P450 gene. The recombinant plasmid of the fragment was named pMD-19-P1F1, and the recombinant plasmid of the reverse fragment was named pMD-19-P2F2.

[0063] 2. Transformation of recombinant plasmid into Escherichia coli

[0064] The recombinant plasmid pMD-19-P1F1 of the forward fragment of the cytochrome P450 gene and th...

Embodiment 3

[0074] Example 3 Construction of RNA interference expression vector

[0075] 1. Enzyme digestion and connection of the target fragment and the vector PFGC5941

[0076] (1) The positive target fragment plasmid pMD-19-P1F1 and vector PFGC5941 of the cytochrome P450 gene extracted in Example 2 were digested with XhoI and NcoI respectively. The digestion system was: Buffer 45 μL, XhoI 1.5 μL, NcoI1.5μL, BSA0.5μL, pMD-19-P1F1 or PFGC594120μL, use ddH 2 O Adjust the total volume of the digestion system to 50μL, put it in a centrifuge tube, and place it in a water bath at 37°C for 4 hours.

[0077] (2) Take out 3-5 μL of the digested product of pMD-19-P1F1 and vector PFGC5941 obtained in step 1 for electrophoresis detection, and run the gel to recover the digested product. The electropherogram of PFGC5941 after digestion with XhoI and NcoI is as follows Figure 4 As shown, the electropherogram of pMD-19-P1F1 digested with XhoI and NcoI is as follows Figure 5 Shown.

[0078] (3) Connect the...

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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 vector, in particular to an RNA interference vector for the cytochrome P450 gene of Bactrocera dorsalis and its construction method and application. Background technique [0002] Bactrocera dorsalis, also known as Oriental fruit fly, 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. , Can harm more than 250 kinds of fruits and vegetables. Bactrocera dorsalis has a wide range of hosts and can harm more than 250 fruits, vegetables and crops such as citrus, mango and starfruit. With climate change, the adjustment of planting structure and the increase of international trade, the damage area has gradually expanded, causing serious economic losses to the fruit and vegetable industry. At prese...

Claims

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Application Information

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Patent Type & Authority Patents(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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