RNA interference vector of bactrocera dorsalis sodium ion channel genes and construction method and application thereof

A technology of sodium ion channel and RNA interference, which is applied in the field of genetic engineering to achieve the effect of reducing the amount of use and broadening the market prospect

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 B. dorsalis sodium channel gene to construct an RNA interference vector to control B. dorsalis

Method used

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  • RNA interference vector of bactrocera dorsalis sodium ion channel genes and construction method and application thereof
  • RNA interference vector of bactrocera dorsalis sodium ion channel genes and construction method and application thereof
  • RNA interference vector of bactrocera dorsalis sodium ion channel genes and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Cloning and recovery and purification of sodium ion channel genes

[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 PrimeScriptTM RT reagent kit with gDNA Eraser reverse transcription kit. For specific steps, please refer to the instructions of the reverse transcription kit.

[0046] 2. Cloning, recovery and purification of the target fragment of the sodium ion channel gene

[0047] (1) Search the full-length sequence of the Bacteralis dorsalis sodium ion channel gene on the NCBI website, compare the conserved sequences, and design the forward...

Embodiment 2

[0056] Embodiment 2 Transformation of recombinant plasmid

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

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

[0059] pMD-19Vector 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 sodium ion channel gene. The recombinant plasmid of the fragment was named pMD-19-C1R1, and the recombinant plasmid of the reverse fragment was named pMD-19-C2R2.

[0063] 2. Transformation of Escherichia coli with recombinant plasmids

[0064] Transform the recombinant plasmid pMD-19-C1R1 of the forward fragment...

Embodiment 3

[0074] Example 3 Construction of RNA interference expression vector

[0075] 1. Ligation of the target fragment with the vector PFGC5941

[0076] (1) Carry out double digestion with XhoI and NcoI of the forward target fragment plasmid pMD-19-C1R1 of the sodium ion channel gene extracted in Example 2 and the carrier PFGC5941 respectively. The enzyme digestion system is: Buffer45μL, XhoI1.5μL, NcoI1.5μL, BSA0.5μL, pMD-19-C1R1 or PFGC594120μL, with ddH 2 O Adjust the total volume of the enzyme digestion system to 50 μL, put it into a centrifuge tube, and bathe in a water bath at 37°C for 4 hours.

[0077] (2) Take 3-5 μL of the digested product of pMD-19-C1R1 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-C1R1 digested by XhoI and NcoI is as follows Figure 5 shown.

[0078...

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Abstract

The invention provides an RNA interference vector of bactrocera dorsalis sodium ion channel 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 sodium ion channel 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 sodium ion channel gene and a construction method and application thereof. Background technique [0002] Bactrocera dorsalis, also known as Bactrocera 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. , 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 veget...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/66A01H5/00
Inventor 冉春岳建苏刘浩强丛林李鸿筠
Owner CITRUS RES INST SOUTHWEST UNIV
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