The rna interference carrier of Bactrocera dorsalis sodium ion channel gene and its construction method and application

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: 2016-06-15
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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  • The rna interference carrier of Bactrocera dorsalis sodium ion channel gene and its construction method and application
  • The rna interference carrier of Bactrocera dorsalis sodium ion channel gene and its construction method and application
  • The rna interference carrier of Bactrocera dorsalis sodium ion channel gene and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Cloning and recovery and purification of the sodium ion channel 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 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 and...

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 550bp, 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) 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.

[0076] (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.

[0077...

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Abstract

The invention discloses an RNA interference carrier of Bactrocera dorsalis sodium ion channel gene, a construction method and application thereof, and belongs to the technical field of genetic engineering. In the present invention, the carrier PFGC5941 is used as the framework, and the forward and reverse specific target fragments of the Bacteralis dorsalis sodium ion channel gene are respectively inserted on both sides of the intron by double enzyme digestion to form a very important region of the RNA interference carrier, and the constructed RNA After the interference expression vector is transformed into the plant, under the drive of its strong promoter CaMV35S, it transcribes to form a "hairpin structure" of double-stranded RNA in the somatic cells, so that the homologous gene fragments in the body after Bactrocera dorsalis eat the plants The normal expression and translation are affected, which interferes with the normal growth and development of insects, achieves the purpose of plant resistance to Bactrocera dorsalis, plays an important role in the control of Bactrocera dorsalis, and reduces the use of chemical pesticides.

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] 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 a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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