Bactrocera dorsalis Taiman gene as well as qRT-PCR (quantitative Reverse Transcription-Polymerase Chain Reaction) detection method and siRNA (small interference Ribonucleic Acid) thereof

A kind of fruit fly, gene technology, applied in its siRNA field, can solve the problems such as unseen, achieve the effect of high silencing efficiency and good application prospect

Inactive Publication Date: 2017-05-17
SOUTHWEST UNIVERSITY
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  • Abstract
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AI Technical Summary

Problems solved by technology

Since humans, higher animals, and plants do not contain juvenile hormone, the juvenile hormone signaling pathway gene is relatively safe as a target for pests. The new insecticides developed based on this approach have great potential in the control of pests, but At present, there are few related studies on the Taiman gene of the Bactrocera dorsalis juvenile hormone pathway, and there is no report on the dsRNA targeting the Taiman gene of the Bactrocera dorsalis juvenile hormone signaling pathway

Method used

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  • Bactrocera dorsalis Taiman gene as well as qRT-PCR (quantitative Reverse Transcription-Polymerase Chain Reaction) detection method and siRNA (small interference Ribonucleic Acid) thereof
  • Bactrocera dorsalis Taiman gene as well as qRT-PCR (quantitative Reverse Transcription-Polymerase Chain Reaction) detection method and siRNA (small interference Ribonucleic Acid) thereof
  • Bactrocera dorsalis Taiman gene as well as qRT-PCR (quantitative Reverse Transcription-Polymerase Chain Reaction) detection method and siRNA (small interference Ribonucleic Acid) thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Embodiment 1, Bacteralis dorsalis Taiman gene alternative splice body sequence cloning

[0026] Follow the steps below:

[0027] 1) Use the RNA extraction kit RNeasy Plus Micro Kit to extract the total RNA of B. dorsalis according to the instructions, and then use the reverse transcription kit Perfectreal time RT reagent to reverse transcribe 1 μg of total RNA into cDNA according to the instructions.

[0028] 2) Using the cDNA obtained above as a template, according to the Bacteralis dorsalis Taiman gene sequence predicted on NCBI and the conserved structural sequence of the gene, the full-length amplification primers were designed in segments, and a total of 5 pairs of segmented amplification primers were designed for full-length amplification. Long amplification: BdTaiS1-F / BdTaiS1-R, BdTaiS2-F / BdTaiS2-R, BdTaiS3-F / BdTaiS3-R, BdTaiS4-F / BdTaiS4-R, BdTaiS5-F / BdTaiS5-R. The PCR conditions were: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 30 s, annealing ...

Embodiment 2

[0043] Embodiment two, the dsRNA of Bactrocera dorsalis Taiman gene

[0044] 1) According to the Bacteralis dorsalis Taiman gene sequence obtained by cloning, design the upstream and downstream primers (sequence containing the T7 promoter) for amplifying the Taiman gene: T7-Tai-core-F (as shown in SEQ ID NO: 16) , T7-Tai-core-R (as shown in SEQID NO: 17), the primer sequences are respectively:

[0045] T7-Tai-core-F: 5'-TAATACGACTCACTATAGGGGCTCTCAATTCGACGACACC-3';

[0046] T7-Tai-core-R: 5'-TAATACGACTCACTATAGGGACCTGACCGTTAAGCAGTGA-3'.

[0047] Use the RNA extraction kit RNeasy Plus Micro Kit to extract the total RNA of Bacteralis dorsalis according to the instructions, and then use the reverse transcription kit Perfect real time RT reagent to reverse transcribe 1 μg of total RNA into cDNA according to the instructions. The reverse transcription system 20 μl, including: total RNA 1 μl (about 1 μg), 5×PrimeScriptBuffer 4 μl, PrimeScriptRT Enzyme Mix I 1 μl, Oligo dT Primer (50...

Embodiment 3

[0055] Embodiment 3, the siRNA of Bactrocera dorsalis Taiman gene

[0056] According to the 5 alternative splicing forms of the Bacteralis dorsalis Taiman gene obtained in Example 1, for the Taiman genes Taiman-A, Taiman-B, Taiman-C, Taiman-D, Taiman-E gene, correspondingly designed 5 specific siRNA sequences and sent them to Guangzhou Ruibo Biological Co., Ltd. for synthesis. The siRNA sequences are as follows:

[0057] siTaiman-A: 5'-GUGUAUCUGCACUACUUAAUA-3', as shown in SEQ ID NO:21;

[0058] siTaiman-B: 5'-UCUAAUGUCGGUCCUGGCGGU-3', as shown in SEQ ID NO:22;

[0059] siTaiman-C: 5'-AAUUCUUUUCAAGGUGGCGGC-3', as shown in SEQ ID NO:23;

[0060] siTaiman-D: 5'-UCAACAUUUACCUGCAGGUAA-3', as shown in SEQ ID NO:24;

[0061] siTaiman-E: 5'-GUAUUGUUGGCUUUGGUGGCA-3', as shown in SEQ ID NO:25;

[0062] siGFP: 5'-GCCACAACGUCUAUAUCAUGG-3', as shown in SEQ ID NO:26.

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Abstract

The invention discloses a bactrocera dorsalis Taiman gene. The nucleotide sequence of the bactrocera dorsalis Taiman gene is shown as SEQ ID NO: 15; siRNA (small interference Ribonucleic Acid) of the bactrocera dorsalis Taiman gene shown as the SEQ ID NO: 15 is provided; and the sequence is shown as SEQ ID NO: 25. The invention further provides a method for carrying out qRT-PCR (quantitative Reverse Transcription-Polymerase Chain Reaction) detection on the gene shown as the SEQ ID NO: 15. Sequences of upstream and downstream primers used for qPCR (quantitative Polymerase Chain Reaction) are shown as SEQ ID NO: 37 and SEQ ID NO: 38. The invention further provides application of the siRNA shown as SEQ ID NO: 25 in aspects of development, metamorphosis and reproduction regulation of bactrocera dorsalis and application of the siRNA to prevention and control of the bactrocera dorsalis or application to preparation of bactrocera dorsalis insecticides. The problem that the bactrocera dorsalis has no effective siRNA of the Taiman gene at present is solved, and the bactrocera dorsalis Taiman gene has a good application prospect in the aspect of researching and developing novel insecticides.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to Bactrocera dorsalis Taiman gene, its qRT-PCR detection method and its siRNA. Background technique [0002] As an important quarantine pest in my country, Bactrocera dorsalis is the most serious hazard in the fruit and vegetable industry. It is also considered to be an important restrictive factor in the export of agricultural products in my country's international trade and the sustainable development of domestic agriculture. Bacteralis dorsalis has a wide range of hosts, strong reproductive capacity and overlapping generations, which greatly increases the difficulty of control. In recent years, chemical control methods have been mainly used for the control of Bactrocera dorsalis. Although it is simple and convenient, long-term use of a single agent, increasing drug resistance is an urgent problem to be solved. [0003] RNA interference (RNA interference, RNAi) is ...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/113C12Q1/68A01N57/16A01P7/04
CPCA01N57/16C07K14/43577C12N15/113C12N2310/14C12Q1/686C12Q2563/107
Inventor 豆威刘鸿王进军岳勇牛金志
Owner SOUTHWEST UNIVERSITY
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