A bactrocera dorsalis Taiman gene, a detecting method of variable spliceosomes thereof and dsRNA of the gene

A technology of Bactrocera dorsalis and genes, applied in the field of dsRNA, can solve problems such as unseen and achieve high silencing efficiency

Inactive Publication Date: 2017-07-04
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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

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  • A bactrocera dorsalis Taiman gene, a detecting method of variable spliceosomes thereof and dsRNA of the gene
  • A bactrocera dorsalis Taiman gene, a detecting method of variable spliceosomes thereof and dsRNA of the gene
  • A bactrocera dorsalis Taiman gene, a detecting method of variable spliceosomes thereof and dsRNA of the gene

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Experimental program
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Effect test

Embodiment 1

[0027] Embodiment 1, Bacteralis dorsalis Taiman gene alternative splice body sequence cloning

[0028] Follow the steps below:

[0029] 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.

[0030] 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

[0045] Embodiment two, the dsRNA of Bactrocera dorsalis Taiman gene

[0046] 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:

[0047] T7-Tai-core-F: 5'-TAATACGACTCACTATAGGGGCTCTCAATTCGACGACACC-3';

[0048] T7-Tai-core-R: 5'-TAATACGACTCACTATAGGGACCTGACCGTTAAGCAGTGA-3'.

[0049] 2) Use the RNA extraction kit RNeasy Plus Micro Kit to extract the total RNA of Bactrocera dorsalis according to the instructions, and then use the reverse transcription kit Perfectreal time RTreagent to reverse transcribe 1 μg of total RNA into cDNA according to the instructions. 20 μl, including: total RNA 1 μl (about 1 μg), 5×PrimeScriptBuffer 4 μl, PrimeScriptRT Enzyme Mix I 1 μl, Oligo dT Primer (50 μM) 1 μl, Random 6mers (100 μM)...

Embodiment 3

[0059] Embodiment 3, the siRNA of Bactrocera dorsalis Taiman gene

[0060] 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:

[0061] siTaiman-A: 5'-GUGUAUCUGCACUACUUAAUA-3', as shown in SEQ ID NO:21;

[0062] siTaiman-B: 5'-UCUAAUGUCGGUCCUGGCGGU-3', as shown in SEQ ID NO:22;

[0063] siTaiman-C: 5'-AAUUCUUUUCAAGGUGGCGGC-3', as shown in SEQ ID NO:23;

[0064] siTaiman-D: 5'-UCAACAUUUACCUGCAGGUAA-3', as shown in SEQ ID NO:24;

[0065] siTaiman-E: 5'-GUAUUGUUGGCUUUGGUGGCA-3', as shown in SEQ ID NO:25;

[0066] siGFP: 5'-GCCACAACGUCUAUAUCAUGG-3', as shown in SEQ ID NO:26.

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Abstract

A bactrocera dorsalis Taiman gene is disclosed. The nucleotide sequence of the gene is shown as SEQ ID NO:11 or 12 or 13 or 14. Segmented amplification primer pairs for amplifying the full-length bactrocera dorsalis Taiman gene are also disclosed. Sequences of the primers are shown as SEQ ID NO:1 to SEQ ID NO:10 in order. The invention also discloses dsRNA of the gene, a synthetic method thereof and applications of the dsRNA. The invention discloses a method of subjecting the gene to qRT-PCR detection. Sequences of primers used in qPCR are shown as SEQ ID NO:27 and SEQ ID NO:28. The invention also discloses methods of performing qRT-PCR detection on the Taiman gene shown as the SEQ ID NO:11, 12, 13 and 14 respectively and primer sequences are shown as SEQ ID NO:29 to SEQ ID NO:36 respectively.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a detection method of Bactrocera dorsalis Taiman gene and its alternative splicing body and dsRNA. 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 i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/11C12N15/113C12N15/10C12Q1/68A01P7/04
CPCC07K14/43577C12N15/10C12N15/11C12N15/113C12N2310/14C12Q1/6888C12Q2531/113C12Q2565/125
Inventor 豆威王进军刘鸿李慧敏蒋红波
Owner SOUTHWEST UNIV
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