Bemisia tabaci defensin gene segment and dsRNA thereof based on gene silencing technology

A technology of whitefly and defensins, applied in the fields of molecular biology and agricultural biology, can solve the problems of unclear molecular mechanism of whitefly virus transmission, less research on functional genes of whitefly, and the prevalence of plant virus diseases, etc., to achieve Reduce mechanical damage, stabilize the double-strand structure, and improve the degradation effect of RNase

Inactive Publication Date: 2015-04-01
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The insect not only feeds on a large amount of plant juice, causing the plants to wither, but also spreads a variety of plant viruses.
However, the molecular mechanism of B. tabaci virus transmission is still unclear, and there are relatively few studies on the functional genes of B. tabaci

Method used

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  • Bemisia tabaci defensin gene segment and dsRNA thereof based on gene silencing technology
  • Bemisia tabaci defensin gene segment and dsRNA thereof based on gene silencing technology
  • Bemisia tabaci defensin gene segment and dsRNA thereof based on gene silencing technology

Examples

Experimental program
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Effect test

preparation example Construction

[0030] The method for synthesizing the whitefly defensin gene dsRNA is to use the constructed pGEM-def plasmid as a template, with the sequence being the upstream primer P3 of SEQ ID NO.5, and the sequence being the downstream primer P4 of SEQ ID NO.6 The dsRNA of the defensin gene of Bemisia tabaci was obtained by PCR amplification. The synthetic method of the dsRNA of described whitefly defensin gene fragment preferably comprises the following steps:

[0031] 1) According to the verified defensin gene fragment sequence, design and synthesize primer P3 with the sequence of SEQ ID NO.5 and primer P4 with the sequence of SEQ ID NO.6, use the constructed pGEM-def plasmid as a template, and use the upstream primer P1 Perform PCR amplification with the downstream primer P4 containing the T7 promoter, the upstream primer P3 containing the T7 promoter, and the downstream primer P2 to obtain two PCR products with the T7 promoter.

[0032] 2) The gel recovery and purification methods...

Embodiment example 1

[0042] Implementation case 1: Obtaining the target fragment of the defensin gene in Bemisia tabaci

[0043] 1.1, TRIzol method to extract total RNA

[0044] (1) Freeze a certain amount ((about 200)) of Bemisia tabaci for 1 min, put it into a 1.5 ml centrifuge tube, add 1 ml of TRIzol reagent, thoroughly grind and homogenate, and let stand at room temperature for 5 min.

[0045] (2) Add 0.2ml chloroform to the centrifuge tube, shake for 15s, transfer the mixture into a TIANGEN centrifuge tube, and let stand for 2min.

[0046] (3) Centrifuge at 12000g for 15min at 4°C, take the supernatant, and transfer it to a new 1.5ml centrifuge tube.

[0047] (4) Add 0.5ml of isopropanol to the centrifuge tube, mix the liquid in the tube gently, and let stand at room temperature for 10 minutes.

[0048] (5) Centrifuge at 12000 g for 10 min at 4°C, and discard the supernatant.

[0049] (6) Add 1ml of 75% ethanol to the centrifuge tube, gently wash the precipitate, centrifuge at 7500g for 5mi...

Embodiment 2

[0067] Example 2: Synthesis and detection of whitefly defensin gene dsRNA

[0068] The method for synthesizing the whitefly defensin gene dsRNA is to use the constructed pGEM-def plasmid as a template, with the sequence being the upstream primer P3 of SEQ ID NO.5, and the sequence being the downstream primer P4 of SEQ ID NO.6 The dsRNA of the defensin gene of Bemisia tabaci was obtained by PCR amplification. The synthetic method of the dsRNA of described whitefly defensin gene fragment preferably comprises the following steps:

[0069] 2.1. Obtaining synthetic dsRNA template

[0070] (1) According to the verified defensin gene fragment sequence, design and synthesize the upstream primer P3 with the sequence of SEQ ID NO.5 and the downstream primer P4 with the sequence of SEQ ID NO.6. Using the constructed pGEM-def plasmid as a template, the upstream primer P1 and the downstream primer P4 containing the T7 promoter, the upstream primer P3 containing the T7 promoter and the do...

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Abstract

The invention relates to molecular biology and agricultural biotechnology, and aims to provide a bemisia tabaci defensin gene segment and dsRNA thereof based on the gene silencing technology. The nucleotide sequence of the bemisia tabaci defensin encoding gene segment is as shown in the SEQ ID NO.1. An improved artificial feeding method is adopted to carry out RNAi interference experiment, and compared with an injection method, the improved artificial feeding method has the advantages that mechanical injury of insect bodies can be reduced, operation is convenient, a true sense of natural feeding is realized, and the method can be widely popularized. The dsRNA of the synthesized defensin gene can be used for effectively silencing the defensin gene, has a stable double-stranded structure, is capable of preventing degradation of RNA enzyme, and can be used for laying a foundation for establishing bemisia tabaci gene function researches by utilizing the RNA interference technology and providing a new experimental means for new strategy of insect control.

Description

technical field [0001] The invention belongs to the fields of molecular biology and agricultural biotechnology. The invention relates to a whitefly defensin gene segment and its dsRNA based on gene silencing technology. Background technique [0002] The whitefly Bemisia tabaci (Gennadius) has become a worldwide invasive and disastrous insect, and it has become a disaster all over the world, causing heavy losses. The co-infestation of Bemisia tabaci shows different damage symptoms to different plants. The damaged leaves of leafy vegetables such as cabbage and cauliflower are shrunk, yellowed, and withered; Tomatoes are affected and the fruit ripens unevenly. The insect not only eats a large amount of plant juice, causing the plants to wither, but also spreads a variety of plant viruses. However, the molecular mechanism of virus transmission by B. tabaci is still unclear, and there are relatively few studies on the functional genes of B. tabaci. [0003] RNAi (RNA interfer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/113C12N15/63C12N15/10A23K1/16
Inventor 陈学新王知知时敏
Owner ZHEJIANG UNIV
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