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A method for gene function verification against Panonychus citrus

A technology of Panonychus citrus and gene function, which is applied in the fields of biochemical equipment and methods, microbial determination/inspection, DNA preparation, etc., can solve the problems such as the difficulty of introducing dsRNA into citrus mites, and the difficulty of dsRNA, and achieve gene silencing efficiency High, wide application range, simple operation effect

Inactive Publication Date: 2018-10-23
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, compared with Panonychus citrus, which is smaller than general insects, it is more difficult to introduce dsRNA by microinjection, and there is no report on introducing dsRNA into Panonychus citrus.

Method used

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  • A method for gene function verification against Panonychus citrus
  • A method for gene function verification against Panonychus citrus
  • A method for gene function verification against Panonychus citrus

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Embodiment Construction

[0024] The sources of chemical reagents used in the embodiments of the present invention are as follows:

[0025] RNeasy Plus Micro Kit (QIAGEN, Germany)

[0026] PrimeSTARMax Premix (Takara, Japan)

[0027] Gel Recovery and Purification Kit (Takara, Japan)

[0028] TranscriptAid T7HighYield Transcription Kit (Thermo fisher Scientific, USA) Perfect real time RT reagent (Takara, Japan)

[0029] qPCR Master Mix (Promega, USA)

[0030] TranscriptAid Enzyme Mix (Thermo fisher Scientific, USA)

[0031] ATP / CTP / GTP / UTP Mix (Thermo Fisher Scientific, USA)

[0032] PrimeScriptRT Enzyme Mix I (Thermo Fisher Scientific, USA)

[0033] Oligo dT Primer (Thermo Fisher Scientific, USA)

[0034] Random 6mers (Thermo fisher Scientific, USA)

[0035] The method for gene function verification for Panonychus citrus, according to the following steps:

[0036] 1. Synthesis of dsRNA:

[0037] 1) Obtain the cDNA sequence of the COPB2 (β subunit of coatomer protein complex) gene (provided b...

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Abstract

The invention relates to a method for gene function identification of orange panonychus citri. The method comprises the following steps: (1) preparing a target gene dsRNA liquid: synthesizing a corresponding dsRNA liquid according to a target gene to be inhibited; (2) preparing a leaf disc by using an orange leaf and washing; (3) drying the leaf disc until the leaf disc obviously shrinks, containing the dsRNA liquid prepared in the step 1 by using a centrifugal tube cover, and floating the leaf disc on the dsRNA liquid until the leaf disc is recovered to the original shape and does not shrink; (4) putting the orange panonychus citri on the floating leaf disc, putting the leaf disc in an incubator and culturing for 60 to 80 hours at a condition that the temperature is 22 to 28 DEG C, the humidity is 55% to 65% and the illumination to darkness ratio is 14h:10h; (5) after the culture is ended, collecting orange panonychus citri bodies, extracting RNA, and detecting the expression condition of the target gene provided in the step 1. The method disclosed by the invention can be used for solving the problem that no effective dsRNA introduction method is provided for the orange panonychus citri at present.

Description

technical field [0001] The invention relates to the fields of growth and development regulation and genetic engineering of insects, in particular to a method for verifying gene functions against Panonychus citrus. Background technique [0002] Panonychus citrus is a worldwide harmful mite, which has a great impact on the yield and quality of citrus. At present, chemical control is still an important measure to control Panonychus citrus. Improper application of chemical pesticides not only affects fruit safety, but also leads to serious drug resistance. Obtaining insect-resistant transgenic materials based on RNA interference technology has been successful in crops such as rice, vegetables, and cotton. [0003] RNA interference (RNAi), an important gene silencing phenomenon discovered in recent years, refers to the highly conserved phenomenon in the evolution process, induced by double-stranded RNA (dsRNA), and the efficient and specific degradation of homologous RNA. dsRNA...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6851C12Q1/6888C12N15/113C12N15/10
CPCC12Q1/6851C12Q2525/207C12Q2545/113
Inventor 王进军廖重宇夏文凯冯英财李刚沈晓敏
Owner SOUTHWEST UNIV