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System construction method for obtaining a large amount of ericerus pela dsRNA in vitro

A construction method and technology of wax worms, applied in the field of system construction for obtaining a large amount of dsRNA from wax worms in vitro, can solve the problems of low dsRNA yield, easy degradation, insufficient transfection methods, etc., and achieve the effect of easy popularization and application

Inactive Publication Date: 2020-09-01
THE RES INST OF RESOURCES INSECTS RIRI OF THE CHINESE ACADEMY OF FORESTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the large amount of absorption on the body surface, dsRNA is unstable and easy to degrade in the outside world. It needs to be sprayed and absorbed on the body surface skin of white wax insects for many times. The yield of dsRNA synthesized by the kit is low (ul measurement), which is not enough for spraying. The transfection method and the synthesis of a large number of kits make the cost of the experiment high. Therefore, in some RNA interference experiments, how to prepare high-yield dsRNA is a problem that needs to be solved by genetic engineering.

Method used

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  • System construction method for obtaining a large amount of ericerus pela dsRNA in vitro
  • System construction method for obtaining a large amount of ericerus pela dsRNA in vitro
  • System construction method for obtaining a large amount of ericerus pela dsRNA in vitro

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Embodiment 1

[0040] A method for obtaining white wax worm double-stranded RNA white wax worm in large quantities in vitro, comprising the steps of:

[0041] In step (1), the total RNA of wax worm is extracted and reverse-transcribed into cDNA, followed by PCR amplification, and the amplified product and the L4440 vector plasmid are respectively digested with SacI and SalI ( image 3 ), the double digested product was ligated overnight at 4°C with T4 ligase to obtain a recombinant plasmid 302a1-L4440 containing the 302a1 gene fragment;

[0042] Step (2), transforming the 302a1-L4440 recombinant plasmid obtained in step (1) into the Escherichia coli competent cell HT115 (DE3) strain, coating a solid medium to screen a single colony, and obtaining 302a1-L4440-HT115 cells;

[0043] In step (3), the 302a1-L4440-HT115 thalline obtained in step (2) is cultured on a liquid shaker to OD 600 =0.38, add IPTG induction, obtain the dsRNA bacterium liquid containing the gene fragment of the wax worm 30...

Embodiment 2

[0046] In step (1), the total RNA of wax worm is extracted and reverse-transcribed into cDNA, followed by PCR amplification, and the amplified product and the L4440 vector plasmid are respectively digested with SacI and SalI ( image 3 ), the double digested product was ligated overnight at 4°C with T4 ligase to obtain a recombinant plasmid 302a1-L4440 containing the 302a1 gene fragment;

[0047] In step (2), the 302a1-L4440 recombinant plasmid obtained in step (1) is transformed into Escherichia coli competent cell HT115 (DE3) strain, and a single colony is screened by coating a solid medium to obtain 302a1-L4440-HT115 bacterium ( Figure 4 );

[0048] In step (3), the 302a1-L4440-HT115 thalline obtained in step (2) is cultured on a liquid shaker to OD 600 =0.42, add IPTG induction for 3.5h, obtain the dsRNA bacterial liquid containing the 302a1 gene fragment of the white wax worm;

[0049] In step (4), the dsRNA bacterial liquid containing the 302a1 gene fragment of the wh...

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Abstract

The invention relates to a system construction method for obtaining a large amount of ericerus pela dsRNA in vitro. The method comprises the following steps: firstly, extracting total RNA of ericeruspela and performing reverse transcription to obtain cDNA, and then, carrying out PCR amplification; respectively carrying out double enzyme digestion on an amplification product and an L4440 vector plasmid; connecting products after double enzyme digestion, and transforming the obtained recombinant plasmid into an Escherichia coli competent cell HT115 (DE3) strain; coating a solid culture medium to screen a single colony, and carrying out liquid shaking culture on the single colony until OD600 is equal to 0.38-0.42; and adding IPTG induction and concentrating the bacterial liquid for RNA interference experiments. Escherichia coli contains a ericerus pela target gene through single-cell cloning, a large amount of dsRNA bacterial liquid containing ericerus pela genes is obtained through metabolism and a large amount of self-propagation replication of escherichia coli, the dsRNA bacterial liquid is sprayed to ericerus pela larvae and enters bodies from the surfaces of the ericerus pela bodies, and the expression quantity of the target genes is effectively interfered.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for constructing a system for obtaining dsRNA in large quantities in vitro. Background technique [0002] White wax worm, Ericerus pela (Chavannes), commonly known as wax worm, white wax worm belongs to Hemiptera (Hemipetera) Coccoidea (Ericerus) in classification (Chavannes, 1819). Cerachinensis is a scale insect with great economic value. The 2nd instar male larvae of Cerachinensis live on the branches of host plants and secrete white waxy coverings. The wax secreted by this insect is called Cerachinensis (Cerachinensis, also known as Cerachinensis ), that is, the secretion of wax worms, which is a Chinese specialty. White wax is a natural polymer compound with high melting point and chemical stability. It has the characteristics of moisture-proof, lubricating, and glossy. It is an important chemical raw material and is widely used in chemical...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/70C12N15/113A01N57/16A01P7/04
CPCC12N15/10C12N15/70C12N15/113A01N57/16C12N2310/14C12N2320/30
Inventor 陈航凌晓霏陆沁张金稳王伟伟柳鹏飞汪伟陈晓鸣
Owner THE RES INST OF RESOURCES INSECTS RIRI OF THE CHINESE ACADEMY OF FORESTRY