Method for controlling CSBV (Chinese bee Sacbrood Virus) by utilizing RNA interference technology

A cystic larvae, RNA interference technology, applied in recombinant DNA technology, DNA/RNA fragments, DNA preparation, etc., to achieve the effect of inhibiting replication, low cost and strong specificity

Active Publication Date: 2014-04-30
FUJIAN AGRI & FORESTRY UNIV
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Problems solved by technology

Chemical drug treatment causes food safety problems of drug residues in bee pr

Method used

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  • Method for controlling CSBV (Chinese bee Sacbrood Virus) by utilizing RNA interference technology
  • Method for controlling CSBV (Chinese bee Sacbrood Virus) by utilizing RNA interference technology
  • Method for controlling CSBV (Chinese bee Sacbrood Virus) by utilizing RNA interference technology

Examples

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Embodiment 1

[0018] Collect larvae or pupae suffering from cystic larvae of Apis mellifera and store them in a -80°C refrigerator.

[0019] Amplification of the RNA-dependent RNA polymerase (RdRp) gene of Chinese sacbrood virus (CSBV) replicator. The total RNA of diseased larvae was extracted by Trizol method, the downstream primers of RdRp gene were used for reverse transcription to synthesize cDNA, and the target gene fragment was amplified by PCR. The gene fragment was connected to the PMD18-T vector, and the positive clones were sent to Huada Gene Company for sequencing.

[0020] Construction of dsRdRp prokaryotic expression vector. Ligate the target fragment amplified in (2) to the L4440 vector under the action of T4 ligase, and ligate overnight at 16°C; transfer the T4 enzyme ligation product into HT115 competent cells, and spread it on the medium containing 100 μg / mL ampicillin Antibiotics on LB solid medium.

[0021] Inducible expression of dsRdRp. Inoculate the positive clones...

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Abstract

The invention relates to a method for controlling CSBV (Chinese Bee Sacbrood Virus) by utilizing an RNA interference technology. The method comprises the following steps: synthesizing dsRdRp through a bacteria prokaryotic induction expression method by utilizing an RNA-dependent RNA polymerase (RdRp) gene which is specific to the CSBV and related to virus replication, and adding double-strand RNA into feed for feeding larva of 1-2 days. The double-strand RNA is expressed by Escherichia coli induction, is low in cost and has a good curing effect for controlling the CSBV. The feeding concentration of dsRdRp is 0.5 microgram/ml, and the pupation rate and eclosion rate of the larva are respectively 70 percent and 55 percent and are obviously improved compared with the pupation rate of 10 percent and the eclosion rate of 0 percent in the larva fed of dsGFP.

Description

technical field [0001] The invention relates to a method for preventing and treating cystic larvae of honeybees by RNA interference technology, and belongs to the field of insect disease prevention and control. Background technique [0002] Chinese bee ( Apis cerana cerana , referred to as the bee), is a native bee species in my country. Honey bees are vulnerable to various diseases, including viruses, bacteria, fungi and protozoa. At present, at least 22 kinds of viral diseases have been found in bees. Among them, cystic larvae is a kind of viral disease with serious damage. Cystic larvae of honey bees was first discovered in Guangdong Province in 1972, and then spread rapidly across the country. After bee larvae are infected by Apis cerevisiae cystic larvae virus, they cannot pupate. A large amount of liquid and virus particles gather in the insect body, and the color of the insect body gradually changes from pearly white to light yellow. After death, the honeybee body...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61P31/14C12N15/70C12N15/113C12N15/10
Inventor 夏晓翠魏太云周冰峰
Owner FUJIAN AGRI & FORESTRY UNIV
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