Optimization of RNA Interference Strategy for lef-11 gene of Bombyx mori Nucleopolyhedrovirus

Tingting Chen1 Zhanqi Dong1 Nan Hu1 Zhigang Hu1 Cheng Lu1,2 Minhui Pan1,2

(1.State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China 400716)
(2.Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Chongqing, China 400716)

【Abstract】[Objective] Gene lef-11 is one of the high conservative late expression factors in Bombyx mori nucleopolyhedrovirus (BmNPV), and contributes to virus replication and proliferation. The effective RNA interference will increase the resistance of host cells to BmNPV. [Methods] Choosing two classical skeletons of sh-RNA-loop and silkworm endogenous miRNA skeleton, we constructed the targeted lef-11 interference vectors: pIZ-shRNA1, pIZ-shRNA2 and pIZ-DsRed-amiR279 and optimized these vectors via different promoters. [Results] First, comparing the interference efficiency of shRNA-based and miRNA-based RNAi vectors on the same site, we found the interference efficiency of pIZ-DsRed-amiR279 on target gene was more than 90%, better than that of shRNA-based RNAi and could be applied for follow-up experiments. Second, we evaluated the antiviral effect of different interference vectors driven by A4, IE1, IE1-295, IE2, IE2-339, hr3A4 and hsp70 promoters and found that the most active promoter had less interference effect on the target gene and IE1 was the optimal among the screened seven promoters, which demonstrated that the accumulation of pre-miRNA did not contribute to interference. [Conclusion] The results showed that the interference effect on the target gene depended on many kinds of factors, such as the interference skeleton, the activity of promoter and the function of target gene.

【Keywords】 Bombyx mori; BmNPV; RNAi; lef-11; vector optimization; promoter;

【DOI】

【Funds】 National Natural Science Foundation of China (31472152) Supported by the Project of National Natural Science Foundation of China(31472152) State of Sericulture Industry Technology System (CARS-22) by the State of Sericulture Industry Technology System(CARS-22)

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(Translated by LU Zufu)

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This Article

ISSN:0001-6209

CN:11-1995/Q

Vol 56, No. 10, Pages 1561-1570

October 2016

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Abstract

  • 1 Materials and methods
  • 2 Results and analysis
  • 3 Discussion
  • References