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;


【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)


    [1]Boden D, Pusch O, Lee F, Tucker L, Ramratnam B. Human immunodeficiency virus type 1 escape from RNA interference. Journal of Virology, 2003, 77(21): 11531-11535.

    [2]Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science, 2002, 296(5567): 550-553.

    [3]Jiang L, Xia QY. The progress and future of enhancing antiviral capacity by transgenic technology in the silkworm Bombyx mori. Insect Biochemistry and Molecular Biology, 2014, 48: 1-7.

    [4]Isobe R, Kojima K, Matsuyama T, Quan GX, Kanda T, Tamura T, Sahara K, Asano SI, Bando H. Use of RNAi technique to confer enhanced resistance to Bm NPV on transgenic silkworms. Archives of Virology, 2004, 149(10): 1931-1940.

    [5]Kanginakudru S, Royer C, Edupalli SV, Jalabert A, Mauchamp B, Chandrashekaraiah, Prasad SV, Chavancy G, Couble P, Nagaraju J. Targeting ie-1 gene by RNAi induces baculoviral resistance in lepidopteran cell lines and in transgenic silkworms. Insect Molecular Biology, 2007, 16(5): 635-644.

    [6]Subbaiah EV, Royer C, Kanginakudru S, Satyavathi VV, Babu AS, Sivaprasad V, Chavancy G, Da Rocha M, Jalabert A, Mauchamp B, Basha I, Couble P, Nagaraju J. Engineering silkworms for resistance to baculovirus through multigene RNA interference. Genetics, 2013, 193(1): 63-75.

    [7]Zhang J, He Q, Zhang CD, Chen XY, Chen XM, Dong ZQ, Li N, Kuang XX, Cao MY, Lu C, Pan MH. Inhibition of Bm NPVreplication in silkworm cells using inducible and regulated artificial micro RNA precursors targeting the essential viral gene lef-11. Antiviral Research, 2014, 104: 143-152.

    [8]Zhou F, Chen RT, Lu Y, Liang S, Wang MX, Miao YG. piggy Bac transposon-derived targeting sh RNA interference against the Bombyx mori nucleopolyhedrovirus(Bm NPV). Molecular Biology Reports, 2014, 41(12): 8247-8254.

    [9]Lebbink RJ, Lowe M, Chan T, Khine H, Wang XY, Mc Manus MT. PolymeraseⅡpromoter strength determines efficacy of micro RNA adapted sh RNAs. PLo S One, 2011, 6(10): e26213.

    [10]Mc Bride JL, Boudreau RL, Harper SQ, Staber PD, Monteys AM, Martins I, Gilmore BL, Burstein H, Peluso RW, Polisky B, Carter BJ, Davidson BL. Artificial mi RNAs mitigate sh RNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(15): 5868-5873.

    [11]MangéA, Couble P, Prudhomme JC. Two alternative promoters drive the expression of the cytoplasmic actin A4gene of Bombyx mori. Gene, 1996, 183(1/2): 191-199.

    [12]Masumoto M, Ohde T, Shiomi K, Yaginuma T, Niimi T. ABaculovirus immediate-early gene, ie1, promoter drives efficient expression of a transgene in both Drosophila melanogaster and Bombyx mori. PLo S One, 2012, 7(11): e49323.

    [13]Ma ZM, Gong CL. Clone and analysis of Bombyx mori nuclear polyhedrosis virus suzhou strain ie-1 promoter sequence. Jiangsu Sericulture, 2001, (3): 1-6. (in Chinese)

    [14]Wang Y, Ye XQ, Wu YL, Gui MY, Zuo ZH. Comparison of four promoters for transient expression of RFP reporter gene in cultured Bombyx mori cells(Bm-e-HNU5). Acta Entomologica Sinca, 2006, 49(2): 167-171. (in Chinese)

    [15]Zhuang LF, Wei H, Lin JR, Zhong BX. Identification of Bombyx mori hsp70 promoter and its function. Chinese Journal of Cell Biology, 2001, 35(5): 503-509. (in Chinese)

    [16]Wang ZX, Zhao L, Li WB. Progress of plant inducible promoter. Soybean Science&Technology, 2011, (3): 5-9. (in Chinese)

    [17]Li HP, Yao XW, Guo N, Yang XM. Experience on analyzing the promoter activity with luciferase and green florence protein. Chinese Journal of Laboratory Diagnosis, 2010, 14(7): 1017-1019. (in Chinese)

    [18]Pan MH, Cai XJ, Liu M, Lv J, Tang H, Tan J, Lu C. Establishment and characterization of an ovarian cell line of the silkworm, Bombyx mori. Tissue and Cell, 2010, 42(1): 42-46.

    [19]Pan MH, Xiao SQ, Chen M, Hong XJ, Lu C. Establishment and characterization of two embryonic cell lines of Bombyx mori. In Vitro Cellular&Developmental Biology-Animal, 2007, 43(2): 101-104.

    [20]Dong ZQ, Zhang J, Chen XM, He Q, Cao MY, Wang L, Li HQ, Xiao WF, Pan CX, Lu C, Pan MH. Bombyx mori nucleopolyhedrovirus ORF79 is a per os infectivity factor associated with the PIF complex. Virus Research, 2014, 184: 62-70.

    [21]Qu J, Ye J, Fang RX. Artificial micro RNA-mediated virus resistance in plants. Journal of Virology, 2007, 81(12): 6690-6699.

    [22]Schwarz DS, Hutvágner G, Du TT, Xu ZS, Aronin N, Zamore PD. Asymmetry in the assembly of the RNAi enzyme complex. Cell, 2003, 115(2): 199-208.

    [23]Song GQ, Sink KC, Walworth AE, Cook MA, Allison RF, Lang GA. Engineering cherry rootstocks with resistance to Prunus necrotic ring spot virus through RNAi-mediated silencing. Plant Biotechnology Journal, 2013, 11(6): 702-708.

    [24]Todd JW, Passarelli AL, Miller LK. Eighteen baculovirus genes, including lef-11, p35, 39K, and p47, support late gene expression. Journal of Virology, 1995, 69(2): 968-974.

    [25]Lin GY, Blissard GW. Analysis of an autographa californica nucleopolyhedrovirus lef-11 knockout: lef-11 is essential for viral DNA replication. Journal of Virology, 2002, 76(6): 2770-2779.

This Article



Vol 56, No. 10, Pages 1561-1570

October 2016


Article Outline



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