Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (3): 581-592.doi: 10.3864/j.issn.0578-1752.2016.03.016

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles     Next Articles

Bac-to-Bac Baculovirus System Facilitates Overexpression of let-7 Cluster MicroRNAs of Silkworm (Bombyx mori)

HE Ting1,2, YIN Quan1,2, WANG Wei1, HUANG Ya-xi1, WU Xiao-yan1, XIA Qing-you1,2, LIU Shi-ping1,2   

  1. 1State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716
    2College of Biotechnology, Southwest University, Chongqing 400716
  • Received:2015-08-24 Online:2016-02-01 Published:2016-02-01

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Abstract: 【Objective】The objective of this study is to construct the Bac-to-Bac baculovirus system overexpressing bmo-let-7, bmo-miR-100, bmo-miR-2795 of bmo-let-7 cluster (bmo-let-7-C), as such will hopefully contribute the future functional study of microRNAs (miRNAs) in the silkworm (Bombyx mori).【Method】The primary precursor of each microRNA in the bmo-let-7-C (pri-let-7, pri-miR-100, pri-miR-2795) and the whole let-7-C sequence were cloned, respectively. Using the gene of red fluorescent protein (RFP) as the reporter and pFastBac1 as the shuttle vector, each cloned miRNA precursor and reporter gene were combined into the baculovirus genome through Tn7 transposons, and thus the recombinant baculovirus plasmids (rBacmids) for each miRNA and the whole cluster were obtained. At 72 h post transfection of these recombinant Bacmids into the cell line of Spodoptera frugiperda (Sf9), the signal of red fluorescent protein was examined under the microscope and the expression of miRNA was evaluated by qRT-PCR. To collect the recombinant baculovirus with infection activity, the Sf9 cell culture was centrifuged at 72 h post transfection, and the supernatant containing the viruses was used to infect the newly cultured Sf9 cells or injected into early 5th instar larval B. mori. Also, the signal of red fluorescent protein and the expression of miRNA were examined.【Result】The primary precursor of each miRNA and the whole let-7-C sequence were successfully combined into the baculovirus genome and the overexpressing vectors for each miRNA and the whole cluster were finally obtained. At 72 h post transfection in the Sf9 cell line, a red fluorescent protein was observed under the microscope and the overexpression of microRNAs were confirmed by qRT-PCR. The recombinant baculovirus with infection activity caused the stronger detection signals of red fluorescent protein and significant overexpression of each miRNA in the newly cultured Sf9 cell. After the injection of recombinant baculovirus into newly ecdysed 5th instar larvae of the B. mori, the infection of virus in various B. mori tissues was determined by real-time PCR and bmo-let-7, bmo-miR-100, and bmo-miR-2795 showed significant up-regulation in the B. mori injected with each Bac-miRNA, but only bmo-miR-2795 was dramatically overexpressed in the B. mori infected with Bac-let-7-C. Further examination revealed the tissue-specific infection of the recombinant baculoviruses and the significant overexpression of miR-2795 was confirmed in the hemocyte, midgut, and fat body.【Conclusion】The baculovirus expression system was successfully used to overexpress the cluster miRNAs of B. mori in the cell line of S. frugiperda (Sf9) and in the larval B. mori body, and might be helpful to the mechanical and functional exploration of let-7 cluster and other miRNAs in this species.

Key words: silkworm (Bombyx mori), microRNA, bmo-let-7 cluster, baculovirus expression system, overexpression

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