利用Bac-to-Bac杆状病毒系统超量表达家蚕let-7簇microRNAs

doi:10.3864/j.issn.0578-1752.2016.03.016

摘要/Abstract

摘要： 【目的】利用Bac-to-Bac杆状病毒表达系统超量表达家蚕（Bombyx mori）let-7 簇（cluster）microRNAs：bmo-let-7、bmo-miR-100和bmo-miR-2795，为研究家蚕microRNA的功能提供参考。【方法】克隆家蚕let-7 miRNA簇（bmo-let-7 cluster，bmo-let-7-C）上各miRNA前体(pri-let-7、pri-miR-100与pri-miR-2795)和bmo-let-7-C全长序列，以红色荧光蛋白(red fluorescent protein，RFP)基因为报告基因，昆虫细胞表达载体pFastBac1为穿梭载体，通过Tn7转座子把目的基因和报告基因转座到杆状病毒A. californica nucleopolyhedrovirus (AcNPV)基因组上，获得各重组杆状病毒质粒（recombinant baculovirus plasmid，rBacmid）：Bac-let-7、Bac-miR-100、Bac-miR-2795和Bac-let-7-C。将重组Bacmid转染草地贪夜蛾（Spodoptera frugiperda）卵巢细胞系Sf9，72 h后用荧光显微镜检查红色荧光蛋白信号，荧光定量PCR检测miRNAs的表达。对转染的Sf9细胞进行、离心、收集上清液，获得具感染力的重组病毒，用于侵染新培养的Sf9细胞和注射家蚕幼虫个体，72 h后用荧光显微镜检查红色荧光蛋白信号，荧光定量PCR检测miRNAs的表达。【结果】成功将bmo-let-7-C上各miRNA前体和bmo-let-7-C全长序列构建到杆状病毒基因组上，获得了各miRNA及bmo-let-7-C的过量表达载体。将各重组过量表达载体分别转染草地贪夜蛾细胞系Sf9，72 h后在显微镜下观察到了红色荧光蛋白信号，荧光定量PCR检测结果表明各miRNA显著过量表达；通过离心收集的各miRNA重组过量表达病毒粒子感染新培养的Sf9细胞72 h后检测到了更强的红色荧光蛋白信号，定量PCR结果表明各miRNA均显著过量表达。将各miRNA的重组病毒注射到家蚕5龄1 d幼虫体内后，均能显著超量表达相应miRNA，但注射Bac-let-7-C后只有miR-2795显著过量表达，并有明显的组织差异性，在丝腺中没有过量表达，在脂肪体、血液和中肠中显著过量表达。【结论】利用杆状病毒过量表达系统在草地贪夜蛾细胞系Sf9和家蚕个体中超量表达了家蚕let-7簇miRNAs，为研究家蚕let-7簇和其他miRNAs的产生机制和功能提供了参考。

关键词: 家蚕, 微小RNA, let-7簇, 杆状病毒表达系统, 过量表达

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

何婷，尹权，王伟，黄亚玺，吴小燕，夏庆友，刘仕平. 利用Bac-to-Bac杆状病毒系统超量表达家蚕let-7簇microRNAs[J]. 中国农业科学, 2016, 49(3): 581-592.

HE Ting, YIN Quan, WANG Wei, HUANG Ya-xi, WU Xiao-yan, XIA Qing-you, LIU Shi-ping. Bac-to-Bac Baculovirus System Facilitates Overexpression of let-7 Cluster MicroRNAs of Silkworm (Bombyx mori)[J]. Scientia Agricultura Sinica, 2016, 49(3): 581-592.

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