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

Abstract

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

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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References

[1] Bartel D P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell,2004, 116(2): 281-297.

[2] Stefani G, Slack F J. Small non-coding RNAs in animal development. Natural Reviews of Molecular and Cellular Biology,2008, 9(3): 219-230.

[3] Bushati N, Cohen S M. microRNA functions. Annual Review of Cellular and Developmental Biology,2007, 23: 175-205.

[4] Ambros V. The functions of animal microRNAs. Nature,2004, 431(7006): 350-355.

[5] Liu S P, Lucas K J, Roy S, Ha J, Raikhel A S. Mosquito-specific microRNA-1174 targets serine hydroxymethyltransferase to control key functions in the gut. Proceedings of the National Academy of Sciences of the United States of America,2014, 111(40): 14460-14465.

[6] Aravin A, Gaidatzis D, Pfeffer S, Lagos-Quintana M, Landgraf P, Iovino N, Morris P, Brownstein M J, Kuramochi-Miyagawa S, Nakano T, Chien M, Russo J J, Ju J, Sheridan R, Sander C, Zavolan M, Tuschl T. A novel class of small RNAs bind to MILI protein in mouse testes. Nature,2006, 442(7099): 203-207.

[7] Miller L K. Baculoviruses for foreign gene expression in insect cells. Biotechnology,1988, 10: 457-465.

[8] Lee R C, Feinbaum R L, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell,1993, 75(5): 843-854.

[9] Reinhart B J, Slack F J, Basson M, Pasquinelli A E, Bettinger J C, Rougvie A E, Horvitz H R, Ruvkun G. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature,2000, 403(6772): 901-906.

[10] Pasquinelli A E, Reinhart B J, Slack F, Martindale M Q, Kuroda M I, Maller B, Hayward D C, Ball E E, Degnan B, Muller P, Spring J, Srinivasan A, Fishman M, Finnerty J, Corbo J, Levine M, Leahy P, Davidson E, Ruvkun G. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature,2000, 408(6808): 86-89.

[11] Grosshans H, Johnson T, Reinert K L, Gerstein M, Slack F J. The temporal patterning microRNA let-7 regulates several transcription factors at the larval to adult transition in C. elegans. Developmental Cell, 2005, 8(3): 321-330.

[12] Sempere L F, Sokol N S, Dubrovsky E B, Berger E M, Ambros V. Temporal regulation of microRNA expression in Drosophila melanogaster mediated by hormonal signals and broad-complex gene activity. Developmental Biology,2003, 259(1): 9-18.

[13] Chawla G, Sokol N S. Hormonal activation of let-7-C microRNAs via EcR is required for adult Drosophila melanogaster morphology and function. Development,2012, 139(10): 1788-1797.

[14] Luhur A, Chawla G, Sokol N S. MicroRNAs as components of systemic signaling pathways in Drosophila melanogaster. Current Topics in Developmental Biology,2013, 105: 97-123.

[15] Liu S P, Li D, Li Q B, Zhao P, Xiang Z H, Xia Q Y. MicroRNAs of Bombyx mori identified by Solexa sequencing. BMC Genomics,2010, 11: 148.

[16] Liu S P, Xia Q Y, Zhao P, Cheng T C, Hong K L, Xiang Z H. Characterization and expression patterns of let-7 microRNA in the silkworm (Bombyx mori). BMC Developmental Biology, 2007, 7: 88.

[17] Yu X M, Zhou Q, Li S C, Luo Q B, Cai Y M, Lin W C, Chen H, Yang Y, Hu S N, Yu J. The silkworm (Bombyx mori) microRNAs and their expressions in multiple developmental stages. Plos One,2008, 3(8): e2997.

[18] Liu S P, Gao S, Zhang D Y, Yin J Y, Xiang Z H, Xia Q Y. MicroRNAs show diverse and dynamic expression patterns in multiple tissues of Bombyx mori. BMC Genomics,2010, 11: 85.

[19] Ling L, Ge X, Li Z, Zeng B, Xu J, Aslam AF, Song Q, Shang P, Huang Y, Tan A. MicroRNA Let-7 regulates molting and metamorphosis in the silkworm, Bombyx mori. Insect Biochemistry and Molecular Biology,2014, 53: 13-21.

[20] Ciccarone V C, Polayes D A, Luckow V A. Generation of recombinant baculovirus DNA in E. coli using a baculovirus shuttle vector. Methods in Molecular Medicine,1998, 13: 213-235.

[21] Lihoradova O A, Ogai I D, Podpisnova M M, Slack J M, Azimova S S. Expression of foreign gene by cysteine proteinase null recombinant baculovirus. Molecular Biology, 2008, 42(2): 328-334.

[22] Guo T Q, Wang J Y, Guo X Y, Wang S P, Lu C D. Transient in vivo gene delivery to the silkworm Bombyx mori by EGT-null recombinant AcNPV using EGFP as a reporter. Archives of Virology,2005, 150(1): 93-105.

[23] Yu X, Hua J, Hu G, Zhang J, Ma L. Expression of recombinant proteins in insect cells by their direct infection with Escherichia coli transformed with baculovirus bacmids. Acta Virologica,2014, 58(1): 61-68.

[24] Huang Y, Zou Q, Shen X J, Yu X L, Wang Z B, Cheng X C. Construction of baculovirus expression vector of miRNAs and its expression in insect cells. Molecular Genetics, Microbiology and Virology,2012, 27(2): 85-90.

[25] Zhang Y, Tian B, Xia H, Guo T, Wang J, Wang S, Wei Z, Lu C. Spread of recombinant Autographa californica nucleopolyhedrovirus in various tissues of silkworm Bombyx mori determined by real-time PCR. Analytical Biochemistry,2008, 373(1): 147-153.

[26] 刘仕平, 夏庆友. Northern杂交检测家蚕microRNA的技术流程. 蚕业科学,2014, 40(4): 724-729.

Liu S P, Xia Q Y. Protocol of Northern blotting hybridization for microRNA detection of silkworm (Bombyx mori). Science of Sericulture, 2014, 40(4): 724-729. (in Chinese)

[27] Xia Q, Guo Y, Zhang Z, Li D, Xuan Z, Li Z, Dai F, Li Y, Cheng D, Li R. Complete resequencing of 40 genomes reveals domestication events and genes in silkworm (Bombyx). Science,2009, 326(5951): 433-436.

[28] Baird G S, Zacharias D A, Tsien R Y. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proceedings of the National Academy of Sciences of the United States of America,2000, 97(22): 11984-11989.

[29] 刘仕平, 黄亚玺, 尹纪云, 吴小燕, 周兰庭, 王伟, 夏庆友. 家蚕Bmyan基因的克隆表达和作为 microRNA 7靶基因的验证. 生物工程学报, 2015, 31(11): 1-11.

Liu S P, Huang Y X, Yin J Y, Wu X Y, Zhou L T, Wang W, Xia Q Y. Cloning and expression profile of Bmyan in the silkworm (Bombyx mori) and experimental validation as one target of microRNA 7. Chinese Journal of Biotechnology, 2015, 31(11): 1-11. (in Chinese)

[30] Heid C A, Stevens J, Livak K J, Williams P M. Real time quantitative PCR. Genome Research,1996, 6(10): 986-994.

[31] Winer J, Jung C K, Shackel I, Williams P M. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Analytical Biochemistry, 1999, 270(1): 41-49.

[32] Barry G F. A broad-host-range shuttle system for gene insertion into the chromosomes of gram-negative bacteria. Gene,1988, 71(1): 75-84.

[33] Liu S P, Zhang L, Li Q B, Zhao P, Duan J, Cheng D D, Xiang Z H, Xia Q Y. MicroRNA expression profiling during the life cycle of the silkworm (Bombyx mori). BMC Genomics,2011, 12: 284.

[34] Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell,1993, 75(5): 855-862.

[35] Jiang J, Ge X, Li Z, Wang Y, Song Q, Stanley D W, Tan A, Huang Y. MicroRNA-281 regulates the expression of ecdysone receptor (EcR) isoform B in the silkworm, Bombyx mori. Insect Biochemistry and Molecular Biology,2013, 43(8): 692-700.

[36] Varghese J, Cohen S M. microRNA miR-14 acts to modulate a positive autoregulatory loop controlling steroid hormone signaling in Drosophila. Genes & Development,2007, 21(18): 2277-2282.

[37] Wu Y C, Chen C H, Mercer A, Sokol N S. Let-7-complex microRNAs regulate the temporal identity of Drosophila mushroom body neurons via chinmo. Developmental Cell, 2012, 23(1): 202-209.

[38] 王颖, 余泽华, 姚汉超, 陶德定, 陈曲侯. 苜蓿银纹夜蛾核型多角体病毒的感染对Sf9细胞周期的影响. 中国病毒学,2002, 17(2): 132-136.

Wang Y, Yu Z H, Yao H C, Tao D D, Chen Q H. Affection of infection of Autographa californica nucleopolyhedrovirus on Sf9 cell cycle. Virologica Sinica, 2002, 17(2): 132-136. (in Chinese)

[39] Singh C P, Singh J, Nagaraju J. bmnpv-miR-3 facilitates BmNPV infection by modulating the expression of viral P6.9 and other late genes in Bombyx mori. Insect Biochemistry and Molecular Biology,2014, 49: 59-69.

[40] Rybak A, Fuchs H, Smirnova L, Brandt C, Pohl E E, Nitsch R, Wulczyn F G. A feedback loop comprising lin-28 and let-7 controls pre-let-7 maturation during neural stem-cell commitment. Nature Cell Biology,2008, 10(8): 987-993.

[41] Newman M A, Thomson J M, Hammond S M. Lin-28 interaction with the Let-7 precursor loop mediates regulated microRNA processing. RNA,2008, 14(8): 1539-1549.

[42] Heo I, Joo C, Cho J, Ha M, Han J, Kim V N. Lin28 mediates the terminal uridylation of let-7 precursor microRNA. Molecular Cell,2008, 32(2): 276-284.

[43] Melton C, Judson R L, Blelloch R. Opposing microRNA families regulate self-renewal in mouse embryonic stem cells. Nature,2010, 463(7281): 621-626.

[44] Li M A, He L. microRNAs as novel regulators of stem cell pluripotency and somatic cell reprogramming. Bioessays,2012, 34(8): 670-680.

[45] Lee Y, Kim M, Han J, Yeom K H, Lee S, Baek S H, Kim V N. MicroRNA genes are transcribed by RNA polymerase II. The EMBO Journal,2004, 23(20): 4051-4060.

[46] Wang G, Wang Y, Shen C, Huang Y W, Huang K, Huang T H, Nephew K P, Li L, Liu Y. RNA polymerase II binding patterns reveal genomic regions involved in microRNA gene regulation. PLoS One,2010, 5(11): e13798.

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

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