利用Bac-to-Bac杆状病毒系统超量表达家蚕let-7簇microRNAs
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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)micro RNAs:bmo-let-7、bmo-mi R-100和bmo-mi R-2795,为研究家蚕micro RNA的功能提供参考。【方法】克隆家蚕let-7 mi RNA簇(bmo-let-7 cluster,bmo-let-7-C)上各mi RNA前体(pri-let-7、pri-mi R-100与pri-mi R-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p Fast Bac1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(Ac NPV)基因组上,获得各重组杆状病毒质粒(recombinant baculovirus plasmid,r Bacmid):Bac-let-7、Bac-mi R-100、Bac-mi R-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mi RNAs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mi RNAs的表达。【结果】成功将bmo-let-7-C上各mi RNA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mi RNA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mi RNA显著过量表达;通过离心收集的各mi RNA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mi RNA均显著过量表达。将各mi RNA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mi RNA,但注射Bac-let-7-C后只有mi R-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mi RNAs,为研究家蚕let-7簇和其他mi RNAs的产生机制和功能提供了参考。
【Objective】The objective of this study is to construct the Bac-to-Bac baculovirus system overexpressing bmo-let-7, bmo-mi R-100, bmo-mi R-2795 of bmo-let-7 cluster(bmo-let-7-C), as such will hopefully contribute the future functional study of micro RNAs(mi RNAs) in the silkworm(Bombyx mori).【Method】The primary precursor of each micro RNA in the bmo-let-7-C(pri-let-7, pri-mi R-100, pri-mi R-2795) and the whole let-7-C sequence were cloned, respectively. Using the gene of red fluorescent protein(RFP) as the reporter and p Fast Bac1 as the shuttle vector, each cloned mi RNA precursor and reporter gene were combined into the baculovirus genome through Tn7 transposons, and thus the recombinant baculovirus plasmids(r Bacmids) for each mi RNA 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 mi RNA was evaluated by q RT-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 mi RNA were examined.【Result】The primary precursor of each mi RNA and the whole let-7-C sequence were successfully combined into the baculovirus genome and the overexpressing vectors for each mi RNA 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 micro RNAs were confirmed by q RT-PCR. The recombinant baculovirus with infection activity caused the stronger detection signals of red fluorescent protein and significant overexpression of each mi RNA 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-mi R-100, and bmo-mi R-2795 showed significant up-regulation in the B. mori injected with each Bac-mi RNA, but only bmo-mi R-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 mi R-2795 was confirmed in the hemocyte, midgut, and fat body.【Conclusion】The baculovirus expression system was successfully used to overexpress the cluster mi RNAs 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 mi RNAs in this species.
【Objective】The objective of this study is to construct the Bac-to-Bac baculovirus system overexpressing bmo-let-7, bmo-mi R-100, bmo-mi R-2795 of bmo-let-7 cluster(bmo-let-7-C), as such will hopefully contribute the future functional study of micro RNAs(mi RNAs) in the silkworm(Bombyx mori).【Method】The primary precursor of each micro RNA in the bmo-let-7-C(pri-let-7, pri-mi R-100, pri-mi R-2795) and the whole let-7-C sequence were cloned, respectively. Using the gene of red fluorescent protein(RFP) as the reporter and p Fast Bac1 as the shuttle vector, each cloned mi RNA precursor and reporter gene were combined into the baculovirus genome through Tn7 transposons, and thus the recombinant baculovirus plasmids(r Bacmids) for each mi RNA 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 mi RNA was evaluated by q RT-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 mi RNA were examined.【Result】The primary precursor of each mi RNA and the whole let-7-C sequence were successfully combined into the baculovirus genome and the overexpressing vectors for each mi RNA 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 micro RNAs were confirmed by q RT-PCR. The recombinant baculovirus with infection activity caused the stronger detection signals of red fluorescent protein and significant overexpression of each mi RNA 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-mi R-100, and bmo-mi R-2795 showed significant up-regulation in the B. mori injected with each Bac-mi RNA, but only bmo-mi R-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 mi R-2795 was confirmed in the hemocyte, midgut, and fat body.【Conclusion】The baculovirus expression system was successfully used to overexpress the cluster mi RNAs 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 mi RNAs in this species.
引文
[1]Bartel D P.Micro RNAs: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.micro RNA functions.Annual Review of Cellular and Developmental Biology,2007,23:175-205.
[4]Ambros V.The functions of animal micro RNAs.Nature,2004,431(7006):350-355.
[5]Liu S P,Lucas K J,Roy S,Ha J,Raikhel A S.Mosquito-specific micro RNA-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 micro RNA 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 micro RNA 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 micro RNAs via Ec R is required for adult Drosophila melanogaster morphology and function.Development,2012,139(10):1788-1797.
[14]Luhur A,Chawla G,Sokol N S.Micro RNAs 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.Micro RNAs 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 micro RNA 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)micro RNAs 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.Micro RNAs 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.Micro RNA 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 Ac NPV 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 mi RNAs 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micro RNA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 micro RNA 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 Ds Red,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micro RNA 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 micro RNA 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.Micro RNA 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.Micro RNA-281 regulates the expression of ecdysone receptor(Ec R)isoform B in the silkworm,Bombyx mori.Insect Biochemistry and Molecular Biology,2013,43(8):692-700.
[36]Varghese J,Cohen S M.micro RNA mi R-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 micro RNAs 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-mi R-3 facilitates Bm NPV 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 micro RNA 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 micro RNA.Molecular Cell,2008,32(2):276-284.
[43]Melton C,Judson R L,Blelloch R.Opposing micro RNA families regulate self-renewal in mouse embryonic stem cells.Nature,2010,463(7281):621-626.
[44]Li M A,He L.micro RNAs 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.Micro RNA 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 micro RNA gene regulation.PLo S One,2010,5(11):e13798.
[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.micro RNA functions.Annual Review of Cellular and Developmental Biology,2007,23:175-205.
[4]Ambros V.The functions of animal micro RNAs.Nature,2004,431(7006):350-355.
[5]Liu S P,Lucas K J,Roy S,Ha J,Raikhel A S.Mosquito-specific micro RNA-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 micro RNA 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 micro RNA 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 micro RNAs via Ec R is required for adult Drosophila melanogaster morphology and function.Development,2012,139(10):1788-1797.
[14]Luhur A,Chawla G,Sokol N S.Micro RNAs 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.Micro RNAs 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 micro RNA 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)micro RNAs 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.Micro RNAs 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.Micro RNA 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 Ac NPV 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 mi RNAs 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micro RNA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 micro RNA 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 Ds Red,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micro RNA 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 micro RNA 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.Micro RNA 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.Micro RNA-281 regulates the expression of ecdysone receptor(Ec R)isoform B in the silkworm,Bombyx mori.Insect Biochemistry and Molecular Biology,2013,43(8):692-700.
[36]Varghese J,Cohen S M.micro RNA mi R-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 micro RNAs 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-mi R-3 facilitates Bm NPV 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 micro RNA 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 micro RNA.Molecular Cell,2008,32(2):276-284.
[43]Melton C,Judson R L,Blelloch R.Opposing micro RNA families regulate self-renewal in mouse embryonic stem cells.Nature,2010,463(7281):621-626.
[44]Li M A,He L.micro RNAs 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.Micro RNA 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 micro RNA gene regulation.PLo S One,2010,5(11):e13798.