应用于pol-miR-26a,26b靶基因检测的psiCHECK-dmrt1-3'UTR报告质粒的建立
|
摘要
为明确牙鲆雄性性别相关关键基因dmrt1 (doublesex and mab-3-related transcription factor 1)是否为pol-miR-26a、pol-miR-26b作用的靶基因,本研究利用PCR技术克隆了dmrt13'UTR区,并成功引入了特殊限制性内切酶PemⅠ和NotⅠ的识别位点;将得到的dmrt1 3'UTR区片段和psiCHECK-2载体进行双酶切、连接后得到野生型重组质粒;并且利用定点诱变法对dmrt1 3'UTR区进行体外定点诱变,将pol-miR-26a、pol-miR-26b识别的靶序列ACTGAA突变为TGACTT,形成突变型重组质粒。经琼脂糖凝胶电泳鉴定、双酶切及测序分析,成功获得了可用于pol-miR-26a、pol-miR-26b的靶基因dmrt1验证的野生型psiCHECK-dmrt1-3'UTR和突变型psiCHECK-mutated dmrt1-3'UTR的报告质粒,为进一步研究pol-miR-26a、pol-miR-26b的靶基因dmrt1鉴定和功能奠定了基础。
To identify whether the key genes dmrt1(Doublesex and mab-3-related transcription factor 1) related to male sex of Paralichthys olivaceus is the target gene of pol-miR-26 a and pol-miR-26 b, in the study, the dmrt1 3'UTR region was cloned by the techniques of PCR, and the recognition sites of specific restriction enzymes PemI and NotI were successfully introduced. The obtained dmrt1 3'UTR region fragment and psiC HECK-2 vector were digested using double enzyme and then they were linked, and the wild type recombinant plasmid was obtained. In vitro site-directed mutagenesis was carried out by site directed mutagenesis for dmrt1 3'UTR region, and the target sequence ACTGAA identified by pol-miR-26 a and pol-miR-26 b was mutated to TGACTT, forming the mutant recombinant plasmid. By agarose gel electrophoresis and double enzyme digestion and sequencing analysis, the wild-type psiC HECK-dmrt1-3'UTR and mutant psiC HECK-mutated dmrt1-3'UTR reporter vectors were successfully obtained. They could be used for verifying the target gene dmrt1 of pol-miR-26 a, pol-miR-26 b, which would lay the foundation for further study on the identification and function of the target gene dmrt1 of pol-miR-26 a and pol-miR-26 b.
To identify whether the key genes dmrt1(Doublesex and mab-3-related transcription factor 1) related to male sex of Paralichthys olivaceus is the target gene of pol-miR-26 a and pol-miR-26 b, in the study, the dmrt1 3'UTR region was cloned by the techniques of PCR, and the recognition sites of specific restriction enzymes PemI and NotI were successfully introduced. The obtained dmrt1 3'UTR region fragment and psiC HECK-2 vector were digested using double enzyme and then they were linked, and the wild type recombinant plasmid was obtained. In vitro site-directed mutagenesis was carried out by site directed mutagenesis for dmrt1 3'UTR region, and the target sequence ACTGAA identified by pol-miR-26 a and pol-miR-26 b was mutated to TGACTT, forming the mutant recombinant plasmid. By agarose gel electrophoresis and double enzyme digestion and sequencing analysis, the wild-type psiC HECK-dmrt1-3'UTR and mutant psiC HECK-mutated dmrt1-3'UTR reporter vectors were successfully obtained. They could be used for verifying the target gene dmrt1 of pol-miR-26 a, pol-miR-26 b, which would lay the foundation for further study on the identification and function of the target gene dmrt1 of pol-miR-26 a and pol-miR-26 b.
引文
Backes C.,Meese E.,Lenhof H.P.,and Keller A.,2010,A dictionary on microRNAs and their putative target pathways,Nucleic Acids Res.,38(13):4476-4486
Bartel D.P.,2004,MicroRNAs:genomics,biogenesis,mechanism,and function,Cell,116(2):281-297
Brennecke J.,Stark A.,Russell R.B.,and Cohen S.M.,2005,Principles of microrna-target recognition,PLo S Biology,3(3):e85
Gu Y.F.,Lei Z.,and Chen X.W.,2014,Differential expression analysis of Paralichthys olivaceus microRNAs in adult ovary and testis by deep sequencing,Gen.Comp.Endocrinol.,204:181-184
Lei L.,Li X.Y.,and Zhang Y.H.,2013,Advances in the study of Dmrt gene in vertebrate sex determination and differentiation,Zhongguo Zuzhi Huaxue Yu Xibao Huaxue Zazhi(Chinese Journal of Histochemistry and Cell Chemistry),22(3):269-276(雷蕾,李忻怡,张育辉,2013,Dmrt基因在脊椎动物性别决定与分化中的研究进展,中国组织化学与细胞化学杂志,22(3):269-276)
Lin F.,Li R.,Pan Z.X.,Zhou B.,Yu D.B.,Wang X.G.,Ma X.S.,Han J.,Shen M.,and Liu H.L.,2012,mi R-26b promotes granulosa cell apoptosis by targeting ATM during follicular atresia in porcine ovary,PLo S One,7(6):e38640
Fu Y.S.,Shi Z.Y.,Wu M.L.,Zhang J.L.,Jia L.,and Chen X.W.,2011,Identification and differential expression of microR-NAs during metamorphosis of the Japanese flounder(Paralichthys olivaceus),PLo S One,6(7):e22957
Luna C.,Li G.,Qiu J.,Epstein D.L.,and Gonzalez P.,2009,Role of mi R-29b on the regulation of the extracellular matrix in human trabecular meshwork cells under chronic oxidative stress,Mol.Vis.,15:2488-2497
Matsuda M.,Nagahama Y.,Shinomiya A.,Sato T.,Matsuda C.,Kobayashi T.,Morrey C.E.,Shibata N.,Asakawa S.,Shimizu N.,Hori H.,Hamaguchi S.,and Sakaizumi M.,2002,DMY is a Y-specific DM-domain gene required for male development in the medaka fish,Nature,417(6888):559-563
Murdock C.,and Wibbels T.,2003,Expression of dmrt1 in a turtle with temperature-dependent sex determination,Cytogenet.Genome.Res.,101(3-4):302-308
Reddy A.M.,Zheng Y.,Jagadeeswaran G.,Macmil S.L.,Graham W.B.,Roe B.A.,Desilva U.,Zhang W.,and Sunkar R.,2009,Cloning characterization and expression analysis of porcine microRNAs,BMC Genomics,10(1):65
Smith C.A.,Roeszler K.N.,Thomas O.,Cummins D.M.,Farlie P.G.,Doran T.J.,and Sinclair A.H.,2009,The avian z-linked gene dmrt1 is required for male sex determination in the chicken,Nature,461(7261):267-271
Sun Z.H.,Liu H.J.,Si F.,Wang Y.F.,and Jiang X.F.,2008,Histological observation of gonadal differentiation incultured Japanese flounder Paralichthys olivaceus,Dalian Shuichan Xueyuan Xuebao(Journal of Dalian Fisheries University),23(6):451-454(孙朝徽,刘海金,司飞,王玉芬,姜秀凤,2008,养殖牙鲆性腺分化的组织学观察,大连海洋大学学报,23(6):451-454)
Wen A.Y.,You F.,Sun P.,Xu D.D.,Wu Z.H.,Ma D.Y.,Li J.,and Zhang P.J.,2010,Cloning of dmrt1 gene and its tissue expression analyses compared with that of P450arom gene in olive flounder(Paralichthys olivaceus),Haiyang Kexue(Marine Sciences),34(11):97-102(文爱韵,尤锋,孙鹏,徐冬冬,吴志昊,马得友,李军,张培军,2010,牙鲆dmrt1基因的克隆及其与P450arom基因的组织表达分析,海洋科学,34(11):97-102)
Wen A.Y.,You F.,Sun P.,Xu D.D.,Wu Z.H.,Ma D.Y.,Li J.,and Zhang P.J.,2011,Study on cloning and expression of gene sox9 in olive fiounder(Paralichthys olivaceus),Haiyang Kexue Jinzhan(Advances in Marine Science),29(1):97-104(文爱韵,尤锋,孙鹏,徐冬冬,吴志昊,马得友,李军,张培军,2011,牙鲆sox9基因的克隆与表达研究,海洋科学进展,29(1):97-104)
You F.,Xu J.H.,Ni J.,Sun W.,Zhu X.P.,Xu D.D.,Xu Y.L.,and Zhang P.J.,2008,Study on artificial induction of mitogynogenetic diploid in Paralichthys olivaceus,Gaojishu Tongxun(Chinese High Technology Letters),18(8):874-880(尤锋,许建和,倪静,孙威,朱香萍,徐冬冬,徐永立,张培军,2008,牙鲆同质雌核发育人工诱导研究,高技术通讯,18(8):874-880)
Bartel D.P.,2004,MicroRNAs:genomics,biogenesis,mechanism,and function,Cell,116(2):281-297
Brennecke J.,Stark A.,Russell R.B.,and Cohen S.M.,2005,Principles of microrna-target recognition,PLo S Biology,3(3):e85
Gu Y.F.,Lei Z.,and Chen X.W.,2014,Differential expression analysis of Paralichthys olivaceus microRNAs in adult ovary and testis by deep sequencing,Gen.Comp.Endocrinol.,204:181-184
Lei L.,Li X.Y.,and Zhang Y.H.,2013,Advances in the study of Dmrt gene in vertebrate sex determination and differentiation,Zhongguo Zuzhi Huaxue Yu Xibao Huaxue Zazhi(Chinese Journal of Histochemistry and Cell Chemistry),22(3):269-276(雷蕾,李忻怡,张育辉,2013,Dmrt基因在脊椎动物性别决定与分化中的研究进展,中国组织化学与细胞化学杂志,22(3):269-276)
Lin F.,Li R.,Pan Z.X.,Zhou B.,Yu D.B.,Wang X.G.,Ma X.S.,Han J.,Shen M.,and Liu H.L.,2012,mi R-26b promotes granulosa cell apoptosis by targeting ATM during follicular atresia in porcine ovary,PLo S One,7(6):e38640
Fu Y.S.,Shi Z.Y.,Wu M.L.,Zhang J.L.,Jia L.,and Chen X.W.,2011,Identification and differential expression of microR-NAs during metamorphosis of the Japanese flounder(Paralichthys olivaceus),PLo S One,6(7):e22957
Luna C.,Li G.,Qiu J.,Epstein D.L.,and Gonzalez P.,2009,Role of mi R-29b on the regulation of the extracellular matrix in human trabecular meshwork cells under chronic oxidative stress,Mol.Vis.,15:2488-2497
Matsuda M.,Nagahama Y.,Shinomiya A.,Sato T.,Matsuda C.,Kobayashi T.,Morrey C.E.,Shibata N.,Asakawa S.,Shimizu N.,Hori H.,Hamaguchi S.,and Sakaizumi M.,2002,DMY is a Y-specific DM-domain gene required for male development in the medaka fish,Nature,417(6888):559-563
Murdock C.,and Wibbels T.,2003,Expression of dmrt1 in a turtle with temperature-dependent sex determination,Cytogenet.Genome.Res.,101(3-4):302-308
Reddy A.M.,Zheng Y.,Jagadeeswaran G.,Macmil S.L.,Graham W.B.,Roe B.A.,Desilva U.,Zhang W.,and Sunkar R.,2009,Cloning characterization and expression analysis of porcine microRNAs,BMC Genomics,10(1):65
Smith C.A.,Roeszler K.N.,Thomas O.,Cummins D.M.,Farlie P.G.,Doran T.J.,and Sinclair A.H.,2009,The avian z-linked gene dmrt1 is required for male sex determination in the chicken,Nature,461(7261):267-271
Sun Z.H.,Liu H.J.,Si F.,Wang Y.F.,and Jiang X.F.,2008,Histological observation of gonadal differentiation incultured Japanese flounder Paralichthys olivaceus,Dalian Shuichan Xueyuan Xuebao(Journal of Dalian Fisheries University),23(6):451-454(孙朝徽,刘海金,司飞,王玉芬,姜秀凤,2008,养殖牙鲆性腺分化的组织学观察,大连海洋大学学报,23(6):451-454)
Wen A.Y.,You F.,Sun P.,Xu D.D.,Wu Z.H.,Ma D.Y.,Li J.,and Zhang P.J.,2010,Cloning of dmrt1 gene and its tissue expression analyses compared with that of P450arom gene in olive flounder(Paralichthys olivaceus),Haiyang Kexue(Marine Sciences),34(11):97-102(文爱韵,尤锋,孙鹏,徐冬冬,吴志昊,马得友,李军,张培军,2010,牙鲆dmrt1基因的克隆及其与P450arom基因的组织表达分析,海洋科学,34(11):97-102)
Wen A.Y.,You F.,Sun P.,Xu D.D.,Wu Z.H.,Ma D.Y.,Li J.,and Zhang P.J.,2011,Study on cloning and expression of gene sox9 in olive fiounder(Paralichthys olivaceus),Haiyang Kexue Jinzhan(Advances in Marine Science),29(1):97-104(文爱韵,尤锋,孙鹏,徐冬冬,吴志昊,马得友,李军,张培军,2011,牙鲆sox9基因的克隆与表达研究,海洋科学进展,29(1):97-104)
You F.,Xu J.H.,Ni J.,Sun W.,Zhu X.P.,Xu D.D.,Xu Y.L.,and Zhang P.J.,2008,Study on artificial induction of mitogynogenetic diploid in Paralichthys olivaceus,Gaojishu Tongxun(Chinese High Technology Letters),18(8):874-880(尤锋,许建和,倪静,孙威,朱香萍,徐冬冬,徐永立,张培军,2008,牙鲆同质雌核发育人工诱导研究,高技术通讯,18(8):874-880)