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.
引文
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