一种具高活性的酪氨酸酶基因启动子片段的分离

一种具高活性的酪氨酸酶基因启动子片段的分离 Isolation of a Hich activity Promoter for Tyrosinase Gene AazazAbderrahmane Aazaz Abderrahmane 1 first-author 王戈林 WANG Ge lin 1 沈萍 SHEN Ping 1 武汉大学生命科学学院 武汉大学生命科学学院 College of Life Sciences, Wuhan University College of Life Sciences, Wuhan University 利用大肠杆菌启动子探测质粒ｐＫＫ２３２－８为载体，经ＨｉｎｄⅢ－ＢａｍＨⅠ完全消化后与ＨｉｎｄⅢ－ＢａｍＨⅠ部分消化的嗜麦芽假单胞菌染色休ＤＮＡ进行体外连接，转化Ｅ．ｃｏｌｉＨＢ１０１感受态细胞，在含氨苄青霉素（Ａｐ）和氯霉素（Ｃｍ）的选择平板上筛选转化子．从随机挑选的５４株转化子中，获得一株抗氯霉素水平达１０００ｍｇ／Ｌ的转化子Ｅ．ｃｏｌｉＰＡＳＩ，所含重组质粒被命名为ｐＡＳＩ．经限制性酶切分析和杂交分析表明，ｐＡＳＩ质粒上插入了一段来源于嗜麦芽假单胞菌染色体的ＤＮＡ片段，其大小为１．４ｋｂ．ＳＤＳ－ＰＡＧＥ分析表明转化子Ｅ．ｃｏｌｉＰＡＳＩ在含Ｃｍ的培养基上，额外表达了一条２２×１０３的ＣＡＴ蛋白质带．将重组质粒ｐＷＳＹ上的嗜麦芽假单胞菌ｍｅｌ基因亚克隆到ｐＡＳＩ上１．４ｋｂ启动子功能片段下游，构建成Ｅ．ｃｏｌｉＡＷＳ工程菌株，使黑色素产率提高了７０．６％． A promoter probe plasmid pKK232 8 was used as a vector which functioned in E.coli. The chromosome DNA of Pseudomonas maltophilia was partially digested by BamHⅠ HindⅢ, recombinated in vitro and transformed into E.coli.The transformants were selected on Ampicillin（Ap） and chloramphenicol（Cm） containing resistance plates. From random seleted 54 strains of transformants we obtained transformant E.coli PASI which level of Cm resistance got to 1 000 mg/L. The recombinant plasmid from E.coliPASI was named pASI. The results of analysis by restriction endonucleases digestion and molecular hybridization showed that recombinant plasmid pASI carried an inserted fragment with size of 1.4 kb from chromosome DNA of P.maltophilia. The result of analysis by SDS PAGE indicated that transformant E.coliPASI expressed an additionaly band of CAT enzyme （22×10 3）. The mel gene on recombinant plasmid pWSY, which is from P.maltophiliaAT18, was subcloned into downstream of the promoter fragment of pASI to construct engineering strain E.coliAWS. The experiment result showed that melanin productivity of E.coliAWS increased 70.6% than that of E.coli WSY strain. 启动子 酪氨酸酶基因 黑色素 嗜麦芽假单胞菌AT18 promoter tyrosinase gene melanin Pseudomanas maltophilia Q554.7 湖北省科委资助的重点项目 1999-02-01 2021-04-01 2