甲基对硫磷水解酶基因的克隆与融合表达  

Cloning,Analysis and Fusion Expression of Methyl Parathion Hydrolase

作　　者：刘智[1] 洪青[1] 徐剑宏[1] 武俊[1] 张晓舟[1] 张小华[1] 马爱芝[1] 朱军[1] 李顺鹏[1] 

机构地区：[1]南京农业大学生命科学学院微生物学系

出　　处：《Acta Genetica Sinica》2003年第11期1020-1026,共7页

基　　金：国家 8 63 ( 2 0 0 1AA2 14 12 1;2 0 0 2AA2 460 81);农业科技跨越计划 (M2 0 0 0 11);国家攻关 ( 2 0 0 2BA5 16A0 1)项目资助~~

摘　　要：利用鸟枪法从甲基对硫磷降解菌DLL 1Peudomonasputida)中克隆了甲基对硫磷水解酶基因 (mpd)片段2 5kb ,并进行了测序。通过软件分析开放阅读框和启动子序列 ,表明该序列中最可能为甲基对硫磷水解酶结构基因的阅读框为 76 9～ 1 794区域。软件分析还表明该水解酶前端 4 5个氨基酸为典型的信号肽结构。通过PCR扩增了mpd结构基因 ,亚克隆到表达载体pET 32a中 ,构建了完整的融合表达载体pET MP。转化大肠杆菌BL2 1 (DE3) ,在IPTG的诱导下 2～ 4h甲基对硫磷水解酶表达可以达到最高水平 ;同时还研究了乳糖的诱导效果 ,2 %乳糖诱导 2h就可以起到很好的效果。通过PCR反应验证了mpd基因定位于DLLMethyl parathion(MP) is a kind of organophosphorous pesticide which was widely used by farmers all over the world in 1990's.It is effective in controlling pesticide but harmful to human beings.Bioremediation is an effective and economic method to treat environment that has been polluted by hazardous organic compounds,so researchers paid much attention in this area.Most of which focused on isolating functional bacteria,studying its degrading mechanism,and cloning degradation related genes.DLL 1 was isolated from soil polluted by MP for a long time and identified as Pseudomonas putida .It could degrade MP to CO 2 and H2 O [9] .DLL E4 with higher degrading ability was obtained by UV and LiCl mutagenesis of DLL 1 [10] .A big plasmid was found in DLL E4 but very difficult to be cured,and mutant M -P + losing activity of MP hydrolase by nitriguanidine mutagenesis also had this plasmid.So shotgun cloning method was chosen to clone the MP degradation gene( mpd ) from DLL E4.Total DNA was digested by Sau3AI,the fragments of 2～4 kb were recovered and inserted in pUC19 at BamHI site.The recombinant plasmids were transferred to DH5α.Six positive clones (pDT1～6) with MP hydrolase activity were selected on LB+MP plate,and pDT3 was chosen to be sequenced for its smallest molecular weight (inserted fragment 2 5 kb).After sequencing and analyzing of the inserted fragment,769～1794 region was considered as the most possible ORF and this region was cloned by PCR and ligated with pET 32a to construct an expressing vector pET MP.pET MP was transferred to E.coli BL21(DE3).The inducing time,concentration of IPTG and lactose were also studied.The result showed that the molecular weight of MP hydrolase was about 36 kDa,the iptimal inducing time and concentration for lactose were 2h and 2%(V/V) respectively. mpd was primary located in chromosome DNA in DLL E4 by PCR.

关 键 词：鸟枪法 甲基对硫磷水解酶基因 基因分析 融合表达 基因定位 

分 类 号：Q785[生物学—分子生物学]