睾丸酮丛毛单胞菌工程菌构建及稳定性分析  

Construction and Stability Analysis of the Engineered Comamonas testosteroni

作　　者：戴艺民[1] 周以飞[2] 陈建秋[2] 林江波[1] 张剑亮[2] 林龙云[2] 潘大仁[2] 

机构地区：[1]福建省农科院甘蔗研究所,漳州363005 [2]福建农林大学作物学院,福州350002

出　　处：《农业生物技术学报》2010年第4期807-814,共8页Journal of Agricultural Biotechnology

基　　金：福建省科技攻关重点项目(No.2003N028);福建省发改委重点项目(闽计投资(2003)170号)共同资助

摘　　要：本研究根据同源重组原理,利用电穿孔法将重组质粒pKtac1-act1和pKtac1-act2分别转化睾丸酮丛毛单胞菌(Comamonas testosteroni),经PCR和Southern blot筛选获得2株睾丸酮丛毛单胞菌基因工程菌。对这两株基因工程菌的关键酶3α-羟基类固醇脱氢酶/碳酰基还原酶(3α-hydroxysteroid dehydrogenase/carbonyl reductase,3α-HSD/CR)表达情况和细菌稳定性的分析结果表明:两株基因工程菌在没有诱导剂诱导的情况下,其3α-HSD/CR的表达量比原始菌提高了近20倍。添加抗生素进行培养,两株工程菌的activator和3α-HSD/CR表达均较稳定;而在不添加抗生素的培养基上培养时,两株工程菌的activator和3α-HSD/CR的表达变化较大,蛋白总体表达水平比添加抗生素时低。研究结果可以初步推断,3α-HSD/CR的表达受activator基因的表达调控。Recombinant plasmids pKtac1-act1 and pKtac1-act2 were integrated into Comamonas testosteroni chromosome by electroporation respectively,and two engineering strains were obtained and identified by the analysis of PCR and Southern blot.Expression and stability of 3α-hydroxysteroid dehydrogenase/carbonyl reductase （3α-HSD/CR） of engineering strains were detected.The results indicated that the expression of 3α-HSD/CR almost 20-folds increased in two engineering strains than that of the wild type without inducing;and the expressions of activator and 3α-HSD/CR were quite high and stable when engineering strains inoculated in LB medium with antibiotic;but unstable when inoculated in LB medium without antibiotic.The preliminary results indicate that the expression of 3α-HSD/CR can be regulated by the activator gene.

关 键 词：睾丸酮丛毛单胞菌 3α-羟基类固醇脱氢酶/碳酰基还原酶(3α-HSD/CR) ACTIVATOR 工程菌 稳定性 

分 类 号：Q78[生物学—分子生物学]