耐盐及苯乙酸、甲基对硫磷降解基因工程菌的构建

Construction of Genetic Engineering Microogranism of Salt Tolerant Degrading Strain

H1 (Halomonassp.)是一株耐高盐浓度 (1 8%NaCl,W V)和降解苯乙酸的菌株 ,pDT3质粒为pUC1 9插入甲基对硫磷水解酶基因 (mpd基因 )构建而成。采用HindⅢ酶切 ,获得含有完整mpd基因片段 ,克隆到广宿主质粒pKT2 3 0和pBBR1 MCS2上 ,构建成质粒pKT MP和pBBR MP。通过三亲杂交 ,在辅助质粒pRK2 0 1 3的帮助下 ,将质粒pKT MP和pBBR MP转移到H1中 ,得到的工程菌H pKT MP和H pBBR MP具有耐盐、降解苯乙酸和水解甲基对硫磷的功能 ,其中H pBBR MP水解酶活性与亲本菌株甲基对硫磷降解菌 (Pseudomonasputida)DLL E4相当 ,而H pKT MP水解酶活性要提高 1倍左右。经过传代试验 。

H1(Halomonas sp.)was a strain of salt tolerance(18% NaCl,W/V) and degr ading phenylacetic acid, plasmid pDT3 was a derivation of pUC19 inserted mpd gene(methyl parathion degrading gene), which is obtained from methyl parathion (MP) degrading strain DLL-E4(Pseudomonas putida). Recombinant plasmids pKT -MP and pBBR-MP were constructed by inserting HindⅢ fragment(which includ e whole mpd gene) of pDT3 into broad host vector pKT230 and pBBR1-MCS2 Wi th the aid of help-plasmid pRK2013, pKT-MP and pBBR-MP were transferred into H1(called tri-parent conjugation) and multi-functioned GEMs H-pKT-MP and H- pBBR-MP were constructed, which were salt tolerant, phenylacetic acid-degradin g and MP-degrading. The activity of MP hydrolase of H-pBBR-MP and H-pKT-MP was 1× and 2× the one of DLL-E4. The MP hydrolase activity of two GEMs were c onstant.