耐盐及苯乙酸、甲基对硫磷降解基因工程菌的构建

H1（Halomonas sp.）是一株耐高盐浓度（18% NaCl, W/V）和降解苯乙酸的菌株，pDT3质粒为pUC19插入甲基对硫磷水解酶基因（mpd基因）构建而成。采用ＨindⅢ酶切，获得含有完整mpd基因片段，克隆到广宿主质粒pKT230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pRK2013的帮助下，将质粒pKTMP和pBBRMP转移到Ｈ１中，得到的工程菌HpKTMP和HpBBRMP具有耐盐、降解苯乙酸和水解甲基对硫磷的功能，其中HpBBRMP水解酶活性与亲本菌株甲基对硫磷降解菌（Pseudomonas putida）DLLE4相当，而HpKTMP水解酶活性要提高1倍左右。经过传代试验，证明了工程菌的稳定性。

Construction of Genetic Engineering Microogranism of Salt Tolerant Degrading Strain

H1 ( Halomonas sp.) was a strain of salt tolerance (18% NaCl, W/V) and degrading 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 Hind III fragment (which include whole mpd gene) of pDT3 into broad host vector pKT230 and pBBR1-MCS2. With 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-degrading and MP-degrading. The activity of MP hydrolase of H-pBBR-MP and H-pKT-MP was 1 x and 2 x the one of DLL-E4. The MP hydrolase activity of two GEMs were constant.