摘要
为了解决嗜水气单胞菌DN322对鱼类的潜在致病性问题,有必要克隆表达该菌的三苯基甲烷染料脱色酶基因tpmD.通过PCR方法获得该基因,并与脱色希瓦氏菌S12的NAD(P)H脱氢酶基因启动子和用于荧光标记的编码CCPGCC的碱基序列融合,将有启动子和无启动子的融合基因片段分别连接到质粒pMD18-T中,转化大肠杆菌E.coliDH5α和E.coliBL21.结果发现,有启动子的融合基因能被E.coli在不加诱导物IPTG的条件下高效正确表达,脱色酶活性甚至超过基因供体菌DN322.将上述两株大肠杆菌工程菌株在自然水体中进行孔雀石绿污染小试处理,60d内在每个350mL反应体系中累积共投加大于10000mg的孔雀石绿,脱色率一直保持在92%以上;细菌数量最后增加了5～10倍.研究结果证明,基因工程菌在不加营养物的条件下也有很强的脱色活性和长期的存活能力;在tpmD基因3’端加入的用于螯合双砷荧光染料的18bp编码氨基酸碱基序列不影响此基因的表达和酶的脱色活性,这将赋予此工程菌可示踪性.
According to the tpmD gene sequence of Aeromonas hydrophila DN322, a set of primers was designed, the forward primer containing a SD sequence before start codon ATG, and the reverse primer containing 18 bp coding for CCPGCC before the stop codon. This CCPGCC tag can covalently bind biarsenical MAPs, i. e. , fluorescein and other dyes with the xanthene backbone derivatized with two As（III） moieties. The PCR product was 919 bp, and named tpmD＇. According to the Shewanella sp. ANA-3 NAD（P） H dehydrogenase gene promoter sequence from 0316 ORF, a set of primers was also designed. A 98 bp promoter （SNDPromoter） was amplified by PCR using ShewaneUa decoloritionis S12 total DNA as template. Through a tri-primer PCR method （TP-PCR）, combination of the 919 bp tpmD＇ and 98 bp promoter was achieved. The 1017 bp PCR product was named SProtpmD＇. The tpmD＇ and the SProtpmD＇ were cloned into the plasmid pMD18-T simple vector and transformed into E. coli DH5α or E. coli BL21. Both E. coli strains carrying the SProtpmD＇ gene expressed deeolorization activity higher than parent strain Aeromonas hydrophila DN322 even without induction with IPTG. Native-PAGE result showed decolorized bands from DN322 or E. coli DH5α/pSPrtpmD＇ or E. coli DH5α/ptpmD＇ in the same position. That means the recombination enzyme has the same apparent molecular weight with parent strain. The deeolorization activity for crystal violet by cell-free soluble protein from E. coli DH5α/ pSPrtpmD＇ was 48 U·mg^-1 protein, while that from DN322 was 35.29 U·mg^-1 protein. The deeolorization from E. coli DH5α/ pMD18-T or E. coli DH5α was 0 U. In a lab scale bioremediation decoloration experiment using E. coli DH5α/ pSPrtpmD＇ or E. coli BL21/pSPrtpmD＇ in natural fishpond water （350 mL in a 1L glass flask） , Malachite Green （MG） was added continually from 20 mg· L^-1 to 700 mg·L^- l , every 2 N 4 days. At the end of the experiment the amount of MG accumulated up to 10000 mg·flask^-1. And about 92% of the added MG
出处
《环境科学学报》
CAS
CSCD
北大核心
2009年第3期527-535,共9页
Acta Scientiae Circumstantiae
基金
国家高技术研究发展计划(863)项目(No.2006AA06Z322)
国家自然科学基金(No.30800031)
广东省科技计划项目(No.2007A020903001)
广东省科技攻关项目(No.2007A020300007)
广东省科学院人才基金项目(No.200601)
野外科学实验站基金项目(No.sytz200710)~~
