• 关键词：嗜水气单胞菌脱色酶基因(tpmD)  三苯基甲烷染料  希瓦氏菌启动子  无诱导表达

• 基金项目：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)

• 卫晋波
• 1. 中国科学院南海海洋研究所, 广州 510301; 2. 广东省微生物研究所菌种保藏与应用重点实验室, 广东省微生物研究所, 广州 510070; 3. 中国科学院研究生院, 北京 100049

• 孙国萍
• 广东省微生物研究所菌种保藏与应用重点实验室, 广东省微生物研究所, 广州 510070

• 任随周
• 广东省微生物研究所菌种保藏与应用重点实验室, 广东省微生物研究所, 广州 510070

• 岑英华
• 广东省微生物研究所菌种保藏与应用重点实验室, 广东省微生物研究所, 广州 510070

• 王燕
• 广东省科技图书馆, 广州 510070

• 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.coli DH5α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.

• Abstract：According to the tpmDgene sequence of Aeromonas hydrophila DN322,a set of primers was designed,the forward primer containing a SDsequence before start codon ATG,and the reverse primer containing 18 bp coding for CCPGCCbefore the stop codon.This CCPGCCtag can covalently bind biarsenical MAPs,i.e.,fluorescein and other dyes with the xanthene backbone derivatized with two As(III) moieties.The PCRproduct was 919 bp,and named tpmD'.According to the Shewanella sp.ANA-3 NAD(P)Hdehydrogenase gene promoter sequence from 0316 ORF,a set of primers was also designed.A 98 bp promoter(SNDPromoter) was amplified by PCRusing Shewanella decoloritionis S12 total DNAas template.Through a tri-primer PCRmethod(TP-PCR),combination of the 919 bp tpmD' and 98 bp promoter was achieved.The 1017 bp PCRproduct was named SProtpmD'.The tpmD' and the SProtpmD' were cloned into the plasmid pMD18-Tsimple vector and transformed into E.coli DH5α or E.coli BL21.Both E.coli strains carrying the SProtpmD' gene expressed decolorization activity higher than parent strain Aeromonas hydrophila DN322 even without induction with IPTG.Native-PAGEresult 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 decolorization 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 decolorization from E.coli DH5α/pMD18-Tor 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 mLin a 1Lglass flask),Malachite Green(MG) was added continually from 20 mg·L^-1 to 700 mg·L^-1,every 2～4 days.At the end of the experiment the amount of MGaccumulated up to 10000 mg·flask^-1.And about 92% of the added MGwas degraded and the engineered bacterial cells were increased 5～10 fold.This result strongly suggests that the engineered E.coli strains can survive in a natural water environment without adding nutriment for more than two months and express high activity for MGdecolorization without IPTGinduction.Based on the decolorization experiment results,it was apparent that the promoter sequence SPr from Shewanella decoloritionis S12 NAD(P)Hdehydrogenase gene was recognized by E.coli cells and the gene under its control is efficiently transcribed and expressed constitutively.This characteristic should be useful for bioremediation in environments polluted by triphenylmethane dyes such as MG.

• Key words：SND Promoter  tpmD  triphenylmethane dyes  non-induction expression

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