呋喃丹降解菌CDS-1的双标记菌株的构建  

Construction of double-labelled carbofuran-degrading bacterium Sphingomonas sp.CDS-1

作　　者：徐剑宏[1] 武俊[1] 洪青[1] 张志琳[1] 李顺鹏[1] 王云端[1] 

机构地区：[1]南京农业大学生命科学学院农业部农业环境微生物工程重点开放实验室,南京210095

出　　处：《微生物学报》2006年第4期613-617,共5页Acta Microbiologica Sinica

基　　金：国家自然科学基金(30400013);江苏省科技厅项目(BE2002345;BE2003343;BG2005322)~~

摘　　要：用Sau3AI消化呋喃丹降解菌Sphingomonassp.CDS-1的基因组DNA,将所得DNA片段与BamHⅠ酶切的启动子探针载体pRobe-GFP酶连后转化E.coliDH5α感受态细胞,在选择性平板上培养,从大约1×104个菌落中筛选到50个含启动子片段的阳性克隆。挑选其中一个发光强度最强的阳性克隆F7,将它的重组质粒pF7用EcoRⅠ和HindⅢ双酶切后得到包含Sphingomonassp.CDS-1启动子和gfp基因的DNA片段,将该片段克隆到广宿主载体pPZP201上,得到pPZP201-gfp质粒。将pPZP201-gfp通过三亲接合转移至Sphingomonassp.CDS-1中得到GFP标记菌株CDS-gfp,经荧光显微镜观察,gfp基因在CDS-gfp中表达量很高。对标记菌株进行连续传代10次(48h/次),发现pPZP201-gfp依然存在,而且发光明显。通过NotⅠ酶切位点把linA基因连接到pUT/mini-Tn5上构建新的转座子载体pUT/mini-Tn5-linA。以pRK600为辅助质粒将pUT/mini-Tn5-linA引入到CDS-1中,linA基因通过转座作用,插入到CDS-gfp的染色体中,得到双标记菌株CDS-GFP-LinA。该菌株是一株能同时降解γ-六六六和呋喃丹的基因工程菌,本研究的结果为研究Sphingomonassp.CDS-1的生态学行为奠定了基础。The genomic DNA of a carbofuran-degrading bacterium Sphingomonas sp. CDS-1 was digested by Sau3Al and ligated to pRobe-GFP digested by BamHI, and the product was transformed to the E. coli DH5α competent cells. Fifty positive clones that could emit green fluorescence under UV were selected from about 1 × 10^4clones grown on selective plates AmpLB. One clone F7 with the strongest fluorescence was selected, the recombinant plasmid pF7 from this clone was digested with EcoR Ⅰ ＆ HindⅢ and the DNA fragment comprising gfp and promoter of Sphingomonas sp. CDS-1 was recovered, which was subsequently cloned into the broad host vector pPZP201 to construct a new plasmid pPZP201-gfp, pPZP201-gfp was introduced into Sphingomonas sp. strain CDS-1 by triparental conjugation to make strain CDS-gfp. gfp was expressed strongly and stably in strain CDS-gfp after 10 times successive re-culturing （48h/time）. The linA gene was inserted into Not Ⅰ -cut transposon vector pUT/mini-Tn5 to construct a new transposon vector pUT/mini-TnS-linA. With the aid of helper plasmid pRK600, pUT/mini-TnS-linA was introduced into CDS-gfp, the dehydrochlorinase gene linA was integrated into the chromosome of CDS-gfp by transposing. The double labelled strain CDS-GFP- LinA was constructed. This strain was also a genetic engineering strain that was able to degrade 7-hexachlorocyclohexane and carbofuran simultaneously. All of these results laid a foundation for the study of ecological performance of Sphingomonas sp. CDS-1.

关 键 词：SPHINGOMONAS sp.CDS-1 呋喃丹 降解 双标记 基因工程菌 

分 类 号：X172[环境科学与工程—环境科学]