呋喃丹降解菌CDS-1的双标记菌株的构建

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。

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

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 DH5alpha competent cells. Fifty positive clones that could emit green fluorescence under UV were selected from about 1 x 10(4) clones 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 & Hind III 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 (48 h/time). The linA gene was inserted into Not I -cut transposon vector pUT/mini-Tn5 to construct a new transposon vector pUT/mini-Tn5-linA. With the aid of helper plasmid pRK600, pUT/mini-Tn5-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 gamma-hexachlorocyclohexane and carbofuran simultaneously. All of these results laid a foundation for the study of ecological performance of Sphingomonas sp. CDS-1.