Pseudomonas putida GM6多聚磷酸盐激酶(ppk)基因的克隆及表达

以一株高效聚磷菌Pseudomonas putida GM6为研究材料.为获得其多聚磷酸盐激酶（polyphosphate kinase,ppk）基因,并验证该基因在磷酸盐转运系统中的作用,根据已报道的ppk基因保守区域设计引物,从其总DNA中成功扩增到ppk基因的部分片段（约528 bp）.随后采用快速染色体步移方法（Self-formed adaptor PCR,SEFA-PCR）技术扩增片段的上下游基因序列,将三个序列拼接,用OMIGA软件分析其ORFs,推测ppk基因全长为2 220 bp（GenBank accession number DQ133537）.构建的多聚磷酸盐激酶表达菌株E. coli BL21（DE3）/ pET29a-ppk经IPTG诱导后3 h时,明显出现分子量约为81 kDa的表达产物.且表达菌株在12 h时的磷去除率高达80%（对照菌株的磷去除率仅为18%）,远高于已报道的40%的去除率.这表明ppk基因在E. coli中的过量表达,导致了E. coli菌体中poly-P的大量聚集,从而大大去除了培养基中的磷酸盐.

CLONING AND EXPRESSION OF POLYPHOSPHATE KINASE GENE FROM PSEUDOMONAS PUTIDA GM6

A strain of high-efficiency phosphorus accumulating bacteria,identified as Pseudomonas putida GM6,was used as research object,from which polyphosphate kinase gene was cloned and its roles in the phosphate transport system verified.A 528-bp fragment of ppk gene was successfully amplified firstly from GM6 genomic DNA using self designed primers corresponding to the well-conserved regions of reported ppk gene sequences.Then,its upstream and downstream sequences were cloned with the technique of self-formed adaptor PCR(SEFA-PCR).Three amplified sequences were put together and analyzed using the OMIGA program(version 2.0),and operon of the complete ppk gene ca.2 220 bp was obtained(it has been deposited in the GenBank database under accession number DQ133537).The constructed recombinant expression strain of ppk gene E.coli BL21(DE3)/pET29appk was induced with IPTG for three hours,and expression product,81 kDa in molecular weight,was observed.The strain removed 80% of the phosphorus in the solution while the CK strain only 18% in 12 h.Its P removing capacity,more than 40%,the so-far reported highest rate,indicates excessive expression of ppk gene in E.coli,which would lead to accumulation of a great deal of poly-P in E.coli in vivo,and removal of a large amount of phosphate from the medium.