大肠杆菌磷酸果糖激酶基因在野生型专性自养嗜酸性氧化硫硫杆菌Tt-Z2中的表达

Expression of phosphofructokinase gene from E.coli in an obligately autotrophic bacterium Acidithiobacillus thiooxidans Tt-Z2 strain

利用PCR技术扩增E .coliK 12磷酸果糖激酶 1基因 (pfkA) ,将该基因与广泛寄主的IncQ质粒pJRD2 15连接构建重组质粒pSDK 1,该质粒可与pfk基因缺陷株E .coliDF10 10互补 .通过接合转移的方式将其导入专性自养极端嗜酸性氧化硫硫杆菌Tt Z2中并得到表达 .pSDK 1在宿主Tt Z2中有较好的稳定性 ,在无选择压力条件下连续传5 0代仍可保留 6 8% .酶活性测定表明 ,pSDK 1的pfkA基因在缺陷株E .coliDF10 10中表达水平略高于出发菌株E .coliK 12 ;而在氧化硫硫杆菌中则以较低水平进行表达 .葡萄糖可促进含pSDK 1的氧化硫硫杆菌Tt Z2的生长 ,而对照菌株的生长则未受明显影响 .说明重组菌可部分利用葡萄糖作为碳源而生长 .

Phosphofructokinase-1 (EC 2.7.1.11) gene ( pfk A) from E.coli K-12 was amplified by PCR and inserted into Bam HI/ Hin dⅢ site of the wide-host-range IncQ plasmid pJRD215 to creat a recombinant plasmid pSDK-1,which can be complement with pfk deficient DF1010 strain. Then the plasmid pSDK-1 was introduced into Acidithiobacillus thiooxidans Tt-Z2 by conjugation and expressed in it. Compared with E.coli K-12,the expression level of pfk A gene was increased in DF1010 strain,but decreased in Tt-Z2 transconjugant. The effect of glucose on the growth of Tt-Z2 (pSDK-1) was studied. In the presence of glucose,the Tt-Z2 transconjugant grow faster than its original strains.