反向PCR法克隆芳香烃龙胆酸降解途径中诱导型龙胆酸1,2-加双氧酶基因  

Cloning and Sequencing of Inducible Enzyme in Aromatic Gentisate Degradative Pathway Using Inverse PCR Method

作　　者：赵渝[1,2] 郭鲁申[3] 徐亚同[1] 陆贻通[4] 高林[3] 

机构地区：[1]华东师范大学资源与环境科学学院 [2]上海师范大学生命与环境科学学院上海200234 [3]上海师范大学生命与环境科学学院 [4]上海交通大学资源与环境系

出　　处：《应用与环境生物学报》2007年第1期87-92,共6页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金资助项目(No.20337010);国家基础研究重大项目(2004CB418503);上海市基础研究重点项目(04JC14051);上海市生态学重点学科建设资助~~

摘　　要：龙胆酸1,2-加双氧酶(GDO)是芳香烃龙胆酸途径的一个关键酶.产碱假单胞菌P25X具有保守性与诱导性各一组GDO同工酶,突变株SNZ28的保守型龙胆酸1,2加双氧酶基因(GDOⅠ)被链霉素/奇霉素抗性基因打断,但在含有龙胆酸盐的基本培养基上,仍能明显观察到GDO的活性.由龙胆酸诱导生长的SNZ28全蛋白二维电泳结果分析,获得了一个与Ralstoniasp.U2的GDO酶有极高同源性的蛋白点.根据对该蛋白点的N端和Q-Tof测序的结果,设计了一对简并引物,克隆了诱导型GDO酶的片段,通过对此片段的分析,设计了逆向PCR引物,利用Southern blot杂交,确定了合适的限制性内切酶(SalⅠ和SphⅠ),以单一酶切的P25X基因组DNA自联后的产物为模板,进行反向PCR.测序表明,获得了一段1047bp与Ralstoniasp.U2.的龙胆酸1,2-加双氧酶具有极高同源性的序列.研究结果揭示了SNZ28中存在诱导型GDO酶.本文中也对不同来源的GDO酶进行了比较研究,其结果为进一步对生物降解基因的进化研究打下了基础.Gentisate 1,2-dioxygenase （GDO） is the key enzyme of the gentisate pathway. Pseudomonas alcaligenes P25X is capable of degrading aromatic hydrocarbon via the gentisate pathway. It has been established that the gentisate pathway harbors isozymes for the gentisate metabolism, one set is constitutively expressed whereas the other set is strictly inducible. A mutant strain of P25X, designated SNZ28, which had the constitutive copy of the gentisate 1,2-dioxygenase gene interrupted by a streptomycin/spectinomycin resistance gene cassette, was found to express gentisate dioxygenase, but only when the cells were induced by gentisate. Proteomic analysis of SNZ28 in response to gentisate induction revealed a protein spot that showed high homology to GDO from Ralstonia sp. U2. A pair of degenerated primers was designed according to the Q-Tof and N-terminal sequencing of this protein spot. Partial GDO Ⅱ PCR product was amplified and cloned into p-GEMT. Inverse PCR approach was employed to sequence the gene encoding the putative inducible GDO Ⅱ. The putative GDO Ⅱ enzyme was also compared with other GDOs. This study provided a basis for further studying of the evolutionary relationship and ancestral relatedness between GDO Ⅱ and GDOI or other GDOs in aromatic hydrocarbon degradation pathway. Fig 4, Tab 2, Ref 16 .

关 键 词：芳香烃 龙胆酸 诱导酶 反向PCR法 

分 类 号：Q78[生物学—分子生物学]