耻垢分枝杆菌烯酰辅酶A水合酶编码基因表达分析及其敲除质粒的构建

Transcription Analysis of Enoyl-Co A Hydratase-encoding Genes in Mycobacterium smegmatis and Construction of Mutagenic Plasmid

胆固醇是某些病原菌胞内生存所必需的碳源和能源物质,而胆固醇的各种代谢酶是潜在的药物作用靶点.烯酰辅酶A水合酶主要参与了脂肪酸的β-氧化循环,但其在胆固醇降解中的作用机理尚不完全清楚.首先对耻垢分枝杆菌(Mycobacterium smegmatis MC2155)进行了全基因组搜索,获得了18个烯酰辅酶A水合酶的候选基因.对在不同甾醇类化合物条件下的转录组测序结果进行了分析,发现18个候选基因差异表达明显,其中位于胆固醇降解基因簇中的MSMEG_5915(ech A19),在胆固醇条件下表达明显上调,MSMEG_5915(ech A19)可能参与了胆固醇的侧链降解.最后,构建了MSMEG_5915的敲除质粒,并对耻垢分枝杆菌进行了基因转化.经PCR快速分子鉴定,MSMEG_5915基因被同源整合于耻垢分枝杆菌染色体上.为进一步揭示MSMEG_5915(ech A19)基因的功能奠定了基础.

Bacterial catabolism of cholesterol plays an important role in Mycobacterium tuberculosis（ Mtb） infection and intracellular survival. Enoyl-Co A hydratase is a key enzyme which involves in β-oxidation of fatty acid. However,the biological function of enoylCo A hydratase in cholesterol side-chain cleavage is still of poorly understood. In this study,genome-wide search for putative enoyl-Co A hydratase encoded genes is conduct and it is found 18 putative genes existed in the genome of M. smegmatis MC2155. The transcription analysis shows that they are differentially expressed,and MSMEG_5915（ echA19） located at cholesterol-degradation gene cluster is up-regulated obviously in cholesterol as compared with pyruvic acid or androstenedione（ AD）. MSMEG_5915（ echA19） are thought to be involved in the side-chain cleavage of cholesterol. In order to understand the exact function of the gene,mutagenic plasmids for targeted gene deletion are constructed by PCR amplification of upstream and downstream regions of the gene MSMEG_5915 using the Sac B counterselection system containing plismid p NIL and p K18 mobsac B. Then,the mutagenic plasmid is electroporated into M. smegmatis cell,the transformants are selected and identified by PCR amplicfication. The results show that homologous recombination is occured.This study can provide the foundation to reveal the function of MSMEG_5915（ echA19） and the molecular mechanism on the cholesterol catabolism.