假单胞菌DLL-E4尿黑酸降解基因的克隆与功能的初步分析  

Cloning and Functional Analysis of Homogentisate Degradation Gene from Pseudomonas putida DLL-E41

作　　者：沈文静[1] 邓海华[1] 曹慧[1] 李晓丹[1] 王世明[1] 崔中利[1] 李顺鹏[1] 

机构地区：[1]南京农业大学生命科学学院微生物学系农业部农业环境微生物工程重点开放实验室,南京210095

出　　处：《生物技术通报》2008年第S1期445-450,共6页Biotechnology Bulletin

基　　金：国家高技术研究发展计划项目("863"项目)(No.2007AA021304);国家自然科学基金(No.3077033);教育部新世纪优秀人才项目资助项目

摘　　要：通过接合转移将质粒pSC123上的转座子Tn5随机插入到DLL-E4基因组DNA中,从大约8,000个突变子中筛选到1株在LB培养基上积累红褐色物质的突变株M18,该突变株不能以L-苯丙氨酸(L-Phenylalanine, Phe)为唯一碳源生长。SEFA-PCR扩增转座子侧翼序列发现其与已报道的尿黑酸1,2-双加氧酶基因hmgA的同源性为92%。将hmgA定向克隆至表达载体pET-29a中,转化至Escherichia coli BL21,经IPTG诱导后可表达分子量约为48kD的蛋白;诱导后转化子粗酶液对尿黑酸有很好的降解效果。将hmgA连入自杀性载体pEX19Gm,通过同源重组整合至M18染色体中,使其恢复了DLL-E4利用Phe的能力,证实了HmgA是尿黑酸苯环裂解酶。A mutant strain which lost L-Phenylalanine(Phe)degrading ability was isolated after transposon mutagenesis with Tn5 and named M18.The mutant accumulated a saturated brown pigment during the growing on LB agar plate. The flanking sequence of Tn5 was amplified with SEFA-PCR and it showing a significant level of homology(up to 92%)to homogentisate 1,2-dioxygenase gene(hmgA)of Pseudomonas putida KT2440.The amplified fragment hmgA was cloned in the proper orientation into the expression vector pET-29a and then transformed into Escherichia coli BL21.A recombinant protein of about 48 kD was expressed and showed high ability of degrading homogentisate induced by IPTG.The hmgA was cloned into suicide vector pEX19Gm and then integrated into the genomic DNA of M18 by a single crossover between the homologous genes and the recombinant restored the degradation ability of Phe.

关 键 词：尿黑酸1 2-双加氧酶 转座子标签法 SEFA-PCR 同源重组 基因敲入 

分 类 号：Q93[生物学—微生物学] Q78