| 假单胞菌DLL-E4尿黑酸降解基因的克隆与功能的初步分析 |
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| 引用本文： | 沈文静,邓海华,曹慧,李晓丹,王世明,崔中利,李顺鹏.假单胞菌DLL-E4尿黑酸降解基因的克隆与功能的初步分析[J].生物技术通报,2008(Z1). |
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| 作者姓名： | 沈文静 邓海华 曹慧 李晓丹 王世明 崔中利 李顺鹏 |
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| 作者单位： | 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 |
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| 基金项目： | 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 |
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| 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。
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| 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 |
Cloning and Functional Analysis of Homogentisate Degradation Gene from Pseudomonas putida DLL-E41 |
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| Sheng Wenjing,Deng Haihua,Cao Hui,Li Xiaodan,Wang Shiming,Cui Zhongli,Li Shunpeng.Cloning and Functional Analysis of Homogentisate Degradation Gene from Pseudomonas putida DLL-E41[J].Biotechnology Bulletin,2008(Z1). |
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| Authors: | Sheng Wenjing Deng Haihua Cao Hui Li Xiaodan Wang Shiming Cui Zhongli Li Shunpeng |
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| Institution: | Key Lab of Microbiology Engineering of Agricultural Environment;Ministry of Agriculture;Nanjing Agricultural University;Nanjing 210095 |
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| Abstract: | 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 t... |
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| Keywords: | SEFA-PCR |
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