阴沟肠杆菌flavocytochrome C基因克隆及序列分析  

Cloning and Sequence Analysis of flavocytochrome C from Enterobacter cloacae NRRL B-414

作　　者：林海龙[1,2] 付畅[1] 郑福帅[1] 任南琪[2] 

机构地区：[1]哈尔滨师范大学生命科学与技术学院,哈尔滨150025 [2]哈尔滨工业大学城市水资源与水环境国家重点实验室,哈尔滨150090

出　　处：《中国农学通报》2011年第18期228-232,共5页Chinese Agricultural Science Bulletin

基　　金：高等学校博士学科点专项科研基金(20102329120002);中国博士后科学基金项目(20090450983);黑龙江省博士后基金项目(LBH-Z08197);黑龙江省教育厅科学技术研究项目(11551141)

摘　　要：为了克隆flavocytochrome C,研究其在阴沟肠杆菌产氢代谢中的作用和机制,利用CODEHOP设计阴沟肠杆菌NRRL B-414的flavocytochrome C简并引物,选用引物FlacJ扩增基因组DNA,分别得到约1800bp左右的PCR产物,该产物连接pMD18-T载体,并克隆转化至E.coli DH5α感受态细胞中,经筛选后测序。该基因片段长1740bp,编码579个氨基酸,G+C含量为58.51%,A+T含量为41.49%,其在GenBank注册号为GU565952。推导的FlacJ扩增产物氨基酸序列与Enterobacter cloacae subsp.cloacaeATCC13047的flavocytochrome C蛋白一致性最高达到95%,573个氨基酸编码序列中仅相差28个氨基酸,表明其为阴沟肠杆菌的flavocytochrome C基因。实验结果表明,试验已成功克隆到阴沟肠杆菌的flavocytochrome C的部分基因,不但增加了该基因的资源,也可为其产氢代谢工程研究提供科学依据和工作基础,同时也再次证明采用CODEHOP设计简并引物进行同源家族基因克隆成功率高。In order to study the function of flavocytochrome C （flac） in the producing-hydrogen metabolism,the authors used CODEHOP to design the degenerate primers of flavocytochrome C,chose one pair of degenerate primers named FlacJ,then used Enterobacter cloacae NRRL B-414 genome DNA as template to make degenerate PCR,got about 1800 bp PCR product,they were transformed into the E.coli DH5α through being linked with pMD18-T vector and sequenced after filtration.The fragment was 1740 bp long,the G＋C content was 58.51%,the A ＋ T content was 41.49%,and its NCBI accepted number was GU565952.Similarity alignment showed that the 1740 bp products were very similar to the flavocytochrome C genes,and shared 95% identity to the large subunit of flavocytochrome C from Enterobacter cloacae subsp.cloacae ATCC 13047,and only had the difference of twenty-eight amino acids.Cloning this fragment would not only enrich the gene resources of flavocytochrome C genes,but also give the scientific warrant for the metabolic engineering research.And it was proved once again that using CODEHOP to design degenerate primers for cloning homologous family genes had a high success rate.

关 键 词：CODEHOP FLAVOCYTOCHROME C 阴沟肠杆菌NRRLB-414 

分 类 号：X172[环境科学与工程—环境科学]