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中国农学通报

中国农学通报 ›› 2011, Vol. 27 ›› Issue (18): 228-232.

所属专题： 生物技术

• 生物技术科学 • 上一篇    下一篇

阴沟肠杆菌flavocytochrome C基因克隆及序列分析

林海龙 付畅 郑副帅 任南琪   

  • 收稿日期:2011-05-05 修回日期:2011-06-08 出版日期:2011-07-25 发布日期:2011-07-25
  • 基金资助:

    高等学校博士学科点专项科研基金;中国博士后基金项目;黑龙江省博士后基金项目;黑龙江省教育厅科学技术研究项目

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

  • Received:2011-05-05 Revised:2011-06-08 Online:2011-07-25 Published:2011-07-25

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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，笔者利用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1740 bp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1740 bp，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. cloacae ATCC 13047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，本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。

关键词: 西部-东部, 西部-东部, 人才需求-供给模型, 平衡点, 动力学行为分析

Abstract:

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 1740 bp PCR product, they were transformed into the E.coli DH5α through being linked with pMD18-T vector and sequenced after filtration. The fragment is 1740 bp long, the G+C content is 58.51%, the A+T content is 41.49%, and its NCBI accepted number is 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 has a high success rate.