摘要
克隆并分析绒毛白蜡NADH还原酶第二亚基(NDHB)基因片段。根据GenBank上已发表的NDHB氨基酸序列,设计合成了一对简并引物,采用RT-PCR技术对绒毛白蜡NADH还原酶第二亚基基因进行扩增,将扩增的PCR产物与pMD18-T连接后转化至E.coli JM109感受态细胞,检测阳性克隆,测序并进行序列分析。克隆的绒毛白蜡NADH还原酶第二亚基基因cDNA序列长893bp,由288个氨基酸残基组成,命名为FvndhB。序列比对结果表明FvndhB与参考的8个物种的NDHB在核酸水平上表现出较高的同源性,其与桑树、银白杨、桉树、烟草、拟南芥、银杏、台东苏铁、日本柳杉NDHB核苷酸序列同源性分别为84.32%、83.88%、83.43%、82.86%、82.18%、78.82%、77.07%、71.23%。利用DNAMAN软件构建的系统进化树显示绒毛白蜡与其他植物亲缘关系较远。成功获得了绒毛白蜡FvndhB基因片段,并对其进行了同源性分析,为进一步克隆绒毛白蜡FvndhB全长基因及分析其表达特性奠定了基础。
To clone and analysis the second subunit of NADH dehydrogenase （NDHB） gene segment. The degenerate primers were designed and synthesized according to the sequence of NDHB amino acid published in GneBank. A novel cDNA encoding the second subunit of NADH dehydrogenase （NDHB） was amplified from Fraxinus velutina Torr by RT-PCR. The PCR products were ligated into the pMD18-T vector, and then transformed into competent cells of E.coli JM109. The positive clone was identified and the sequence was sequenced and analyzed. NDHB gene segment was obtained which was 893 bp in length and coding 288 amino acid, and it was designated as FvndhB. Sequence alignment showed that FvndhB presented high homology with other NDHBs at the level of nucleic acid. Compared with the corresponding region of Ginkgo biloba, Cryptomeria japonica, Eucalyptus spp, Nicotiana tabacum, Morus alba, Populus alba, Arabidopsis thaliana, Cycas taitungensis, the nucleotide sequence homology was 84.32%, 83.88%, 83.43%, 82.86%, 82.18%, 78.82%, 77.07% and 71.23% respectively. A further analysis of phylogenetic tree was constructed by the software of DNAMAN. The result showed that Fraxinus velutina Ton＂ has a far genetic relation with other plants. In this research, FvndhB gene segment was obtained, and the phylogenetic analysis was researched. It established basis on cloning the span of FvndhB and studying the characteristics of FvndhB gene expression in Fraxinus velutina Torr.
出处
《中国农学通报》
CSCD
北大核心
2009年第6期40-46,共7页
Chinese Agricultural Science Bulletin
基金
国家林业局林业科技支撑计划项目(2006BAD01A1603)
关键词
绒毛白蜡
ndhB
基因克隆
序列分析
Fraxinus velutina TorT, ndhB, gene cloning, sequence analysis
