烟草脱水素基因NtDEH1的克隆及研究

Cloning and Analysis of Dehydrin Gene NtDEH1 in Nicotiana tabacum

为研究脱水素在植物早期胚胎发育中的作用和机理,采用显微操作技术获得烟草卵细胞并构建其c DNA文库,从中筛选到一个脱水素基因NtDEH1。通过RACE技术获得该基因全长,基因组测序结合生物信息学分析表明该基因拥有一段长度为651 bp的完整开放阅读框和一段535 bp的内含子,编码由217个氨基酸残基组成的蛋白质,其理论等电点为5.27,属于酸性蛋白,且具有一定的亲水性。氨基酸序列比对分析发现在许多物种比如绒毛状烟草、林烟草、甜辣椒、番茄、马铃薯和丹参中都具有与NtDEH1蛋白非常类似的保守的同源序列,均具有SK2型脱水素特征。借助Genome walking技术获取NtDEH1基因约1 706 bp的5'侧翼序列,使用小叶烟草瞬时表达系统检测到其具有较强的启动子活性。该研究为进一步了解脱水素基因NtDEH1在植物生殖发育中的具体功能打下基础。

To study the function and mechanism of dehydrin in early embryogenesis of plant,egg cells were isolated from tobacco to construct a c DNA library,and a gene named NtDEH1 were found. The full sequence of NtDEH1 were obtained by RACE,genome cloning and bioinformatics analysis showed that NtDEH1 included a 651 bp CDS region and a 535 bp intron. NtDEH1 which contained 217 amino acids were hydrophilic and acidic,the theory isoelectric point was 5. 27. The amino acid homology of NtDEH1 compared Nicotiana tabacum to Nicotiana tomentosiformis,Nicotiana sylvestris,Capsicum annuum,Solanum tuberosum,Solanum lycopersicum and Salvia miltiorrhiza showed that NtDEH1 homologous sequences in all these species were conserved and belonged to SK2 type. 5＇ flanking sequence of NtDEH1 gene was also obtained by genome-walking,and the promoter activity of 5＇ flanking sequence was checked by transient gene expression. The result laid a foundation in researching the role of NtDEH1 gene in plant reproduction.