烟草脱水素基因NtDEH1的克隆及研究

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。

Cloning and Analysis of Dehydrin Gene NtDEH1 in Nicotiana tabacum

To study the function and mechanism of dehydrin in early embryogenesis of plant,egg cells were iso-lated from tobacco to construct a cDNA library,and a gene named NtDEH1 were found.The full sequence of Nt-DEH1 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 tomen-tosiformis,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′flan-king sequence of NtDEH1 gene was also obtained by genome-walking,and the promoter activity of 5′flanking se-quence was checked by transient gene expression.The result laid a foundation in researching the role of NtDEH1 gene in plant reproduction.