基于天麻转录组数据,设计特异引物,克隆天麻DRP1E(dynamin-related protein 1E)基因,命名为GeDRP1E(GenBase登录号为C_AA018577.1),借助ExPASy、ClustalW、MEGA等软件对基因进行生物信息学分析,利用拟南芥、马铃薯遗传转化体系获得转基因植株,并对转基因拟南芥株高、结实率,转基因马铃薯微型薯的块茎长、宽、重量和淀粉含量等农艺性状进行检测和分析,初步解析GeDRP1E基因的功能.结果显示,GeDRP1E基因ORF全长1 899 bp、编码632个氨基酸残基,相对分子质量为69.90 kDa,分子式为C3079H4973N883O933S19;理论等电点为7.27,不稳定系数为43.34,亲水性平均指数为-0.259,预测属于不稳定亲水性蛋白.GeDRP1E不存在跨膜结构,无信号肽,定位于细胞质.系统进化树显示,GeDRP1E与其他种类植物DRP1E蛋白之间具有较高的同源性,其中与铁皮石斛DcDRP1E(XP_020689662.1)同源性最高,为90.05％.运用双酶切法构建植物表达载体pCambia1300-35Spro-GeDRP1E,并通过农杆菌介导转化法获得了 GeDRP1E基因的拟南芥互补突变体、马铃薯过表达株系.与拟南芥AtDRP1E基因突变体相比,GeDRP1E基因互补突变体株高、结实率表型得以恢复.与野生型马铃薯相比,GeDRP1E基因过表达株系微型薯体积、重量显著增大,淀粉含量显著增高.初步推测GeDRP1E很可能参与调节线粒体的形态,从而影响拟南芥植株、马铃薯微型薯的生长发育.上述研究结果为深入阐明天麻块茎生长发育的分子机制奠定了基础.
Abstract
The gene GeDRPlE encoding dynamin-related protein 1E in Gastrodia elata was cloned by specific primers which were designed based on the transcriptome data of G.elata.Bioinformatics analysis on GeDRP1E gene was carried out by using ExPASy,ClustalW,MEGA,etc.Positive transgenic Arabidopsis plant and potato minituber were obtained with the genetic transformation system of Arabidopsis and potato.The plant height and seed setting rate of transgenic Arabidopsis,and agronomic characters,such as size,weight and starch content of potato minituber of transgenic potato were tested and analyzed.And GeDRP1E gene function was preliminarily investigated.The results showed that the open reading frame of GeDRP1E gene was 1 899 bp in length and 632 amino acids residues were encoded,with a relative molecular weight of 69.90 kDa and a molecular formula of C3079H4973N883O933S19.It was predicted that the theoretical isoelectric point was 7.27,the instability coefficient was 43.34,and the average hydrophilicity index was-0.259,which was indicative of an unstable hydrophilic protein.GeDRP1E has no transmembrane structure and signal peptide,and was localized in the cytoplasm.The phylogenetic tree showed that GeDRPlE was highly homologous with DRP1E proteins of other plant species,among which GeDRP1E had the highest homology with DcDRPlE(XP_020689662.1)in Dendrobium candidum,reaching 90.05％.GeDRP1E plant expression vector pCambial300-35Spro-GeDRP1E was constructed by double digests,and Arabidopsis complementary mutant and potato overexpression strain of GeDRP1E gene were obtained by Agrobacterium-mediated gene transformation.Compared with the Arabidopsis AtDRP1E mutant,the height and seed setting rate of the GeDRP1E complementation mutant were rescued.The minituber of GeDRP1E overexpression potato had larger size,heavier weight and higher starch content,comparing to wild-type potato.It was preliminarily induced that GeDRP1E was involved in mitochondrial morphology regulation,which related to the growth and development of Arabidopsis plants and potato miniature.The research results laid a foundation for further elucidating the molecular mechanisms underlying the growth and development of G.elata tuber development.
关键词
天麻/DRP1E基因/遗传转化/功能分析/块茎发育
Key words
Gastrodia elata/DRP1E gene/genetic transformation/functional analysis/tuber development
维普
万方数据