橡胶树胞质型谷胱甘肽还原酶基因的克隆与表达分析

根据橡胶树GR1基因（Hb GR1）部分序列设计特异引物,运用RACE和RT-PCR技术克隆Hb GR1全长c DNA序列;运用DNAMAN、MEGA 6.06、Prot Param及Signal P 4.1 Server等生物信息学软件对Hb GR1序列、GR1系统进化关系及Hb GR1的基本理化性质和亚细胞定位等进行分析;利用实时荧光定量PCR技术研究Hb GR1的表达模式;构建原核表达载体p EASYE1-Hb GR1,并将其转入大肠杆菌BL21（DE3）,用IPTG诱导融合蛋白原核表达。获得2 082 bp的Hb GR1全长c DNA,其中5′非编码区293 bp,3′非编码区298 bp,开放阅读框长1 491 bp,共编码496个氨基酸,其编码的蛋白分子质量约为53.68 k Da,理论等电点p I为6.18。序列分析发现Hb GR1无信号肽,在氨基酸水平上与其他植物GR1具有很高的同源性,包含植物GR典型的NADH结合结构域、二聚体结构域和高度保守的GGTCV[I/L]RGCVPKK[I/L]LVY基序。对植物GR进行系统进化分析表明,Hb GR1属于双子叶植物GR分枝,与同属大戟科的蓖麻亲缘关系最近。实时荧光定量PCR结果表明Hb GR1在橡胶树胶乳、叶片、树皮和花中均表达;Hb GR1表达受死皮、乙烯、茉莉酸、过氧化氢和伤害调控。SDS-PAGE电泳结果表明重组质粒p EASY-E1-Hb GR1在大肠杆菌BL21（DE3）中有效表达一个分子量约为55 k Da的融合蛋白。

Molecular Cloning and Expression Analysis of a Cytosolic Glutathione Reductase Gene from Hevea brasiliensis

The specifi c primers were designed according to EST sequence of Hb GR1. The full-length c DNA of Hb GR1 was cloned using RACE and RT-PCR technology. The sequences of Hb GR1, phylogenetic relationship with plant cytosolic GRs, properties and subcellular localization of Hb GR1 were analyzed by a variety of bioinformatics softwares such as DNAMAN, MEGA 6.06, Prot Param and Signal P 4.1 Server, etc. The expression profi les of Hb GR1 were analyzed by real-time PCR method. The recombinant plasmid p EASY-E1-Hb GR1 containing the coding sequence of Hb GR1 was constructed and expressed via inducing with IPTG in E. coli BL21（DE3）. The full-length c DNA of Hb GR1 was 2 082 bp in length, containing a 1 491-bp open reading frame fl anked by a 293-bp 5′-UTR and a 298-bp 3′-UTR. Hb GR1 encoded a polypeptide of 496 amino acids with a calculated molecular weight of 53.68 k Da and a PI of 6.18. Being high homology with other plant cytosolic GRs, Hb GR1, without signal peptide sequence, contained a NADH binding domain, a dimerization domain and a highly conserved motif GGTCV[I/L]RGCVPKK[I/L]LVY. Phylogenetic analysis of plant GRs showed that Hb GR1 located in the dicot branch and was closely related to that of Rc GR1, which also belongs to Euphorbiaceae. Real-time PCR analysis indicated that Hb GR1 was ubiquitously expressed in latex, leaves, barks and fl owers. The expression of Hb GR1 was regulated by ethylene, jasmonic acid, hydrogen peroxide and wounding treatments and tapping panel dryness（TPD）. The SDS-PAGE analyses indicated that the recombinant plasmid p EASY-E1-Hb GR1, was effectively expressed in E. coil BL21（DE3） and the molecular weight was approximately 55 k Da.