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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。
According to the partial sequence of Hb GR1 gene of rubber tree, specific primers were designed and the full-length cDNA sequence of Hb GR1 was cloned by RACE and RT-PCR. The DNA sequence of Hb GR1 was cloned by bioinformatics software such as DNAMAN, MEGA 6.06, Prot Param and Signal P 4.1 Server GR1 sequence, the evolutionary relationship of GR1 system and the basic physical and chemical properties and subcellular localization of Hb GR1. The expression pattern of Hb GR1 was studied by real-time fluorescence quantitative PCR. The prokaryotic expression vector pEASYE1-Hb GR1 was constructed and transferred into Escherichia coli BL21 (DE3) was used to induce prokaryotic expression of the fusion protein with IPTG. The full-length cDNA of Hb GR1 with 2 082 bp was obtained. The 5 ’non-coding region 293 bp, the 3’ non-coding region 298 bp, the open reading frame 1 491 bp, encoding a total of 496 amino acids. Is 53.68 kDa, the theoretical isoelectric point p I is 6.18. Sequence analysis revealed that Hb GR1 has no signal peptide and has high homology with other plant GR1 at the amino acid level, including the typical NADH binding domain, dimer domain and highly conserved GGTCV [I / L] RGCVPKK [I / L] LVY motif. Phylogenetic analysis of plant GR showed that Hb GR1 belongs to the dicotyledonous GR branch, which is closest to castor in the genus Euphorbiaceae. Real-time quantitative PCR results showed that Hb GR1 was expressed in rubber latex, leaves, bark and flowers; Hb GR1 expression was regulated by dead skin, ethylene, jasmonic acid, hydrogen peroxide and injury. The result of SDS-PAGE showed that the recombinant plasmid pASY-E1-Hb GR1 efficiently expressed a fusion protein with a molecular weight of about 55 kDa in E.coli BL21 (DE3).