滇龙胆异戊烯基焦磷酸异构酶基因的克隆与表达分析
Cloning and expression analysis of isopentenyl pyrophosphate isomerase gene in Gentiana rigescens
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摘要: 根据药用植物滇龙胆转录组异戊烯基焦磷酸异构酶基因(GrIDI)序列, 应用 RT-PCR 技术从滇龙胆幼叶克隆该基因开放阅读框(ORF) , 获得 GrIDI1 和 GrIDI2 两条序列, 其 GenBank 登录号分别为KM879183 和 KM879184。 生物信息学分析表明 GrIDI1 基因 ORF 长 708 bp, 编码 235 氨基酸; GrIDI1 蛋白相对分子质量为 27.10 ku, 理论 pI 为 5.01。 该蛋白属于 I 型异戊烯基焦磷酸异构酶家族成员, 可能定位于细胞质。该蛋白无信号肽, 为亲水稳定蛋白, 主要由 α- 螺旋(45.53%)和无规则卷曲(39.57%)构成。 该蛋白具有其他 IDI 蛋白的活性位点、 金属结合位点和保守结构域(NUDIX 羟化酶结构域) 。 GrIDI1 蛋白与黄龙胆 GlIDI蛋白亲缘关系最近。 原核表达结果表明, GrIDI1 基因在大肠杆菌中表达的重组蛋白相对分子质量约为 53.11ku, 与预期大小一致。 组织特异性表达分析结果表明 GrIDI1 基因主要在叶中表达。Abstract: The ORF of isopentenyl pyrophosphate isomerase gene(GrIDI)was cloned from young leaves of Gentiana rigescens by RT-PCR technology according to the GrIDI sequence of transcriptome of medicinal plant G. rigescens. As a result, two sequences GrIDI1 and GrIDI2 with their GenBank accession numbers KM879183 and KM879184 were obtained. Sequence analysis showed that GrIDI1 gene had a ORF of 708 bp coding for 235 amino acids. Its relative molecular weight was 27.10 ku with the theoretical isoelectric point of 5.01. GrIDI1 was a member of IDI superfamily I and may localize in cytoplasm. GrIDI1 was a hydropholic stable protein without signal peptide and composed of mainly α-helix(45.53%)and random coils(39.57%). The active sites, metal binding sites and domain of NUDIX hydroxylase in other IDI proteins all existed in GrIDI1. GrIDI1 protein was close to GlIDI in G. lute. The results of prokaryotic expression of GrIDI1 gene in E. coli showed that the recombinant protein was approximately 53.11 ku, which was consistent with the anticipated size. The tissue-specific expression results indicated that GrIDI1 gene primarily expressed in leaf.