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

根据药用植物滇龙胆转录组异戊烯基焦磷酸异构酶基因(Gr IDI)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Gr IDI1和Gr IDI2两条序列,其Gen Bank登录号分别为KM879183和KM879184。生物信息学分析表明Gr IDI1基因ORF长708 bp,编码235氨基酸;Gr IDI1蛋白相对分子质量为27.10 ku,理论p I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)。Gr IDI1蛋白与黄龙胆Gl IDI蛋白亲缘关系最近。原核表达结果表明,Gr IDI1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Gr IDI1基因主要在叶中表达。

The ORF of isopentenyl pyrophosphate isomerase gene （ GrlDI ） was cloned from young leaves of Gentiana rigescens by RT-PCR technology according to the GrlDI sequence of transcriptome of medicinal plant G. rigescens. As a result, two sequences GrlDI1 and GrlDI2 with their GenBank accession numbers KM879183 and KM879184 were obtained. Sequence analysis showed that GrlDI1 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 ct-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 GIIDI in G. lute. The results of prokaryotic expression of GrlDI1 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 GrlDI1 gene primarily expressed in leaf.