滇龙胆香叶醇10-羟化酶基因的克隆与表达分析

Cloning and Expression Analysis of Geraniol10-hydroxylase Gene in Gentiana rigescens

【目的】克隆滇龙胆香叶醇10-羟化酶基因Gr G10H(geraniol 10-hydroxylase),分析其生物信息学特征、基因结构、蛋白表达和组织表达情况,为龙胆苦苷生物合成途径解析提供理论依据。【方法】通过RT-PCR及基因克隆方法,从滇龙胆总RNA中克隆得到基因Gr G10H及其基因组序列;使用生物信息学方法分析其DNA序列及其编码蛋白特性,使用Clustal X2.1软件对Gr G10H蛋白质及其同源序列做序列比对,使用MEGA7.0软件进行系统发育树构建;利用定量PCR技术分析Gr G10H基因在滇龙胆根茎叶中的表达水平。【结果】使用RT-PCR方法从滇龙胆叶片中克隆得Gr G10H基因全长为1834 bp,包含2外显子和1内含子,ORF1548 bp,将序列提交至Gen Bank,得到序列号KJ418410。生物信息学分析表明,该基因编码515个氨基酸,分子量为57.74 k D,理论等电点为9.02;该蛋白属于细胞色素P450超家族成员,定位于内质网,其N端含一跨膜螺旋(21~43),其中1~20氨基酸残基位于膜内,44~515位于膜外。该蛋白无信号肽,为亲水稳定蛋白,主要由无规则卷曲(44.85%)和α-螺旋(40.58%)构成。Gr G10H蛋白与川西獐牙菜Sm G10H蛋白具有较高的相似性(87%),且亲缘关系最近。原核表达结果表明,Gr G10H基因在大肠杆菌中表达的重组蛋白相对分子质量约为83.74 k D(含GST标签26.00 k D),与预期蛋白大小一致。组织特异性表达分析结果表明Gr G10H基因主要在叶中表达。【结论】克隆到滇龙胆Gr G10H基因,并成功在大肠杆菌中表达,推测其在主要叶片中起作用。

[ Objective] The objective of this study is to clone gentian geraniol 10-hydroxylase gene GrGIOH, to analyze its bioinformatics char- acters, gene structure, prokaryotie expression and tissue expression, and to provide a theoretical base for elucidation of getiopicroside biosyn- thesis pathway. [ Method] RT-PCR and gene cloning were adopted to isolate the gene GrGIOH from the leaves of Gentiana rigescens. Bioin- formatics tools were used to analyze the characters of both DNA sequence and its coding protein. Clustal X2.1 software was used to make the multiple sequence alignments between the GrG10H protein and their homologous sequences, and the phylogenetic tree of homologous species was constructed by MEGA6.0. The real-time fluorescent quantitative RT-PCR was applied to analyze the expression of GrGIOH in root, stem and leaf. [ Result] GrGIOH gene was obtained from G. rigescens leaves by RT-PCR, and was deposited into GenBank （accession number： KJ418410）. The results of bioinformatics analysis showed that GrGIOH gene had a length of 1834 bp containing two exons and one intron. Its ORF （open reading frame） was 1548 bp coding for 515 amino acids. Its relative molecular weight was 57.74 kD with the isoelectric point of 9.02. GIGIOH protein was the member of cytochrome 17450 superfamily and may localize in endoplasmic reticulum; there was a trans- membrane region in N-terminal （21 -43 ）, while the first 20 amino acids resided within the membrane and that of 44 -515 were out of membrane; GrG10H was a hydrophilic stable protein without signal peptide and composed of mainly a-helix （40.58%） and irregular coils （44. 85%）. GrG10H protein was close to SmG10H of Swertia mussotii. The results of prokaryotic expression of GrGIOH gene in E, coli showed that the recombinant protein was approximately 83.74 kD （ containing GST tag protein 26.00 kD）, which was consistent with the theoretical size. The tissue-specific expression results indicated that GrGIOH gene was primarily expressed in leaf. [ Conclusion ] GrGIOH gene was cloned and expressed in E. coli. successfully, and it may mainly tunctioned in leaves.