滇龙胆香叶醇10-羟化酶基因的克隆与表达分析

发布时间：2019-01-19 09:37

【摘要】：【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。
[Abstract]:[objective] to clone the gene Gr G10H (geraniol 10-hydroxylase) and analyze its bioinformatics characteristics, gene structure, protein expression and tissue expression. [methods] the gene Gr G10H and its genomic sequence were cloned from the total RNA of Gentiana by RT-PCR and gene cloning. The DNA sequence and its encoding protein were analyzed by bioinformatics method, the sequence alignment of Gr G10H protein and its homologous sequence was made by Clustal X2.1 software, and the phylogenetic tree was constructed by MEGA7.0 software. Quantitative PCR technique was used to analyze the expression of Gr G10H gene in the rhizome leaves of Gentiana yunnanensis. [results] the Gr G10H gene was cloned from the leaves of Dian Gentiana by RT-PCR method. The full length of Gr G10H gene was 1834 bp, with exon 2 and intron 1, ORF1548 bp,. Submit the sequence to the Gen Bank, to get the sequence number KJ418410. Bioinformatics analysis showed that the gene encodes 515 amino acids with molecular weight of 57.74 KD and theoretical isoelectric point of 9.02; The protein belongs to the cytochrome P450 superfamily and is located in the endoplasmic reticulum. The N-terminal of the protein contains a transmembrane helix (21 / 43), in which the 1o 20 amino acid residues are located in the membrane and 44 / 515 are located outside the membrane. The protein is a hydrophilic stable protein, which is composed of irregular curl (44.85%) and 伪 -helix (40.58%). The Gr G10H protein is similar to Sm G10H protein (87%). And the kinship is closest. The results of prokaryotic expression showed that the relative molecular weight of the recombinant protein expressed by Gr G10H in E. coli was about 83.74 k D (with GST tag 26.00 k D), and the expected protein size was the same. The results of tissue specific expression analysis showed that Gr G10H gene was mainly expressed in leaves. [conclusion] Gr G10H gene was cloned and successfully expressed in Escherichia coli.
【作者单位】：玉溪师范学院资源环境学院;云南省农业科学院药用植物研究所;
【基金】：云南省教育厅重点项目(2015Z171) 云南省大学生创新项目(201511390005)
【分类号】：S567.239

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