天麻中糖基转移酶基因GeGT1的克隆及原核表达分析

Cloning and prokaryotic expression of glycosyltransferase GeGT1 from Gastrodia elata

目的克隆天麻糖基转移酶基因(glycosyltransferase,Ge GT1)的全长cDNA序列,进行序列分析和原核表达分析,并探究该基因在天麻不同器官中的表达规律。方法在天麻转录组数据库中通过注释和比对,用PrimerPremier5.0软件设计基因特异性引物。通过反转录聚合酶链式扩增(reverse transcription-polymerase chain reaction,RT-PCR)方法从天麻块茎中克隆得到Ge GT1全长cDNA序列,利用生物信息学软件预测基因编码蛋白的特征;利用Clustal W和MEGA6.06软件构建GeGT1的系统进化树;构建原核表达载体pET-28a-GeGT1,转化至大肠杆菌Transetta(DE3)进行诱导表达;通过实时荧光定量PCR(quantitative real-time PCR,q RT-PCR)技术分析基因表达模式。结果Ge GT1基因开放阅读框(open reading frame,ORF)全长1503 bp,编码500个氨基酸,蛋白相对分子质量为55494.33,理论等电点为5.51,为稳定的酸性蛋白。襄襆襈襌襐襒襔襠襢襦襨襶襼覊覌覚覜覦覨覮覰覸覺覼覾览觊角觔觜膜结构域,无信号肽,为非分泌蛋白。通过序列同源性分析,GeGT1与铁皮石斛具有较高的同源性。系统进化树分析证实了兰科植物糖基转移酶基因的同源性。SDS-PAGE结果显示,诱导表达蛋白为63000,与预期蛋白大小一致。基因表达结果显示,Ge GT1基因的表达水平在花中最高,其次是块茎、茎。结论首次克隆并分析了Ge GT1基因,建立了原核表达体系,为进一步研究天麻活性成分合成的分子机制奠定基础。

Objective To clone and analyze the full-length c DNA sequence of Ge GT1,performed sequence analysis and prokaryotic expression analysis,and to explore the expression law of this gene in different organs of Gastrodia elata.Methods Gene specific primers were designed by primer premier 5.0 software through annotation and comparison in G elata transcriptome database.The full-length cDNA sequence of Ge GT1 was cloned from the tuber of G.elata by reverse transcription-polymerase chain reaction(RT-PCR),and the characteristics of the gene-encoded protein were predicted by bioinformatics software.The phylogenetic tree of GeGT1 was constructed by Clustal W and MEGA 6.06 software.Prokaryotic expression vector p ET-28 a-GeGT1 was constructed and converted to E.coli Transetta(DE3)for induced expression.The gene expression patterns were analyzed by quantitative real-time PCR(qRT-PCR).Results The open reading frame(ORF)of Ge GT1 gene was 1503 bp,encoding 500 amino acids,the relative molecular mass of the protein was 55494.33,and the theoretical isoelectric point was 5.51.It was a stable acidic protein.The protein secondary structure was composed of 49%random coils,28.2%α-helices,and 22.8%extended chains.GeGT1 belonged to the GTB-type super family,without signal peptide,and was a non-secreted protein.Through sequence homology analysis,Ge GT1 and Dendrobium candidum had high homology.Phylogenetic tree analysis confirmed the homology of glycosyltransferase genes in orchidaceae.SDS-PAGE showed that the induced expression protein was 63000 consistent with the expected protein size.The results of gene expression showed that the expression level of Ge GT1 gene was different in different parts of G.elata,the highest in flower,the second in tuber,and the lowest in stem.Conclusion In this study,the Ge GT1 gene of G.elata was cloned and analyzed for the first time,and established a prokaryotic expression system,which laid a foundation for further study on the molecular mechanism of G.elata active components synthesis.