连翘FsUGT83A1基因的克隆、生物信息学和表达分析

Cloning, Bioinformatic and Expression Analysis of FsUGT83A1 Gene in Forsythia suspensa

目的:克隆连翘糖基转移酶基因FsUGT83A1,并对其进行生物信息学和组织表达分析,了解其与连翘苷合成的相关性。方法:基于连翘基因组和转录组数据,利用RT-PCR扩增FsUGT83A1全长cDNA并进行生物信息学分析;运用实时荧光定量PCR检测基因在不同组织中的表达;采用HPLC测定成分在不同组织中的含量。结果:FsUGT83A1基因的蛋白质编码区(CDS)全长为1 365 bp,编码一个由454个氨基酸组成的蛋白,该蛋白为亲水性非跨膜蛋白,亚细胞定位主要在细胞质中。多序列比对分析表明,该基因具有UGT特有的保守序列PSPG-box;系统进化分析表明,FsUGT83A1与拟南芥UGT83A1聚在一个亚家族,与同科植物油橄榄UGT83A1的同源性最高。该基因在叶中的表达显著高于花、果等组织。HPLC定量分析表明,连翘苷在叶中的含量显著高于其他组织。FsUGT83A1的组织表达与连翘苷的组织分布具有显著相关性,推测该基因可能是调控连翘苷糖基化合成过程的功能基因。结论:FsUGT83A1为首次从连翘中克隆得到的UGT基因,为进一步研究该基因的功能及调控机制提供了研究基础。

Objective: To clone the glycosyltransferase genes FsUGT83A1 of Forsythia suspensa and analyze the bioinformatics and relative expression in different tissue, to investigate its correlation with forsythin synthesis.Methods: Based on the genome and transcriptome data of Forsythia suspensa,the FsUGT83A1 full length cDNA was amplified by RT-PCR and the bioinformatics was analyzed.Real-time quantitative PCR was used to detect the gene expression in different tissues.HPLC was used to determine the content in different tissues.Results: The Coding sequence(CDS) full length of FsUGT83A1 gene was 1 365 bp, encoding 454 amino acids.The protein was a hydrophilic non-transmembrane protein, with a likely subcellular localization in the cytoplasm.The multiple sequence alignment indicated that FsUGT83A1 had the typical conserved domains of PSPG-box of UGT genes.Phylogenetic tree analysis showed that FsUGT83A1 was classified as the same subfamily as the UGT83A1 of Arabidopsis thaliana,and showed the highest homologies with the UGT83A1 of same family plant Olea europaea.The expression of FsUGT83A1 in leaves was significantly higher than that in flowers, fruits and other tissues.HPLC quantitative analysis indicated that the content of forsythin in leaves was higher than that in other tissues.The tissue expression of FsUGT83A1 was significantly correlated with the tissue distribution of forsythin, suggesting that FsUGT83A1 may be a functional gene regulating the glycosylation and synthesis process of forsythin.Conclusion: FsUGT83A1 is the first UGT gene cloned from Forsythia suspensa,the study provides important basic information for the further study of the function and regulatory mechanism of the gene.