生物技术通报2024，Vol.40Issue(4)：255-263,9.DOI:10.13560/j.cnki.biotech.bull.1985.2023-1074
芦笋皂苷合成相关糖基转移酶基因克隆及原核表达分析
Cloning and Prokaryotic Expression Analysis of Asparagus Saponin Synthesis Related Glycosyltransferase Genes
摘要
Abstract
[Objective]This study aims to clone the sterol glycosyltransferase gene AoSGT1 from Asparagus officinalis,to assess its catalytic properties,and to elucidate its role in the biosynthesis of asparagus saponins and their metabolic regulation.[Method]We designed specific primers based on asparagus transcriptome data to amplify the complete open reading frame(ORF)of AoSGT1.The gene was sequenced and analyzed through bioinformatics,and its expression profile was investigated via quantitative real-time PCR(RT-qPCR).We also constructed a pGEX-4T-3-AoSGT1 expression vector,expressed it in Escherichia coli BL21(DE3),and induced the recombinant protein production.[Result]AoSGT1 has a length of 1 800 base pairs,encoding 599 amino acids,with a relative molecular weight of 66.72 kD.It is a hydrophilic protein with no transmembrane domains or signal peptides.The phylogenetic analysis revealed a close homology between AoSGT1 and Dioscorea zingiberensis Dz3GT2,indicating their membership in the UGT80B1 subfamily.Multiple sequence alignment revealed that the protein sequence contained conserved domains"PSBD Box"and"PSPG Box"of sterol glycosyltransferase,indicating its potential glycosylation activity at the 3β-OH position of steroidal compounds.RT-qPCR results showed that AoSGT1 was highly expressed in the roots,while expression in stems and flowers was relatively low.Additionally,SDS-PAGE results revealed that the target protein was efficiently expressed in a soluble form within E.coli,matching the predicted size.[Conclusion]The AoSGT1 gene was successfully cloned and demonstrated to exhibit tissue-specific expression in asparagus,suggesting its potential involvement in the biosynthesis of steroidal saponins in asparagus.Additionally,heterologous expression of the target protein in E.coli was successfully achieved.关键词
芦笋/甾体皂苷/甾醇糖基转移酶/基因克隆/生物信息学分析/RT-qPCR/原核表达/转录组Key words
Asparagus officinalis/steroidal saponins/sterol glycosyltransferase/gene cloning/bioinformatics analysis/RT-qPCR/prokaryotic expression/transcriptome引用本文复制引用
钟匀,林春,刘正杰,董陈文华,毛自朝,李兴玉..芦笋皂苷合成相关糖基转移酶基因克隆及原核表达分析[J].生物技术通报,2024,40(4):255-263,9.基金项目
云南省外专引智项目(202105AO130001),云南省农业联合专项重点项目(202101BD070001-027,202301BD070001-018),云南省基础研究计划面上项目(202201AT070254) （202105AO130001）
