芦笋皂苷合成相关糖基转移酶基因克隆及原核表达分析OA北大核心CSTPCD

[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.