Study on functional genomics of metabolite-related genes by immochemical method in root culture systems

1.发根农杆菌介导法是一种获得大量转基因根克隆的高通量转化系统。细菌Ri质粒中的rol基因簇负责毛状根的诱导。我们开发了用于毛状根激活的ROL型二进制载体(HR-AT)。基因表达数据表明这些载体在植物中起作用。2.首次从茄子中成功克隆了类固醇糖基转移酶基因。茄科植物中含有具有多种生物学和药理活性的甾体皂苷。糖基化是皂苷生物合成的最后一步，其糖链结构被认为对其活性具有重要影响。在筛选了几个茄子糖基转移酶(GTS)的基因片段后，从尖叶茄子中获得了三个全长cDNA候选基因。重组GT在大肠杆菌中以GST融合蛋白的形式表达。其中一个GT，命名为SaG4A，对一些甾体皂苷元和螺茄烷型生物碱具有糖受体专一性。3.为了高通量地筛选基于免疫化学方法的根无性系，确定了检测甾体皂苷(针叶皂苷)的条件。结果表明，在最佳条件下，可检测到2μg以上的麻黄皂苷。重组毛状根培养物中SaG4A的RNAi显示皂苷含量降低，表明SaG4A是植物中的一种功能性类固醇糖基转移酶。

1.Agrobacterium rhizogenes-mediated transformation is a high-throughput system for obtaining large numbers of transgenic root clones. The rol-gene cluster in the bacterial Ri plasmid is responsible for hairy root induction. We developed rol-type binary vectors for hairy-root activation(HR-AT). Gene expression data suggest that these vectors work in planta.2.We succeeded in cloning a cDNA for steroid glycosyltransferase from Solanum sp., for the first time. The Solanaceae contain steroid saponins with diverse biological and pharmacological activities. Glycosylation is the final step in the biosynthesis of saponins, and their sugar chain structure is considered important for their activities. After screening gene fragments for putative glycosyltransferases(GTs) from several Solanum sp., three candidates for full-length cDNAs were obtained from S.aculeatissimum. Recombinant GTs were expressed in E.coli as GST fusion proteins. One of the GTs, named SaG4A, showed sugar acceptor specificity for some steroidal sapogenins, as well as spirosolane-type alkaloids. The glucose moiety might be transferred to the 3-hydroxy position of the sapogenin.3.For high-throughput screening of root clones based on immnochemical method, condition for detecting steroidal saponin (aculeatiside) was determined. As a result, more than 2μg of aculeatiside was detected in a best condition. RNAi of SaG4A in recombinant hairy root cultures showed a decreased saponin content, suggesting that SaG4A is a functional steroid glycosyltransferase in planta.