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

1.发根农杆菌介导的转化是一种高通量的获得大量转基因根克隆的系统。细菌Ri质粒中的rol基因簇负责毛状根的诱导。我们开发了罗尔型二元载体的毛根激活（HR-AT）。基因表达数据表明，这些载体在植物中的工作。2.我们成功地克隆了一个cDNA类固醇糖基转移酶从茄属，第一次茄科植物中含有甾体皂苷类化合物，具有多种生物活性和药理活性。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很重要。在从几种茄属物种中筛选推定的糖基转移酶（GT）的基因片段后，从S. aculeatissimum获得了全长cDNA的三个候选物。重组GT在大肠杆菌中表达为GST融合蛋白。其中一个GT，命名为SaG 4A，显示糖受体特异性的一些甾体皂苷元，以及螺茄型生物碱。葡萄糖部分可能被转移到皂苷元的3-羟基位置。3.为了高通量筛选基于免疫化学方法的根克隆，确定了检测甾体皂苷的条件。结果表明，在最佳条件下，刺五加苷的含量可达2μg以上。在重组毛状根培养物中，SaG 4A的RNAi显示皂苷含量降低，表明SaG 4A是植物中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.