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

1.根农杆菌介导转化是获得大量转基因根无性系的高通量系统。细菌Ri质粒中的roll -基因簇负责毛状根的诱导。我们开发了毛根激活（HR-AT）的卷型二元载体。基因表达数据表明这些载体在植物中起作用。首次从茄属植物中成功克隆了类固醇糖基转移酶cDNA。茄科植物含有甾类皂苷，具有多种生物学和药理活性。糖基化是皂素生物合成的最后一步，其糖链结构被认为对其活性很重要。从几种茄属植物中筛选推测的糖基转移酶（GTs）基因片段后，从茄属植物中获得了3个全长cdna候选基因。重组GTs以GST融合蛋白的形式在大肠杆菌中表达。其中一种被命名为SaG4A的GTs对某些甾体皂原素和螺罗茄碱类生物碱表现出糖受体特异性。葡萄糖部分可能被转移到皂苷元的3-羟基位置。为了利用免疫化学方法对根无性系进行高通量筛选，确定了甾体皂苷的检测条件。结果表明，在最佳条件下，马鞭草苷的检出量大于2μg。重组毛状根培养中SaG4A的RNAi显示皂苷含量降低，表明SaG4A在植物中是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.