| Project/Area Number |
15510189
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Living organism molecular science
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| Research Institution | RIKEN |
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Principal Investigator |
MURANAKA Toshiya RIKEN, Laboratory for Biochemical Resources, Laboratory head, バイオケミカルリソース研究チーム, チームリーダー (60342862)
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| Co-Investigator(Kenkyū-buntansha) |
TANAKA Hiroyuki Kyushu University, Graduate school of medical science, Associate professor, 大学院・薬学研究院, 助教授 (30253470)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2004: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2003: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | hairy root / steroidal saponin / steroidal sapogenin / Solanum aculeatissimum / gllycosyltransferase / glucosyltransferase / RNAi / plant tissue culture / アクティベーションタギング / ステロール / ステロイド |
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| Research Abstract |
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.
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