2005 Fiscal Year Final Research Report Summary
Tracking of petal cell maturing process by single-cell based metabolic analysis
| Project/Area Number |
16510161
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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 | Osaka University |
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Principal Investigator |
KAJIYAMA Shin'ichiro Osaka University, Graduate school of Engineering, Specially Appointed Researcher, 大学院工学研究科, 特任研究員 (20243496)
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| Project Period (FY) |
2004 – 2005
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| Keywords | Single-cell / metabolic analysis / profiling / petal cell / laser cell-processing / microsampling / RT-PCR / Nanoflow LC_MS |
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| Research Abstract |
In this study, the petal cell maturing process of Torenia plant was investigated by using laser single-cell sampling and analysis technique. In the first year, sample preparation method and laser irradiation condition, and nano-flow LC-MS analysis condition of single-cell origin samples were investigated. Through this investigation, the facile analytical method for petal cell pigment was established. In second year, the quantitative analysis of mRNA concerning petal pigment biosynthesis was investigated. In particular, novel high sensitive quantitative mRNA analysis based on the ESI-MS was developed. In this method, the reporter molecule such as FAM (fluorescein phosphoramidite) was attached to the 5' end of the PCR primer and according to the PCR polymerizing reaction, the reporter molecule and the 5'end nucleotide was released. By quantify the released molecule using ESI-MS, the amount of targeted mRNA in the sample was estimated. The FAM attached nucleotide (FAM-A, FAM-T, FAM-G, FAM-C) was completely separated and quantified by LC-ESI-MS. Using this method, we are succeeded in the estimation of CHS and GADPH quantities in single petal cell of Torenia plant.
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