オオムギ子葉細胞における遺伝子発現解析のための基礎的研究
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概要
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[Synopsis] Among glass pipettes prepared with a needle-drawing apparatus, those with tip diameter of 2.5-3.0μm were found suitable for sucking up cytoplasmic contents of barley coleoptile cells. The volume of sucked up cytoplasmic contents was proportional to the number of microsucked cells and the volume of cytoplasmic content of single cell was estimated to be approximately 2.8×10^<-2> nl from the regression line. The effect of microsuction of cytoplasm from targetted cell on the survival rate of adjacent cells was determined by cytoplasmic streaming and stainability with fluorescein diacetate. Neighboring cells of microsucked cell showed normal cytoplasmic streaming and fluoresced as those located far from the microsucked cell, suggesting that microsuction could be used for sucking up cellular contents from a specific cell without causing major damage to adjacent cells. To prove that the extracted cytoplasm contains RNA, the extracts were hybridized with EcoRI digested-pGR221 which contains 18s rRNA sequence of barley coleoptile cells. Positive hybridization signal was observed indicating that the extracts contained at least rRNA of the cells. Total RNAs were then extracted from control and powdery mildew-inoculated barley leaves and mRNA was isolated by using oligo(dT) latex particles. The mRNAs were transcribed to cDNA for differentiating mRNA specific to infected cells. One of clones which were expressed in both the control and infected leaves, pCDNA 10, was used for Northern hybridization with the extracted cytoplasmic contents. Since a positive signal was observed, its sequence was determined. These results suggested that genes expressed in a particular cell could be specified by the procedure developed in this study. However, in view of scanty amounts of mRNA in cells, it is necessary to amplify them by some measures such as in vitro or in situ PCR. As an approach to in situ PCR, a procedure for pricking introduction of FITC-labeled bovine albumine was established.
- 近畿大学農学総合研究所の論文
- 1997-03-00
近畿大学農学総合研究所 | 論文
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