| Summary: | 碩士 === 國立臺灣大學 === 植物學研究所 === 89 === Escherichia coli cells were transformed with pUC8 (pUC8 vector only), pUC-FL (rice class I low molecular mass heat shock protein Oshsp16.9 cDNA ORF inserted into pUC8), and pUC-C108 plasmids (DNA fragment containing C-terminal 108 amino acids of Oshsp16.9 cDNA ORF inserted into pUC8) previously in our laboratory and the recombinant proteins were expressed. In the present study, the ultrastructural changes of the transformed E. coli cells affected by heat shock were studied with electron microscopy. Meanwhile, immunolocalization and the movement of Oshsp16.9 or its fragment in E. coli cells affected by heat shock were also reported.
Escherichia coli grown under normal environment, cells was enclosed by cell wall with dense ectoplasm and more or less light endoplasm, in which nucleoid region located. With IPTG induction or heat treatment, some dark regions were found in ectoplasm. Having been transferred back to 37℃ after heat shock, about 27% of pUC-FL cells pretreated with IPTG induction recovered to normal. Cells without IPTG pretreatment, only had 0~8% of recovery. These results are in agreement with the thermoprotection of Oshsp16.9.
Immunolocalization of Oshsp16.9 expressed in E. coli after IPTG induction were found both in pUC-FL and pUC-C108 transformed clones. Quantitative comparison of immuno-gold particles in cells under different treatments showed results as follows: there was a distinct difference in Oshsp16.9 expression of cells with and without IPTG induction; there was no conspicuous movement of Oshsp16.9 in normal or high temperature environment; and the density of gold particles in dark regions was higher than that in other regions.
In the second part of this thesis, class I low molecular mass heat shock protein induced by azetidine were precipitated with mitochondria by subcellular fraction were investigated. In this issue, immunolocalization of soybean class I low molecular mass heat shock protein induced by azetidine were performed. Most of the class I low molecular mass heat shock protein accumulated in the cytoplasm and the less around mitochondria of soybean seedlings pretreated by azetidine. When seedlings pretreated with azetidine were transferred back to normal condition and their heat shock proteins would move to cytosol. When soybean seedlings pretreated with azetidine were shifted to heat shock of 40℃, the class I heat shock proteins were observed more abundant in cytosol.
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