| Summary: | 碩士 === 國立臺灣大學 === 分子與細胞生物學研究所 === 101 === Both the cpHsc70 (chloroplast heat shock cognate proteins 70 kD) and Hsp93 (heat shock proteins 93 kD) chaperone motor proteins have been shown to be important for driving precursor translocation from cytosol into the chloroplast stroma. However, their functional mechanisms are not known and it has only been shown that both proteins can bind transit peptides in vitro. Our in vivo import time course experiments showed that cpHsc70 may have an additional role in binding to the mature region of precursors. My thesis studies are consisted of two parts. First, to directly test if cpHsc70 can bind to the mature region of precursors during import, I performed side-directed mutagenesis of predicted Hsp70 binding sites in the mature regions and in vitro binding assays. I found that some of the mutants indeed have reduced binding to cpHsc70. These mutants will be used in chloroplast import experiments to test if they really have reduced import and whether the reduction is due to disrupted interaction with cpHsc70. Second, all Hsp70s require specific co-chaperone J proteins to recruit them to specific processes. The J protein that recruits cpHsc70 to the chloroplast translocon remains to be identified. I therefore characterized a family of three small J proteins, DJC23 and its two homologues DJC24 and DJC66. Knockout mutants were obtained for DJC23 and DJC66 but not for DJC24. Double mutants of djc23 djc66 were generated. However, so far no obvious difference was observed for protein import efficiencies among chloroplasts from wild-type, single and double mutant plants. I have also tested heat stress tolerance of the mutants and found no clear difference to the wild type. DJC24-RNAi plants are being generated. Plants with djc23 djc66 double mutation and DJC24 knocked down will be generated and their phenotypes in protein import or other aspects of chloroplast biogenesis can then be investigated.
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