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標題: | cpHsc70 作為葉綠體蛋白運輸馬達的機制 Mechanism of cpHsc70 as a Chloroplast Protein Import Motor |
作者: | Po-Kai Huang 黃柏凱 |
指導教授: | 李秀敏(Hsou-min Li) |
關鍵字: | 葉綠體,熱休克蛋白 70,胞內運輸, Chloroplast,Hsp70,Intracellular trafficking, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 葉綠體熱休克同源蛋白70 (chloroplast heat shock cognate proteins 70 kD, cpHsc70) 和熱休克蛋白93 (Hsp93) 這兩個運輸馬達在前驅蛋白 (precursor protein)由細胞質進到葉綠體基質 (stroma) 的過程中扮演了重要的角色。不過目前只知道這兩個蛋白質會在生物體外的實驗條件下和前驅蛋白上的導引訊息 (transit peptide) 進行結合。在連續時間點取樣的實驗裡,我們發現在生物體內的實驗條件下,cpHsc70或許也會和前驅蛋白的成熟區位 (mature region) 進行結合。我的論文由兩個部分所組成。首先,為了要測試cpHsc70是否會在蛋白質運輸的時候去結合mature region,我在cpHsc70可能的結合位上進行了位置導向的突變 (site-directed mutagenesis) ,並且使用這些突變蛋白去進行結合試驗 (binding assays) 。我發現有部分的突變蛋白的確對cpHsc70有較差的結合力。這些突變蛋白將會被用來進行葉綠體蛋白質運輸試驗並測試是否會造成運輸效率的下降、以及此下降的現象是否起因於較差的cpHsc70結合力。第二部分則是:熱休克蛋白70家族 (heat shock proteins 70 kD, Hsp70) 執行專一的生理活動時必須有專一的J protein來召集Hsp70到執行功能的位置。召集cpHsc70到葉綠體運輸複合體,以幫助蛋白質運輸的J protein目前仍然是未知的。因此,我分析了葉綠體DJC23家族 (含DJC23、DJC24、及DJC66) 的特性。我們已經得到DJC23以及DJC66的基因敲除 (knockout) 株,不過並沒有得到DJC24的敲除株。我也製造了djc23 djc66的雙重突變株。不過,目前為止我並沒有觀察到野生型、單一基因突變、以及雙重基因突變的葉綠體之間有任何蛋白質運輸效率上的顯著差異。此外,我也進行了熱壓力耐受度 (heat stress tolerance) 的測試,但也沒有發現野生型和突變型之間有任何的差異。 DJC24-RNAi植物目前正在著手製造中。我將在djc23 djc66的雙重突變中以DJC24-RNAi降低DJC24的量、並且觀察這些植株是否有蛋白質運輸或者其他葉綠體功能的缺失,以了解DJC23這個J protein家族的生理功能。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/61887 |
全文授權: | 有償授權 |
顯示於系所單位: | 分子與細胞生物學研究所 |
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