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研究生:黃仲義
研究生(外文):Chung-Yi Huang
論文名稱:篩選和鑑定水稻第一族低分子量熱休克蛋白質基因的新成員
論文名稱(外文):Isolation and Characterization of New Members of class I Low-Molecular-Weight Heat Shock Proteins in Rice
指導教授:林秋榮林秋榮引用關係
指導教授(外文):Chu-Yung Lin
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:64
中文關鍵詞:熱休克蛋白質低分子量熱休克蛋白質
外文關鍵詞:heat shock proteinshspHSPLMW HSP
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水稻在熱休克處理下(41℃),所產生的第一族低分子量熱休克蛋白質(low molecular weight heat shock protein, LMW HSP)所生成的複合體(heat shock protein complex) 經由雙向電泳(2D-PAGE)及其Western blot抗體分析得知水稻第一族LMW HSP約有8、9個成員。目前本研究室分離並鑑定出6個成員,分別為Oshsp16.9A (pTS1)、Oshsp16.9B、Oshsp16.9C、Oshsp17.3 (pTS3)、Oshsp17.7和Oshsp18.0 (pYL)。本研究的目的是希望從水稻基因庫中篩選出其它的第一族LMW HSP基因的新成員。
首先,以Lambda ZAP載體(EcoRI/CIAP-treated)建構水稻基因庫,然後以既有的水稻第一族LMW HSP基因最具保留性的片段C108(編譯pTS1的C端108個胺基酸的核甘酸序列)和具有基因專一性之六個基因的 3’untranslated region (3’-UTR)分別當探針進行篩選,共挑選得到18個會與C108探針雜合但不會與3’-UTR探針雜合的溶菌斑。經限制酵素圖譜分析並定序後,最後只篩選到一個完整的水稻第一族LMW HSP基因,定為Oshsp17.9,此為第七個成員。
Oshsp17.9與位於chromosome 1的Oshsp16.9A、B、C三個基因分別有76.6%,77.3%及75.3%的相同性(identity);而與位於chromosome 3的Oshsp17.3、Oshsp17.7、Oshsp18.0三個基因有較高的相同性,分別為91.8%,90.8%及93.2%的。經由染色體定位(chromosome mapping)得知Oshsp17.9亦位於chromosome 3。在Oshsp17.9基因的5’上游處分別具有TATA box和heat shock elements (5’-nGAAn-3’)的相似序列。以primer extension方法定出轉錄起始點,位於TATA box下游第25個核甘酸A處,亦即轉譯起始點上游第108個核甘酸處。以Oshsp17.9基因的3’-UTR當探針,水稻在41℃處理下,30分鐘即可見Oshsp17.9基因轉錄的mRNA累積,一小時即達最高量,其後遞減。Oshsp17.9蛋白質分子量17.9KD ,pI值5.9。利用2D-PAGE及其Western blot分析重組的Oshsp17.9蛋白質進一步證實Oshsp17.9確實為水稻第一族低分子量熱休克蛋白質基因的新成員。

There are 8 or 9 members of rice Class I low molecular weight heat shock protein (LMW HSP) genes according to 2D-PAGE and subsequent Western blotting analysis of the LMW HSP complex. Six members of Class I LMW HSP, Oshsp16.9A (pTS1), Oshsp16.9B, Oshsp16.9C, Oshsp17.3 (pTS3), Oshsp17.7 and Oshsp18.0 (pYL) have been isolated and characterized in previous study. This study was aimed to isolated and characterized the new members of rice Class I LMW HSP.
Firstly, rice genomic DNA digested with EcoRI was ligated in λ ZAP vector (EcoRI/CIAP-treated) to construct the rice genomic library. Using the conserved DNA fragment encoding C-terminal 108 amino acids (C108) of Oshsp16.9A and gene specific fragment, 3’-untranslated regions (3’-UTR) of the 6 members as probes respectively, 18 positive clones were isolated. After restriction enzyme mapping and sequence analysis, only one genomic clone (designated as R2.6) was identified, which contained a putative new member of class I LMW HSP gene assigned as Oshsp17.9.
Comparing the nucleotide sequence of Oshsp17.9 with Oshsp16.9A, Oshsp16.9B and Oshsp16.9C located on chromosome 1, the identities are 76.6%, 77.3% and 75.3%, respectively. On the other hand Oshsp17.9 shows higher identity of 91.8%, 90.8% and 93.2% with Oshsp17.3, Oshsp17.7 and Oshsp18.0, respectively. Several heat shock elements and one TATA box were deduced from 5’ upstream region of Oshsp17.9. The transcription initiation site of Oshsp17.9 was located on the 108th base upstream from the start codon and the 25th base downstream from the TATA box.
Using 3’-UTR as probe, the transcription of Oshsp17.9 was induced after 41℃ heat shock treatment for 30 minutes, and reached to the highest level in one hour.
Expression of Oshsp17.9 gene in E. coli was analyzed by western blot analysis after 2D-PAGE. We identified Oshsp17.9 as a new member of rice Class I LMW HSP with the molecular weight of 17.9 kD and pI value of 5.9.

目 錄
縮寫對照表
中文摘要------------------1
英文摘要------------------3
簡介--------------------5
材料與方法-----------------8
結果--------------------25
討論--------------------30
參考文獻------------------33

附錄

參考文獻
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