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標題: | 水稻低分子量熱休克蛋白質cDNA clones之篩選與定序 Screening and Sequencing of Rice Low Molecular Weight Heat Shock Proteins cDNA Clones |
作者: | 曾冬筍 |
出版年 : | 1990 |
學位: | 碩士 |
摘要: | 以pCE53作為DNA探針(pCE53為大豆15-18kd低分子量熱休克蛋白質cDNA clone;由Dr. Joe L. Key, Department of Botany, the University of Georgia Athens, G.A. USA所贈),從水稻熱休克蛋白質cDNA庫中,共篩選到6個λclones。其中4個,cDNA inserts大小分別是0.5, 0.8, 0.7, 0.7kb。 以水稻RNA blot分析,顯示這幾個clones對水稻熱休克RNA (HS RNA)有專一性的雜合反應,而且這些雜合的熱休克RNA大略都是0.9kb。 這四個λ clones在次選殖(subcloned)到pGEM 3Z DNA之後,選擇了pTS1及pTS4 (cDNA inserts分別是0.8及0.5kb),進行hybrid-select in vitro translation以及cDNA定序等實驗。 pTS1及pTS4 hybrid-select in vitro translation的結果得到了同一群由7個蛋白質所組成的16-20kd的低分子量熱休克蛋白質,以及另一個30kd的熱休克蛋白質。 而pTS1及pTS4 cDNA序列比對的結果,彼此有70%的同源性(homology)。同時,這兩個clones,在DNA序列上,也與大豆LMW hsp class I基因有68%以上的同源性。 pTS1的胺基酸序列與GmHSP 18.5-C及Wheat C5-8分別有69.5%,80.4%的相同(idenity);而pTS1的hydropathy profile也顯示與其他已知的低分子量熱休克蛋白質有相似的圖形(pattern),不過pTS1的厭水性區域(hydrophobic domains)較多。 從以上的實驗結果,我們可以肯定pTS1及pTS4確實是水稻低分子量熱休克蛋白質cDNA clones。 We have constructed a cDNA library from heat shocked rice seedlings; six HS spcific cDNA clones were isolated with pCE53 as a DNA probe (a low molecular weight soybean heat shock protein cDNA clone provided by Dr. Joe L. Key, Botany Dept., The Univ. of Georgia, Athens,GA. U.S.A.). The cDNA inserts of 4 λ clones were identified as 0.5, 0.8, 0.7, 0.7 kb long respectively; the Northern blot analysis indicated that these clones hybridized specifically to RNA from 41℃ treated rice seedlings,and no detectable signals appeared on RNA from controle (28℃) seedlings. The λ clones subsequently subcloned to pGEM 3Z were designated as pTS1, pTS2, pTS3, pTS4 (with 0.8, 0.7, 0.7, 0.5 kb cDNA inserts respectively). The hybrid-select in vitro translation of 41℃ poly(A)+RNA with pTS1 and pTS4,showed that these two clones hybrid-selected a group of HS poly(A)+ RNA,and translated to 7 peptides in the range of 16-20 kd, and one with 30 kd. The nucleotides of pTS4 and the full length cDNA clone -- pTS1 were sequenced with Sanger's dideoxynucleotide chain termination method, and the results showed 70% identity between these two cDNAs; also both clones have a high homology (above 68%) with soybean LMW hsp class I genes. The derived amino acid sequence of pTS1 showed 69.5% idenity with GmHSP 18.5-C and 80.4% idenity with wheat C 5-8 , the hydropathy profile of pTS1 showed a similar but more hydrophobic pattern with other LMW HSPs。 |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75748 |
全文授權: | 未授權 |
顯示於系所單位: | 植物科學研究所 |
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