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標題: | 阿拉伯芥中酵母菌SAC1同源性基因之分析 Characterization of an Arabidopsis Gene with Homology to Yeast SAC1 Gene |
作者: | Hwang-Long Tsay 蔡皇龍 |
出版年 : | 2001 |
學位: | 碩士 |
摘要: | 阿拉伯芥多澱粉突變株sex4,將突變基因定位於第三號染色體m457、g4125標誌區中,在此標誌區中發現有一AccI限制脢切位在野生型及突變體中出現多型性的變異,而此變異落在SAC1-like基因座上。本論文分別選殖包含完整編碼區域不包含5'-或3'-端非轉譯區之SAC1-like基因genomic DNA片段,轉殖於突變株,在20株轉殖株中並未發現回復野生型之植株。另一方面利用RNAi的方式擬阻斷正常植株中SAC1-like基因的表現,觀察SAC1-like基因突變後的表現型,在73株轉殖株中未能觀察到外表型發生變化之突變株。由阿拉伯芥cDNA基因庫中選殖出SAC1-like基因的cDNA clone,並轉接於表現載體pGEX3KS大量表現蛋白質,並製備抗體,以SAC1-like抗體偵測SAC1-like基因在突變株中表現量較野生型低,並觀察植株中有同源蛋白質,分析SAC1-like蛋白質氨基酸序列,並於DataBase中發現,阿拉伯芥中還有K2A18.9與AtG5與SAC1-like有極高的相似性,且皆具有SAC1-like domain。由於SAC1-like基因在阿拉伯芥中之功能並不十分清楚,但在酵母菌中SAC1基因則參與訊息傳遞,將阿拉伯芥之SAC1-like基因之cDNA轉化至△SAC1缺失之酵母菌體中,結果阿拉伯芥之SAC1-like基因可以補償酵母菌SAC1基因突變之缺失,暗示阿拉伯芥之SAC1-like基因在植物中扮演類似SAC1的角色。 Arabidopsis sex4 mutant has increased starch content in leaves. An AccI restriction fragment length polymorphism (RFLP) between the wild type and sex4 was detected by BAC clone F26O13. The altered AccI site was located in the gene F26O13.100 named SAC1-like gene, which has homology to yeast SAC1 gene. Transgenic study showed that a genomic DNA fragment containing SAC1-like coding region but without 5'- or 3'-untranslated region failed to complement the starch excess phenotype of sex4 mutant. Using a dsRNA interference approach 73 transgenic Arabidopsis was generated. None of these transgenic plants has starch excess phenotype. The cDNA encoding the SAC1-like protein was isolated and was predicted 597 amino acids with three transmembrane domains. SAC1-like protein over-expressed in Escherichia coli was used to prepare antibody. Western analysis indicated that the SAC1-like protein was reduced in the mutant. Analysis of the primary sequence of the cDNAs revealed that the SAC1-like protein has a high degree of similarity to K2A18.9 and AtG5 of Arabidopsis. Thus, there are three SAC1-like proteins present in Arabidopsis but the function of them was unclear. For physiological functional analysis, we knocked out the SAC1 gene of the yeast strain S1560. Arabidopsis SAC1-like cDNA was over-expressed in yeast upon a galactose induction. We showed that Arabidopsis SAC1-like cDNA can complement the yeast Δsac1 mutant, suggesting that Arabidopsis SAC1-like gene may serve similar biological function of yeast SAC1 gene in plants. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75230 |
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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