鄭石通臺灣大學:植物科學研究所劉朴桓Liu, Pu-HuanPu-HuanLiu2010-05-112018-07-062010-05-112018-07-062008U0001-2901200800195400http://ntur.lib.ntu.edu.tw//handle/246246/181911OSAG78為文心蘭之老化相關基因OSAGs（senescence-associated genes），以35S啟動子將OSAG78於阿拉伯芥中過量表現，其轉殖株具有體型較小、葉型變圓、莖變粗、雄蕊和雌蕊及花軸較粗短的外表型。本研究旨在探討過量表現OSAG78基因對於阿拉伯芥轉殖株之影響與其作用機制，期能應用於產生新品系文心蘭。拉伯芥中與OSAG78蛋白質序列相似的其他基因受老化刺激的表現模式與OSAG78迥異，顯示OSAG78具有特殊的功能性。分析成熟與幼嫩簇生葉的生長比率發現，35S::OSAG78轉殖株具有生長不同的現象，可能與吉貝素(GA)之缺乏有關。然而，GA3之處理雖然可使轉殖株之開花時間提前，對植株之外表型及花期之延緩則無影響。惟經由GC-MS分析結果，轉殖株中的GA4與GA7含量的確較野生型阿拉伯芥為低；且轉殖株中GA20ox1與GA2ox1表現量下降，而GA3ox1表現量略微上升，顯示GA生合成途徑受影響。將相同生長階段之轉殖株與野生型阿拉伯芥離體葉片進行暗處理，分析葉綠素衰退情形與葉片老化之指標基因AtSAG12等表現量之結果，證實轉殖株的確具有特別之能力，且可延緩經由乙烯所誘導的現象，然而對於離層酸之誘導作用抑制效果並不顯著，對吉貝素之抑制的反應亦較不敏感。經由粗萃取蛋白質活性分析，則發現轉殖株之phospholipase A活性較野生型阿拉伯芥為高。而以大腸桿菌表現GST:OSAG78之重組蛋白質，亦發現OSAG78具有較高的lipase與phospholipase A的活性。且GST:OSAG78重組蛋白質大量累積在不可溶蛋白質沈澱物中，此一結果證實OSAG78蛋白質的性質與比對資料中極性趨向脂溶性之蛋白質類似。續以GFP融合於OSAG78蛋白質的C端並使其短暫表現於洋蔥之表皮細胞中，則證實OSAG78融合蛋白質表現在細胞膜附近。綜合本研究之試驗結果推測，OSAG78影響阿拉伯芥老化之作用乃因其過量表現lipase與phospholipase A活性，使得SAGs表現降低進而改變訊息傳導途徑所致。OSAG78 is a senescence-associated gene cloned from Oncidium flower and was predicted as a lipid acyl-hydrolase protein. The full-length cDNA of OSAG78 was isolated and its ectopic expression of OSAG78, driven by 35S promoter in Arabidopsis, was analyzed. Compared with wild-type plants, the transgenic plants display smaller body size, stiffer inflorescence stem, thicker leaves, shorter siliques and more round-shaped flowers. Moreover, the 35S::OSAG78 transgenic lines display different flowering time. The mechanism of over-expressing OSAG78 in Arabidopsis is further studied in this research. According to RT-PCR assays, OSAG78 was regulated either during flowing time or after ethylene treatment in Oncidium. We found that OSAG78 showed the different expression pattern comparing to patatin-like proteins in Arabidopsis. Based on the leaf trichome distribution and phenotype of 35S::OSAG78 transgenic lines, the effects of OSAG78 in Arabidopsis were associated with an increase in the length of developmental phases of plant. It was assumed that the growth retardation may caused by GA deficiency or blocking in GA responses. The contents of bioactive GA4 and GA7, quantified by GC-MS, as well as the levels of AtGA20ox1 and AtGA2ox1 transcripts, quantified by real-time PCR, were lower than those in the wild-type plants. These results suggest that OSAG78 accumulation affects GA metabolism through the repression of biosynthetic steps catalyzed by GA 20-oxidase. he content of chlorophyll and the expression of AtSAG in the detached leaves of transgenic and wild-type plants were investigated. In the leaves with or without ethylene, the expression of AtSAG was lower in transgenic lines than in wild-type plant. However, effects in transgenic plant were not observed after ABA treatment, and the application of GA and BAP decreases the expression of AtSAG12 in both transgenic and wild-type plants. Therefore, there should be another pathway to display effects of 35S::OSAG78 transgenic lines. By analysis of protein crude extracts, phospholipase A activity was higher in transgenic lines than in wild-type plants. Furthermore, a recombinant GST:OSAG78 fusion protein that overexpressed in Escherichia coli was accumulated in the insoluable protein pellet displaying lipase and phospholipase A activity. This result agrees with the previous prediction that OSAG78 protein is hydrophobic. To confirm the membrane association of OSAG78, a OSAG78:GFP fusion polypeptide was transiently expressed in onion (Allium cepa) epidermal cells, indicating that OSAG78 is localized at the cytoplasmic membrane. These results point out that over-expression of membrane-associated patatin protein OSAG78 that had phospholipase A activity affected GA biosynthesis in plant.口試委員會審定一章 前言…………………………………………………………… 1心蘭切花老化………………………………………………………… 1由突變株研究老化相關機制………………………………………… 1老化相關之植物荷爾蒙……………………………………………… 2花老化基因釣取與研究……………………………………………… 3量表現OSAG78於阿拉伯芥…………………………………………… 4atatin蛋白質之生理特性……………………………………………… 5他植物中的patatin-like 蛋白質 ………………………………… 6LA與訊息傳導…………………………………………………………… 7A生合成途徑…………………………………………………………… 8究目的與方向………………………………………………………… 9二章 材料與方法…………………………………………………… 10、材料………………………………………………………………… 10、常用實驗方法……………………………………………………… 10、阿拉伯芥葉片生長統計…………………………………………… 13、內生性GA含量測定………………………………………………… 13、葉綠素含量測量…………………………………………………… 14、RNA萃取…………………………………………………………… 14、RT-PCR和real-time PCR………………………………………… 15、質體構築與挑選…………………………………………………… 17、植物基因組DNA抽取……………………………………………… 18、阿拉伯芥花序浸潤轉殖與轉殖株篩選…………………………… 18一、基因槍暫時性表現OSAG78融合綠色螢光蛋白………………… 20二、暗處理誘導老化與施予植物荷爾蒙…………………………… 21三、植物蛋白質萃取………………………………………………… 21四、融合蛋白質表現………………………………………………… 22五、蛋白質活性測試………………………………………………… 27六、GUS組織染色分析……………………………………………… 28七、文心蘭轉殖……………………………………………………… 28三章 結果…………………………………………………………… 30SAG78為老化抑制基因………………………………………………… 30SAG78 在其他植物中的相似基因…………………………………… 30拉伯芥OSAG78 homolog與細胞分裂素反應基因分析……………… 315S::OSAG78轉殖株具有生長遲緩的生理現象……………………… 31量表現OSAG78造成阿拉伯芥內生性GA含量下降…………………… 325S::OSAG78轉殖株具有延緩老化的生理現象……………………… 325S::OSAG78轉殖株花期延長並非GA缺乏所致……………………… 33A的缺乏並非造成35S::OSAG78轉殖株延緩葉片老化的主要原因… 335S::OSAG78轉殖株可減緩乙烯所誘導的老化現象………………… 34量表現OSAG78無法延緩離層酸所誘導的老化……………………… 35SAG78位於細胞膜附近………………………………………………… 35殖株粗萃取蛋白質具有較高的lipase與phospholipase A活性……36SAG78表現蛋白質具有lipase與phospholipase A活性………………36心蘭轉殖……………………………………………………………… 37四章 討論…………………………………………………………… 39SAG78在植物老化中扮演的角色……………………………………… 39SAG78可能與養分累積有關…………………………………………… 40量表現OSAG78抑制GA生合成途徑造成生長遲緩…………………… 415S::OSAG78轉殖株花期延長與延緩老化機制相關………………… 43量表現OSAG78推測可延緩文心蘭切花老化………………………… 43SAG78具有PLA酵素活性與延緩老化相關…………………………… 44論……………………………………………………………………… 46表……………………………………………………………………… 47錄……………………………………………………………………… 70考文獻………………………………………………………………… 71application/pdf15309179 bytesapplication/pdfen-US文心蘭吉貝素磷脂酶阿拉伯芥OncidiumArabidopsisgibberellinphospholipidhttp://ntur.lib.ntu.edu.tw/bitstream/246246/181911/1/ntu-97-R91226022-1.pdf