黃鵬林Huang, Pung-Ling臺灣大學:園藝學研究所林孟均Lin, Meng-JinMeng-JinLin2010-05-052018-06-292010-05-052018-06-292008U0001-2407200811370600http://ntur.lib.ntu.edu.tw//handle/246246/180866為了瞭解香蕉ABC (ATP-Binding Cassette) 轉運蛋白 (transporter) MhPDR1及MhPDR2的功能，本研究以CaMV 35S啟動子過量表達MhPDR1及MhPDR2，經農桿菌共培養法轉殖至菸草中，得到1棵Nt-MhPDR1轉殖株及6棵Nt-MhPDR2轉殖株，以GUS活性組織化學染色法分析，結果顯示所有轉殖株皆有GUS之活性表達。以MhPDR cDNA為探針，以菸草轉殖株Nt-MhPDR2基因組DNA進行南方氏雜交分析，結果顯示轉殖株Nt-MhPDR2-2~6皆出現預期的片段，證實篩選到過量表達MhPDR1及MhPDR2的轉殖株。 與非轉殖株相較，菸草轉殖株Nt-MhPDR1及Nt-MhPDR2具有較好的生長勢，進行礦物元素分析，結果顯示，轉殖株會累積較高濃度的鎂、鐵、鈣，及累積較低濃度的錳。葉綠素含量分析顯示，轉殖株Nt-MhPDR2，具有較高濃度的葉綠素a及胡蘿蔔素。以重金屬處理，結果顯示，轉殖株Nt-MhPDR1及Nt-MhPDR2對於鋁、銅及鎘離子具有較高的抗性，進一步分析顯示，轉殖株會累積較低含量的重金屬鋁、銅及鎘離子。推測香蕉ABC轉運蛋白MhPDR1及MhPDR2與鎂、鐵、鈣及錳的運移有關，因而促使轉殖株生長旺盛；另一方面，推測MhPDR1及MhPDR2扮演著將鋁、銅及鎘離子輸出幫浦 (efflux pump) 的角色，因此可增加植物細胞對重金屬的抗性。 為了瞭解香蕉ABC轉運蛋白MhPDR1及MhPDR2細胞中的表達位置，將香蕉ABC轉運蛋白MhPDR1及MhPDR2構築於綠色螢光 (Green Fluorescent Protein, GFP) 表達載體，並以基因槍短暫性表達於洋蔥表皮細胞，結果顯示，MhPDR1及MhPDR2均表達於原生質膜 (plasma membrane )，證實香蕉ABC轉運蛋白MhPDR1及MhPDR2為膜蛋白 (membrane protein)。To understand the function of banana ABC (ATP-Binding cassette) transporter MhPDR1 and MhPDR2, CaMV 35S promoter was used to overexpress MhPDR1 and MhPDR2 in Nicotiana tabacum. The overexpressing plasmids were transferred to tobacco through Agrobacterium–mediated method for stable gene expression. One and six independeutly transgenic plants, for MhPDR1 and MhPDR2, respectively, were obtained and designated as Nt-MhPDR1-1 and Nt-MhPDR2-1~6. The results of β-glucuonidase (GUS) activity analysis showed that all of the transgenic lines expressed GUS. Southern hybridization analysis of genomic DNA from Nt-MhPDR2 transformants was further carried out to demonstrate the integration of foreign gene into the genome of transgenic plants. In contrast with untransformed plants, phenotype of tobacco transgenic lines of Nt-MhPDR1 and Nt-MhPDR2 showed higher growth potential. Mineral element content analysis showed that transgenic lines accumulated higher concentrations of magnesium, iron, and calcium, and accumulated lower concentrations of manganese. Chlorophyll content analysis showed that transgenic lines of Nt-MhPDR2 accumulate higher concentrations of chlorophyll a and carotenoids. The treatment of leaves of Nt-MhPDR2 with heavy metals indicated that transgenic lines had higher tolerance to aluminum, copper and cadmium ion. Besides, heavy metal content analysis showed that transgenic lines accumulated lower concentrations of aluminum, copper and cadmium. Therefore, we propose the possibility that ABC transporter MhPDR1 and MhPDR2 in banana contributes to strong growth potential of transgenic lines by pumping transport of magnesium, iron, calcium and manganese. On the other hand, MhPDR1 and MhPDR2 may also play the role of ion efflux pump for aluminium, copper and cadmium, leading to those transgenic lines achieve the tolerance of heavy metals. To investigate the localization of ABC transporter MhPDR1 and MhPDR2 in banana, we fused MhPDR1 and MhPDR2 with the green fluorescent protein (GFP). The fusing plasmids were transferred to onion epidermal cells via particle bombardment for transient assay. The green fluorescence of MhPDR1 and MhPDR2 were found mainly at the plasma membrane. The results suggest that ABC transporter MhPDR1 and MhPDR2 in banana are localized at the plasma membrane.中文摘要_________________________________________________1bstract_________________________________________________2、前言_________________________________________________4、前人研究_____________________________________________5、ABC (ATP-Binding Cassette) 轉運蛋白 (transporter)____5、植物ABC轉運蛋白分類__________________________________6、植物ABC轉運蛋白基因表現與生理活性____________________6一)MRP次家族____________________________________________6二)MDR/PGP次家族________________________________________9三)PDR次家族___________________________________________10四)ATM次家族___________________________________________12、植物重金屬轉運蛋白相關研究__________________________13一)ZIP轉運蛋白_________________________________________13二)Nramp轉運蛋白_______________________________________14、香蕉ABC轉運蛋白_____________________________________15一)Mh-ABC1基因啟動子分析_______________________________15二)香蕉ABC轉運蛋白cDNA選殖_____________________________15、材料與方法__________________________________________16、試驗材料____________________________________________16一)香蕉ABC轉運蛋白cDNA_________________________________16二)質體材料____________________________________________16三)植物材料____________________________________________17、試驗方法____________________________________________17一)香蕉ABC轉運蛋白cDNA之構築___________________________17三)基因過量表達 (overexpression) 載體之構築____________20四)農桿菌之轉型________________________________________24五)阿拉伯芥之基因轉殖、篩選及分析______________________30六)菸草之基因轉殖、篩選及分析__________________________31七)蛋白質定位 (localization) 分析載體之構築____________32八)南方氏雜交分析 (Southern hybridization analysis)____39九)基因過量表現轉殖株分析______________________________40十)基因槍 (particle bombardment) 暫時性表達 (transient expression)_____________________________________________45、結果________________________________________________47、過量表達MhPDR基因菸草轉殖株之確認___________________47、菸草轉殖株Nt-MhPDR1及Nt-MhPDR2生長差異之分析________47一)轉殖株Nt-MhPDR1及Nt-MhPDR2外觀形態分析______________47二)轉殖株Nt-MhPDR1及Nt-MhPDR2礦物元素分析______________53三)轉殖株MhPDR1及MhPDR2葉綠素含量測定__________________53、過量表達MhPDR2之菸草轉殖株Nt-MhPDR2抗重金屬之分析___63一)轉殖株Nt-MhPDR2葉片抗重金屬分析_____________________63二)轉殖株Nt-MhPDR2鋁含量分析___________________________63三)轉殖株Nt-MhPDR2銅含量分析___________________________63四)轉殖株Nt-MhPDR2鎘含量分析___________________________69、MhPDR1及MhPDR2蛋白質定位分析________________________69、討論________________________________________________73、香蕉與菸草轉運蛋白基因同源性________________________73、香蕉ABC轉運蛋白MhPDR與植株生長之關係________________73、過量表達MhPDR1及MhPDR2基因礦物元素累積之影響________74、菸草轉殖株Nt-MhPDR2抗重金屬之分析___________________75一)轉殖株Nt-MhPDR2對重金屬鋁離子之抗性_________________75二)轉殖株Nt-MhPDR2對重金屬銅離子之抗性_________________76三)轉殖株Nt-MhPDR2對重金屬鎘離子之抗性_________________77、MhPDR1及MhPDR2蛋白質定位分析________________________77、結語________________________________________________79考文獻________________________________________________80application/pdf2417732 bytesapplication/pdfen-US香蕉ABC轉運蛋白過量表達重金屬蛋白質定位綠色螢光蛋白BananaABC transporteroverexpressionheavy metalProtein localizationGreen fluorescence protein.thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/180866/1/ntu-97-R95628103-1.pdf