請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/20833
標題: 利用BN-PAGE比較分析綠竹筍Bambusa oldhamii和冬筍Phyllostachys edulis粒線體的超級複合體
Comparative analysis of mitochondrial supercomplexes from summer bamboo Bambusa oldhamii and winter bamboo Phyllostachys edulis by BN-PAGE
作者: 吳苑菁
Wu, Yuan Ching
關鍵字: ArrayBamboo
Blue native PAGE
Mitochondria
Supercomplex
出版社: 生命科學系
摘要: 竹在亞洲是很重要的經濟作物之一,其新生的幼芽生長非常快速,雖然機制還不清楚,但可猜想到一定是需要很大的能量去支持其快速的生長。爲了瞭解竹筍能量的代謝,將綠竹筍和冬筍的粒線體萃取出來並且比較兩者之間的差異。首先,利用氧電極去測定其呼吸速率,分別加入呼吸鏈複合體I的受質NADH和複合體II的受質succinate,發現不論是用NADH或是succinate當作受質,綠竹筍的呼吸速率皆比冬筍來的高,尤其是綠竹筍Complex I的呼吸速率比冬筍高兩倍。此外,又加入複合體 I 的抑制劑rotenone後,發現綠竹筍複合體I的活性是綠竹筍的兩倍,但是兩者間external/internal NADH dehydrogenase活性則沒有差異。再者,利用一维BN-PAGE (blue-native polyacrylamide gel electrophoresis) 和二維BN-PAGE或Tricine-SDS-PAGE及膠內活性染色 (in-gel activity staining) 分析方法,發現介面活性劑digitonin可以溶解出較多粒腺體內膜上的超級複合體 (supercomplex) 和複合體。分析用digitonin溶解後的粒線體蛋白質組成,發現綠竹筍和冬筍皆有將近87%的複合體I組成超級複合體。在複合體III部分,綠竹筍有將近60%的複合體III組成超級複合體,冬筍則只有50%複合體III組成超級複合體。另外,兩種竹筍皆有將近75%的複合體V組成超級複合體。綠竹筍中的超級複合體I+III2和I+F1的含量比冬筍來的多,此結果也許可以解釋爲何綠竹筍用NADH當受質時呼吸速率較高。在綠竹筍和冬筍中可以發現三個嶄新的超級複合體I+III2+V2、I+III2+V和I+F1還未發現於別的物種中。 此外,利用一维CN-PAGE (clear-native polyacrylamide gel electrophoresis) 再配合二維native-PAGE、膠內活性染色去分析超氧歧化酶 (superoxide dismutase, SOD) 的異構酶,發現在綠竹筍中有四個Mn-SOD和三個Cu/Zn-SOD,在冬筍中則有兩個Mn-SOD和三個Cu/Zn-SOD。最後,利用質譜儀 (matrix assisted laser desorption/ ionization- Time of flight mass spectrometer, MALDI-TOF MS)進一步鑑定SOD的確切身分。
Bamboo, an important cash crop in Asia, is one of the fastest growing plants in this planet as a new shoot may grow rapidly over 3 feet per day. However, the mechanism of this burst of rapid growth is unclear. It was supposed that the rapid growth requires a huge amount of energy supplement. In order to understand how the energy metabolizes in young bamboo shoots, I intend to work on the isolated mitohcondria where the energy is mainly produced. Mitochondria isolated from young bamboo shoots of B. oldhamii and P. edulis were therefore compared. Firstly, using either succinate or NADH as substrate, the mitochondrial respiration rates of B. oldhamii were higher than those of P. edulis. B. oldhamii possessed about 2-fold complex I activity but similar external/internal NADH dehydrogenase activity as compared to P. edulis. Furthermore, the assembly of functional mitochondrial respiratory supercomplexes was studied using 1-D/2-D BN-PAGE, 1-D/2-D Tricine-SDS-PAGE, and in-gel activity staining with addition of digitonin. In both species, almost 87% of total complex I was assembled into supercomplexes. In B. oldhamii, about 40% of complex III was assembled into supercomplexes, but in P. edulis, about 50% was assembled into supercomplexes. Moreover, in assembling complex V, both species performed in a similar manner that 75% of complex V was assembled into supercomplexes. The amounts of the supercomplexes I+III2 and I+F1 in B. oldhamii were more than those in P. edulis. It may explain why the respiration rates using NADH as substrate in B. oldhamii were higher than those in P. edulis. In bamboo mitochondria, the novel supercomplexes I+III2+V2, I+III2+V, and I+F1 were be identified and never found in other species. Identifying the mitochondrial SOD isoenzymes using 1-D CN-PAGE, 2-D native-PAGE, and in-gel activity staining, it was found that there were four Mn-SODs and three Cu/Zn-SODs in B. oldhamii whereas two Mn-SODs and three Cu/Zn-SODs in P. edulis. Two SOD protein spots excised from 2-D gels were further analyzed by MALDI-TOF MS.
URI: http://hdl.handle.net/11455/20833
顯示於類別:生命科學系所

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