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研究生:劉念慈
研究生(外文):Nien-Tzu Liu
論文名稱:醇溶蛋白米粒突變品系之蛋白質體學研究
論文名稱(外文):Proteomic analysis of rice prolamin mutants
指導教授:王國祥
指導教授(外文):Co-Shine Wang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:日文
論文頁數:70
中文關鍵詞:醇溶蛋白蛋白質體學米粒
外文關鍵詞:prolaminproteomicrice
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水稻 (Oryza sativa L.) 台農67號利用疊氮化鈉 (NaN3) 造成基因突變,產生許多不同性狀的突變品系。本實驗室目前進行醇溶蛋白米粒突變品系之蛋白質體學研究。水稻米粒儲藏性蛋白質突變品系SA1443,總蛋白質量較突變親台農67號約多20 %,而且西方轉漬法 (Western blot) 的結果顯示,突變品系SA1443的16-kDa的醇溶蛋白含量為突變親台農67號的2.3倍。為了找尋改變的原因,我們利用二維電泳技術,分離不同時期的水稻種子蛋白質,比較突變親台農67號和突變品系之間的差異,從而找出6個突變品系專一的蛋白質 (mutant-specific protein) 和8個突變品系缺乏的蛋白質 (mutant-deficient protein)。其中,開花後8天的穀粒具有差異的蛋白質包括了4個突變品系專一的蛋白質及6個突變品系缺乏的蛋白質。使用ESI-Q-TOF MS/MS,成功鑑定出其中5個:ADP-glucose pyrophosphorylase (AGPase) 及RAB24是突變品系缺乏的蛋白質;Malate dehydrogena (MDH)、Triosephosphate isomerase (TIM) 及另一個RAB24同型異構物是突變品系專一的蛋白質。Rab24是逆境所誘導出來的抗氧化的酵素。MDH、TIM和AGPase為參與碳水化合物代謝的酵素。RT-PCR的結果顯示在突變品系中MDH及TIM mRNA表現量較突變親台農67號高,而AGPase mRNA表現量較突變親台農67號低,顯示出突變品系之米粒的澱粉和醣代謝發生改變。MDH、TIM和AGPase的酵素活性測試,也符合此推測,而另一參與澱粉合成的Granule bound starch synthase (GBSS) 酵素活性測試結果,SA1443較台農67號高。因為醇溶蛋白含較多白胺酸 (Leucine)、麩胺酸 (Glutamate) 及醯胺麩胺酸 (Glutamine)、天門冬酸 (Aspartate) 及醯胺天門冬酸 (Asparagine),所以進一步以RT-PCR分析Isocitrate dehydrogenase (IDH)、Aspartate aminotransferase (AspAT) 及Glutamate dehydrogenase (GDH) mRNA表現量。結果顯示突變品系AspAT mRNA表現量較突變親台農67號高,IDH及GDH mRNA表現量與突變親台農67號差不多。綜合以上的結果,我們對突變品系中醇溶蛋白與總蛋白質量增加的現象提出一個假說。突變品系SA1443的種子,由於AGPase酵素活性降低而GBSS活性增加,不會改變澱粉的含量,但TIM活性增加,促進了檸檬酸循環的代謝,也會使丙酮酸 (pyruvate) 的量增加,進而造成白胺酸含量增加。同時,由於MDH及AspAT含量增加,會增加相關胺基酸的生合成增加。然而,GDH和IDH mRNA表現量並沒增加,顯示突變品系中檸檬酸循環的某些代謝步驟可能存有偏好性。
Oryza sativa L. cv. Tainung No.67 seeds treated with sodium azide (NaN3) have generated a large number of rice mutants with genetic diversity. We have worked on rice prolamin mutants using the proteomic approach. The content of total protein in mutant SA1443 has 20 % higher than wild type. Western blot analysis of total seed protein revealed that 16-kDa prolamin content in SA1443 has 2.3-fold higher than wild type. Differences in protein content and pattern of various developing seeds between mutants and wild type were analyzed by two-dimensional gel electrophoresis. Six mutant-specific proteins and eight mutant-deficient proteins have thus been identified. Of these, four mutant-specific proteins and six mutant-deficient proteins that accumulated at 8 DAF seeds were chosen for further investigation. Using ESI-Q-TOF MS/MS, five proteins have been successfully identified: two mutant-deficient proteins, ADP-glucose pyrophosphorylase(AGPase)and RAB24 and three mutant-specific proteins including malate dehydrogenase(MDH, triosephosphate isomerase(TIM)and an isoform of Rab24. While Rab24 is a stress-inducible antioxidant enzyme, MDH、TIM and AGPase are a group of enzymes that participate in carbohydrate metabolism. RT-PCR was used to reveal that the levels of MDH and TIM mRNA increased whereas the level of AGPase mRNA decreased suggesting changes in carbohydrate metabolism. Changes in activity of these enzyme also correlated with their mRNA levels. Granule bound starch synthase (GBSS), an enzyme associated with in starch synthsis, increased its activity in mutant SA1443 when compared to wild -type. Because of prolamin riched in Leucine, Glutamate, Glutamine, Aspartate and Asparagine, RT-PCR was also used to analyze the mRNA level of Isocitrate dehydrogenase (IDH), Aspartate aminotransferase (AspAT) and Glutamate dehydrogenase (GDH). The result revealed that AspAT mRNA increased its level in mutant but the levels of IDH and GDH kept the same as wild-type. Together, we propose a hypothesis of flux interchange between carbohydrate and protein metabolism. In high prolamin mutant seeds, the decrease of AGPase and increase of GBSS may not change the metabolism of starch synthesis. The increase of TIM promotes metabolism in citric acid cycle and also results in the increase of Leucine content. In addition, the levels of MDH and AspAT increase promotes the synthesis of relevant amino acids whereas GDH and IDH mRNA do not change in mRNA levels, suggesting that the preference of metabolic flux in citric acid cycle.
壹、中文摘要-------------------------------------------------1
貳、英文摘要-------------------------------------------------2
参、文獻整理-------------------------------------------------3
一、植物種子蛋白質-------------------------------------------3
二、醇溶蛋白為水稻種子的主要儲藏性蛋白質之一-----------------4
三、水稻儲藏性白質之合成與累積-------------------------------5
四、水稻醇溶蛋白的種類及分子生物學上的研究-------------------6
五、水稻醇溶蛋白的營養價值與消化特性-------------------------7
六、穀纇儲藏性蛋白質和其他蛋白質突變株之相關研究-------------8
七、利用蛋白質體學的方法研究穀類儲藏性蛋白質-----------------9
肆、材料與方法----------------------------------------------13
一、材料-------------------------------------------------13
二、蛋白質的萃取、定量和電泳-----------------------------13
三、蛋白質的染色-----------------------------------------17
四、蛋白質質譜分析及相似性蛋白質之比對-------------------18
五、西方轉漬法確認醇溶蛋白的量---------------------------18
六、RT-PCR-----------------------------------------------19
七、酵素活性分析-----------------------------------------22
八、水稻榖粒水解胺基酸組成分析---------------------25
九、澱粉含量測試-----------------------------------------25
十、澱粉中直鏈澱粉的含量測試-----------------------------25
伍、結果---------------------------------------------------27
一、突變親台農67號與突變品SA1443外表型之差異-------------27
二、水稻醇溶蛋白的萃取與定量-----------------------------27
三、水稻種子發育時期蛋白質之生合成-----------------------29
四、水稻不同時期突變親台農67號與突變品系SA1443差異性蛋白質
之比較-----------------------------------------------29
五、蛋白質質譜分析及相似蛋白質之比對---------------------30
六、水稻突變親台農67號與其他突變品系進行RT-PCR分析-------31
七、突變親台農67號與突變品系SA1443之AGPase、TIM及MDH酵素活
性比較------ --------------------------------------- 32
八、突變親台農67號及突變品SA1443之澱粉中直鏈澱粉的含量比較32
陸、討論---------------------------------------------------34
柒、參考文獻------------------------------------------------39
捌、圖表----------------------------------------------------48
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