鄭石通Jeng, Shih-Tong臺灣大學:植物科學研究所林盈仲Lin, Ying-ChungYing-ChungLin2010-05-112018-07-062010-05-112018-07-062008U0001-2207200817075400http://ntur.lib.ntu.edu.tw//handle/246246/181928澱粉分支酶(Starch branching enzyme, SBE)的酵素功能為催化α-(1→6)糖苷鍵的形成，前人透過比對胺基酸序列將SBE分類成SBEI、SBEII和SBEIII，而在阿拉伯芥中經過胺基酸序列比對後發現有SBE2.1、SBE2.2和AtSBEIII三種可能具有SBE酵素活性的蛋白質。前人研究中發現當阿拉伯芥失去AtSBEIII酵素活性不會影響澱粉的性質與結構，所以AtSBEIII不參與澱粉合成。若阿拉伯芥失去SBE2.1或SBE2.2的酵素活性，僅能在支鏈澱粉的鏈長分佈發現些許的改變，然而一旦阿拉伯芥同時失去SBE2.1與SBE2.2的酵素活性，則無法累積澱粉，也就是在葉片中觀察不到任何澱粉粒的形成，也無法知道SBE對於阿拉伯芥的影響。我們實驗的策略為利用干擾性核醣核酸(RNAi)的方式同時降低SBE2.1與SBE2.2在阿拉伯芥中的活性，卻不完全去除他們的活性。在SBE RNAi轉植株中SBE2.1的活性下降至20~30%，SBE2.2下降至70~80%；透過總澱粉含量發現在SBE RNAi轉殖株較野生型阿拉伯芥中含量下降，且轉殖株的植物外型上較野生型阿拉伯芥略小，可得知SBE RNAi轉殖株內SBE的活性不足以提供植株進行正常澱粉的合成。以高效陰離子交換層析搭配脈衝安培檢測(HPAEC-PAD)檢測支鏈澱粉鏈長分佈後發現SBE RNAi轉殖株在鏈長DP 6-10的比例下降，DP 26-35的比例稍微上升，而DP 11-25的比例沒有明顯改變，可得知轉殖株中支鏈澱粉平均鏈長上升。偏光顯微鏡下看見SBE RNAi轉殖株的澱粉粒偏光性質大幅下降；以示差掃描分析儀(DSC)檢測糊化溫度，發現SBE RNAi轉殖株的糊化溫度範圍變大，也就是糊化終止溫度扣除糊化起使溫度的數值上升，代表著轉殖株澱粉粒內部結構較不整齊。Starch branching enzymes catalyze the formation of theα-1,6 linkages within the starch polymer. In regard to their peptide sequences, these SBEs are categorized into three classes named SBEI, SBEII, and SBEIII. Three starch branching enzymes, SBE2.1, SBE2.2, and AtSBEIII, have been found in the genome of Arabidopsis thaliana. In the previous studies, no impact on starch structure was observed in the null AtsbeIII mutants, while only slight modifications in amylopectin structure were detected in sbe2.1 and sbe2.2. In stead of knocking out both SBE2.1 and SBE2.2, which leads to no starch accumulation, the strategy adopted in this study is to knock down both SBE 2.1 and SBE 2.2 in order to analyze the effects of SBE on the characteristics of starch granules. The mutants, resulted from RNA interference, decrease the activity of SBE 2.1 to 20~30% and SBE2.2 to 70~80%, compared to those of wild type. On the whole, the decrease of SBE activity may have more severe effects on SBE RNAi mutants than that of sbe 2.1 or sbe 2.2. In this regard, the dwarf mutants, namely SBE RNAi mutants, contain less amount of starch than that of wild type. Starch granules from plants with SBE RNAi mutants show slight difference from those from wild type regarding to morphology as well as diameter. In terms of amylopectin structure, the distribution of chain lengths of amylopectin from SBE RNAi mutants are reduced at DP 6-10 and are comparably high at DP 26-35, compared to those of wild type. The thermal behaviors of the starches isolated from SBE RNAi mutants have greater gelatinization temperature range than those of wild type, indicating the starch structure from SBE RNAi mutants is less regular.中文摘要……………………………………………………………………..….… 3文摘要…………………………………………………………………………… 4一章 前言粉分子與結構………………………………………………………………..5粉的糊化……………………………………………………………………..6粉合成相關酵素……………………………………………………………..8tarch branching enzyme的功能……………………………………………9物種中已完成的SBE研究………………………………………………10二章 材料與方法、材料……………………………………………………………………….12、載體構築………………………………………………………………….12、阿拉伯芥轉殖…………………………………………………………….15、檢測轉殖植物…………………………………………………………….16、澱粉特性檢測…………………………………………………………….23三章 結果體構築………………………………………………………………………27測轉殖株Genomic DNA…………………………………………………..27BE RNAi轉殖株中SBE2.1與SBE2.2的mRNA含量下降……………..28BE RNAi轉殖株中SBE2.1與SBE2.2的蛋白質活性下降……………...29殖株的外表型………………………………………………………………29鏈澱粉鏈長分佈……………………………………………………………30粉粒外觀與粒徑……………………………………………………………30粉十字偏光特性……………………………………………………………31粉熱性質分析………………………………………………………….…..31四章 討論鏈澱粉的鏈長分佈…………………………………………………………33粉粒糊化溫度………………………………………………………………34表………………………………………………………………………………….37考文獻…………………………………………………………………………….50錄………………………………………………………………………………….57135 bytestext/htmlen-US澱粉分支酶干擾性核醣核酸支鏈澱粉鏈長分佈糊化溫度RNA interferenceStarch branching enzymegelatinization temperaturehttp://ntur.lib.ntu.edu.tw/bitstream/246246/181928/1/index.html