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標題: | 玉米澱粉代謝相關酵素表現之組織特異性研究 Differential Expression of Starch Metabolism Related Enzymes in Maize Tissues |
作者: | Yi-Lin Chen 陳怡伶 |
出版年 : | 2000 |
學位: | 碩士 |
摘要: | 為瞭解阿拉伯芥葉綠體型phosphoglucoisomerase (PGI)酵素在澱粉合成過程中所扮演的角色,本論文第一部份實驗進行葉片中PGI酵素的純化。經DEAE chromatography、Gel Filtation、FPLC HAP chromatography等純化步驟後,將所得次分子單元體60 KDa之蛋白質進行N端胺基酸定序,與NCBI資料庫進行比對後,意外地,發現此蛋白質並非阿拉伯芥的PGI酵素,而與阿拉伯芥基因庫中的putative PDI (Protein Disulfide Isomerase)有94%的相似度,顯示此蛋白質可能為阿拉伯芥中尚未被發現及研究的PDI蛋白質。第二部分實驗中探討澱粉代謝相關酵素在玉米不同組織間的表現;實驗之初,檢測玉米幼苗的初生葉和第一片葉片中PGI、phosphoglucomutase (PGM)、α-amylase (AA)、β-amylase (BA)、starch phosphorylase (SP)等酵素活性,實驗結果顯示,無論初生葉或第一片葉片,上述酵素的總活性並無明顯的晝夜變化;收集生長於晝夜週期或完全黑暗條件下之根尖、根體、芽鞘、初生葉和第一片葉片,分析其PGI、PGM、total amylase、AA、BA、SP等酵素活性,以及觀察PGI、PGM、total amylases、SP、ADPglucose pyrophosphorylase (ADGase)、soluble starch synthase (SSS)等同功?模式。分析結果顯示,不同的生長條件對同種組織中之酵素活性沒有顯著影響,而不同組織之酵素活性則有較大差異,根尖部位的PGI、PGM、AA及SP活性較其他組織高出許多。根尖組織的PGI和PGM之同功?模式與其他組織相同,雖然幾乎偵測不到β-amylase活性,但有明顯的α-amylase活性。根尖及第一片葉片中均具有細胞質型和質體型的SP酵素,且同功?表現的模式相同,而根體的SP活性低於其他組織,且只具有細胞質型的同功?表現;各組織均有質體型的ADGase,只有根尖組織中缺乏ADGase-3同功?;至於SSS依組織的不同,同功?的模式和活性各異,根尖與第一片葉片的同功?模式大致相同,而與根體則有明顯的差異。推測根尖組織中表現較強的SSS活性,應是造成其澱粉含量較根體為高的主要因素之一。由於根尖細胞中缺乏ADGase-3酵素,因此推測在根尖細胞之細胞質由韌皮部獲得蔗糖後,可能是經由UDGase及ADGase的作用在細胞質中形成ADP-glucose,經由ADP-glucose transporter運送到澱粉體中,再經SS的作用形成澱粉,根尖組織之澱粉合成途徑可能與地上部組織大不相同。根尖細胞具有很強的α-amylase活性,經水漂洗後雖仍保留含澱粉粒的根冠細胞,但α-amylase的活性會完全地喪失,由此可推測α-amylase主要位於根冠外層鬆散的細胞中,而漂洗根尖的處理並不會降低SP及SSS的活性強度,可知根尖之SP及SSS主要位於澱粉粒存在年輕的根冠細胞;據此可推論根冠在向前端生長時,不斷地合成澱粉,其細胞漸漸成熟脫落時,則以α-amylase分解澱粉供應細胞維生之用。 In order to understand the role of plastid phospho-glucoisomerase (PGI) in the pathway of starch biosynthesis, PGI was tried to be purified from arabidopsis leaves. By passing DEAE, gel filtration and hydroxyapatite chromatography, a major protein having 60 kDa of molecular weight was purified and subjected to amino acid sequencing. According to the sequence of N-terminus, the 60 kDa protein was not PGI but a putative PDI (protein disulfide isomerase). The differential expression of enzymes regarding starch metabolism in maize tissues was studied in the second part of the thesis. No matter the seedlings were grown in the Day/Night cycle or in darkness, there was no significant difference in the activities of PGI, PGM (phosphoglucomutase), α-amylase (AA), β-amylase (BA) and starch phosphorylase (SP) in the same type of tissues. Among various tissues of seedlings, the strength of enzyme activity was rather different, and the root tip possessed much higher activities of PGI, PGM, AA and SP. By using iodine staining, it was observed that root tip apparently accumulated much more starch than other tissues such as root proper. Isozyme pattern of each enzyme was analyzed, the patterns of PGI and PGM of root tip were all similar in all tissues examined. Beta-amylase was the major type of amylases in all tissues but it was not present in root tip. Both cytosolic and plastid forms of SP were present in root tip and the first leaf, but the plastid form was absent in root proper. All tissues examined expressed plastid form of ADP-glucose pyrophosphorylase (ADGase), but root tip was deficient in ADGase-3. The isozyme patterns of soluble starch synthase (SS) of root tip and the first leaf were similar, but the root proper has no activities of SS-1, SS-2, SS-3 and SS-5. So, the difference in starch content between root tip and root proper could be attributed to the high activity of SS in root tip. It is suggested that ADPglucose is synthesized from sucrose via the action of cytosolic UDGase and ADGase, and then transported into amyloplast in where ADPglucose is consumed to form starch just like the pathway occurs in the developing endosperm of cereal plants. On the other hand, α-amylase which is responsible for starch degradation was located at root cap. The removal of the outer loosened layers of root cap resulted in the loss of α-amylase, but the activity of SP and SS in root rip were not affected. So, it is obvious that starch is synthesized by SS in the young root cap cells and broken down by α-amylase in the aging cap cells. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75123 |
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