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標題: | 阿拉伯芥質體型α-amylase功能之分析 Functional analysis of Arabidopsis plastidial α-amylase |
作者: | Te-Lung Tsai 蔡德龍 |
出版年 : | 2001 |
學位: | 碩士 |
摘要: | Ataamy1是近年來在阿拉伯芥中發現的一個可能於葉綠體內具有α-amylase功能的基因序列,為確認阿拉伯芥Ataamy1是否真的具有α-amylase的酵素活性,本實驗利用載體讓Ataamy1 cDNA在大腸桿菌中大量表現,得到Ataamy1蛋白質進行澱粉水解反應和產物分析,結果顯示Ataamy1確實有α-amylase的功能,它能將澱粉分解為單醣、雙醣和多醣等分子。阿位伯芥多澱粉突變株sex4在種皮及葉子上都有澱粉的累積現象,且葉片中的Ataamy1蛋白質表現量及活性較野生型為低。Sex4基因位於第三條染色體上,而Ataamy1基因位於第一條染色體上,故Sex4基因並不是Ataamy1基因。為瞭解Ataamy1基因在阿拉伯芥中扮演的生理功能,將Ataamy1 cDNA接在35S啟動子之後(Chen),利用農桿菌轉殖入突變株sex4及野生型中,結果得到sex4型轉殖株中之T65和T66,其葉片、種皮不再有累積澱粉的現象,葉片之澱粉含量及α-amylase蛋白質的表現亦回復正常,顯示α-amylase在阿拉伯芥植株中應該具有正常分解澱粉之功能。但α-amylase基因轉殖到野生型植株時,檢測到co-suppression之轉殖株T18,其Ataamy1蛋白質量明顯降低,且無α-amylase活性;另一轉殖株T39,α-amylase活性明顯較高,但無論T18或T39,其葉片的澱粉含量並未因α-amylase的減弱或增高而有累積或降低之現象。由以上結果α-amylase可使突變株sex4中澱粉含量回復到野生型的水準。但α-amylase在野生型中大量表現或受抑制(co-suppression)時,對於澱粉含量變化的影響卻極有限,顯示在植物中影響澱粉分解之機制極為複雜,在阿拉伯芥中應該還存在著其他調控或改變α-amylase功能之表現及暫存性澱粉含量的基因。 Ataamy1 is a putative plastidial α-amylase gene of Arabidopsis possessing a starch associated domain and a conserved amylase domain. The locus of Ataamy1 was mapped to chromosome 1. The full Ataamy1 and its α-amylase domain were expressed in Escherichia coli, and the enzymatic activity of the expressed proteins was examined. Based on the end products, either Ataamy1 or the α-amylase domain alone does function as α-amylase in vitro. Arabidopsis sex4 is a starch excess mutant having starch accumulated in leaves and the seed coat. The activity of plastidial α-amylase is much lower in sex4 than in wild type. SEX4 was localized at chromosome 3 and does not encode the plastidial α-amylase. To study whether the reduction of the plastidial α-amylase activity is involved in the sex4 phenotype, the Ataamy1 cDNA directed by CaMV35S promoter was transformed into both sex4 mutant and wild type plants. Transformants with the sex4 genetic background, T65 and T66, were examined and their starch content and α-amylase protein level were closed to wild type. It is suggested that the 35S promoter-driven Ataamy1 encoded an active chloroplast α-amylase and that in turn restored sex4 phenotype to wild type. On the other hand, when Ataamy1 was transformed into wild type, over-expression (T39) and co-suppression (T18) transformants were obtained. Both transgenic plants examined gave the phenotype of wild type no matter how their Ataamy1 α-amylase level is. The results indicated that the starch content of Arabidopsis leaves is complicatedly regulated, and the α-amylase activity is certainly not the sole factor. The functions of the plastidial α-amylase in starch degradation and the regulation of starch content are discussed. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75234 |
全文授權: | 未授權 |
顯示於系所單位: | 植物科學研究所 |
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