玉米β-amylase基因之選殖與特性分析

Abstract

植物體的β-amylase是重要的澱粉降解酵素之一，在試管中表現外切水解澱粉，產生麥芽糖的生化性質；但在萌芽的玉米種子中，β-amylase只會在不含澱粉的糊粉層細胞中表現，此種酵素與受質位置背離的現象，使β-amylase的生理功能難有定論。?了能夠更深入地瞭解玉米β-amylase基因表現的特性，本實驗中首先建構genomic library，自基因庫中篩選得到完整β-amylase基因，完成核酸定序，並由序列資料得知β-amylase基因序列的結構；另外，也利用短暫表現系統進行?動子的活性分析。可惜的是所嘗試的三種短暫表現系統，均無法獲得成功的表現結果。?期對於玉米β-amylase基因的?動子有進一步的瞭解，利用網路資源與文獻資料做進一步的序列分析。搜尋比對的結果，在玉米β-amylase?動子序列內找出了一些可能參與基因調控的cis-elements；在與水稻、大麥?動子序列進行的排列比對結果中，也發現了TATA box附近與5'-UTR中具有高相似性的區域。這些搜尋結果，有助於未來玉米β-amylase之相關研究工作的設計，並可提供解讀實驗結果之佐證。

β-amylase is one of the prevalent starch degrading enzymes in plants. It exhibits an exo-amylolytic activity and releases β-maltose molecules from starch in vitro. In germinating maize kernels, however, β-amylase is expressed in the aleurone cells, which contain no starch for its exo-amylolytic action. Difference in the localization of enzyme and substrate molecules makes it difficult to understand the physiological functions of β-amylase in vivo. To further investigate its functions, maize genomic libraries were constructed, and a full-length β-amylase gene was cloned and sequenced. The primary structure of β-amylase was also deduced from the sequence data. β-amylase promoter activity analysis was also undertaken with transient expression systems. Three different systems were tested, but proved unsuccessful. The promoter sequences were also analyzed by searching through internet resources and research articles. Possible cis-elements in promoter region were found by database searching. Moderate degrees of homology were also found by computer-aided alignment around TATA box and 5'-UTR region among β-amylase promoters of maize, barley, and rice. These results could provide insights into related research and data interpretation on β-amylase in the future.