玉米β-澱粉水解?基因?動子活性之研究

Abstract

β-澱粉水解?是植物體中重要的澱粉水解酵素之一。本論文旨在探討玉米β-澱粉水解??動子序列對於基因表現的影響。授粉前的玉米子房壁、萌芽後3天之初生葉及糊粉層等組織均能活潑表現β-澱粉水解?基因，是適合建立電穿孔及粒子槍短暫表現系統的好材料。將全長之β-澱粉水解??動子接上GUS報告基因，pZmBAP::GUS，以粒子槍轉殖入玉米及菸草葉片中，結果在葉肉細胞中可偵測到GUS基因的活性表現，惟在菸草的表皮上只有保衛細胞才會表現GUS活性。本論文進一步進行?動子刪除之實驗，構築pZmBA783P::GUS、pZmBA539P::GUS、pZmBA492P::GUS、pZmBA366P::GUS及pZmBA50P::GUS等具不同長度之?動子序列的載體，並以粒子槍打入玉米初生葉；結果顯示β-澱粉水解??動子在-1571?-783及-539?-366兩段序列中可能有負調節因數的存在，而在-783?-539之間則可能存在正調節因數。在GUS基因前放入β-澱粉水解?基因的表現子I（exon I）與插入子I（intron I）並不會加強基因的表現能力；?動子序列中若只包含TATA box的短片段序列，反而有最強的GUS活性表現。除了短暫基因表現之測試外，亦將β-澱粉水解?全長之?動子序列，接上GUS報告基因，用農桿菌轉殖到菸草中，得到穩定之轉殖株，在轉殖株之葉片及癒傷組織中可偵測到GUS基因表現，顯示玉米β-澱粉水解??動子和水稻?動子之表現特性有所差異。本文就玉米與水稻?動子序列做比對，並討論兩者之cis -acting element的差別。

Beta-amylase is one of the most important starch hydrolases in plants. In maize, β-amylase gene is actively expressed in young ovary wall, germinating aleurone layers and 2.5 to 3-days-old primary leaves. This indicated that these three types of tissues are suitable for building up the transient expression systems. A pZmBAP::GUS having GUS gene directed by β-amylase promoter(1.5kb) was constructed and transferred into maize and tobacco leaves by bombardment. GUS activity was detected in mesophyll of both maize and tobacco. Besides, GUS activity was also present in tobacco guard cells but not in the regular epidermal cells. Various chimeric constructs, pZmBA783P::GUS, pZmBA539P::GUS, pZmBA492P::GUS, pZmBA366P::GUS and pZmBA50P::GUS, contained different length of β-amylase promoter were constructed. After bombardment and incubation, GUS activity in maize leaves was measured and normalized by internal Luciferase control. It was suggested that negative regulators might exist in the sequences between -1571?-783 and -539?-366, and sequences between -738 ? -539 might possess positive cis-elements. The shortest promoter consisting of TATA box examined gave the strongest activity in leaves. In addition to transient expression, GUS gene followed the full-length β-amylase promoter was transformed into tobacco mediated by Agrobacterium. GUS activity was detected in leaves and calli of the transgenic tobacco plants. It is known that GUS gene was not expressed in transgenic tobacco under the direction of rice promoter. The comparison of promoter sequences of maize and rice β-amylase genes were discussed.