甘藷葉部澱粉分支酶基因之概日韻律表現及啟動子序列分析

Abstract

澱粉分支酶 (starch branching enzyme；SBE) 為負責支鏈澱粉 (amylopectin)分支鏈形成的重要酵素。在16小時光照/ 8小時黑暗 (16L/ 8D) 生長環境中，甘藷葉部的 SBE 基因表現呈現規律性的變化，其轉錄產物 (transcript) 於光照時期進入黑暗時期後達到最高 (即開燈後16小時)，當植株進入黑暗週期時基因表現開始下降。當植株自16L/ 8D 的生長環境移入連續光照 (constitutive light；LL)的環境下， SBE 基因的表現週期相較於16L/ 8D環境中生長之植株的基因表現週期略為縮短，但基因表現仍維持相似之規律性變化，此結果顯示甘藷葉部 SBE 基因規律性的表現是受內在生物時鐘所調控而產生之概日韻律現象。然而在甘藷葉部之 SBE 基因之概日韻律為開燈後16小時累積到最高值，負責直鏈澱粉 (amylose) 生合成的澱粉生合成酵素 (granule-bound starch synthase I；GBSSI) 的概日韻律表現則是在開燈後4小時累積到最高，兩基因在概日韻律的表現是有差異的。為探討 SBE 基因表現受生物時鐘的調控機制，以 genomic PCR walking 的方式由基因組 DNA (Genomic DNA) 中將 SBE 基因的啟動子片段選殖出來，經調控單元 (cis-acting elements) 的分析後發現，在該片段中具有數個與概日韻律調控之調控單元。

Starch branching enzyme (SBE) is the key enzyme for amylopectin synthesis. Gene expression of SBE was fluctuated during 16 hr light and 8 hr dark photoperiod cycles (16L/ 8D). In 16L/ 8D condition, the peak of SBE transcript accumulation was near the end of day time. Since the oscillation of SBE expression was also presented when the plants were transferred to constitutive light photoperiod (LL), it were suggested that the expression of SBE gene in leaves during diurnal cycles was controlled by a biological clock(s). The period length of SBE gene expression in LL was slightly shorter than that in 16L/ 8D. Both SBE and granule-bound starch synthase I (GBSSI) gene expression were circadian regulated, however, the expression of SBE gene presented the different diurnal rhythm from that of GBSSI. To understand the mechanism of SBE transcription in diurnal cycles, an effort was made to isolate and characterize the SBE promoter. The result showed several circadian clock-related and light regulation-related cis-acting elements were located in the SBE promoter sequence.