阿拉伯芥金屬螯合素合成酶Thr 49突變株活性分析及轉殖株之鎘耐受性

Abstract

植物金屬螫合素合成酶 (phtychelatin synthase, PCS, EC2.3.2.15) 與植物對重金屬的耐受性有高度相關。目前的研究中已經發現，PCS的活性調控可能是藉由轉譯後修飾 (post-translational modification) 而非轉錄後修飾層級。利用點突變技術和大腸桿菌 (E. coli) 表現系統，已經證實Thr 49上的磷酸化修飾對阿拉伯芥 (Arabisopsis thaliana) PCS (AtPCS1) 的活性為正向調控。Thr 49可能會與鄰近的Arg 183形成離子鍵，固定活性反應區的立體結構，因而影響了PCS的催化活性。製作阿拉伯芥PCS (T49A, T49E) 補償性轉植株。以抗生素篩選、蛋白表現量、半定量RT-PCR及IPCR等方式檢測，選出穩定表現之T4植株。以不同濃度的鎘處理，並利用植株根長及新鮮重量來觀察轉植株對鎘耐受度的差異。結果顯示，在50 μM鎘處理下，Thr 49的突變性阿拉伯芥PCS補償性轉植株對鎘的耐受度較野生型及阿拉伯芥PCS補償性轉植株差。為了研究Thr 49和Arg 183在生理條件下對PCS影響，本論文利用阿拉伯芥PCS補償性轉植株證實Thr 49對於阿拉伯芥耐受鎘的能力有重大的影響。

Phytochelatin synthase (PCS, EC2.3.2.15) plays important roles in the sequestration of heavy metals in plants. Previous studies demonstrated that phytochelatin synthase (PCS, EC 2.3.2.15) may be regulated at post-translational modifications. By using mutagenesis and E. coli expression system, we had demonstrated that phosphorylation on Thr 49 might play an important role in the formation of the active site through the interaction with Arg183. In order to reveal the physiological role of Thr 49 and Arg 183, we constructed pCAMBIA vectors containing AtPCS1 promoter sequence and its cDNA and selected several transgenic lines that expressed normal or mutated AtPCS1. The expression level and the copy number of AtPCS1 (T49A, T49E) was determined by semi-quantitative RT-PCR, western blotting, and nested IPCR. At modest Cd concentrations, wild type Arabidopsis (ecotype Columbia-0) and AtPCS1 complementary lines were more tolerant to Cd that AtPCS1-T49A and AtPCS1-T49E complementary lines. These data indicated that Thr 49 is important for Arabidopsis to resist the presence of Cd.