台灣留學生出席國際會議補助

2010年7月30日 星期五

Production of a1-2 knockdown lines in an a1-3 background to assess the role of the cnd41 aspartyl protease in leaf senescence

論文發表人: 戴雨豆 / 加州州立大學長堤分校生物所碩士班

 

http://www.lyon.edu/photosynthesis/

 

植物老化是一種很嚴謹且規律的過程. 在植物老化過程中,葉綠體中的蛋白質會被分解並轉送到植物其他新生部位如果實和種子. 研究顯示菸草蛋白CND41在植物老化過程中扮演很重要的腳色. CND41是葉綠體DNA結合蛋白並含有一個蛋白質水解酵素功能區塊. 阿拉伯芥中發現三個CND41 異物種同源基因A1-1, A1-2 A1-3.研究顯示 A1-2 A1-3基因在較老的植物葉片中有較高的表現因此被視為跟植物老化有關聯但是a1-2 a1-3單基因突變植物體並沒有明顯的植物老化現象產生,其原因有可能是A1-2 A1-3基因互為互補基因. 為了能了解A1-2A1-3基因的功能,我們利用RNAi的技術去產生一個雙重基因突變(a1-2KD/a1-3KO)阿拉伯芥植株並去研究這個雙重突變植物體是否對植物的老化現象產生影響. 我們選擇使用RNAi的技術去獲得雙重突變植物體的原因是因為A1-2A1-3基因是相鄰基因,因此我們無法利用傳統育種的方式來取得雙重突變植物體. 這個研究分成兩個部分,第一個部分包含了RNAi質體的設計和構築以及基因轉植植株的生成.第二個部分則是研究雙重基因突變植株(a1-2KD/a1-3KO)是否可以延遲植物的老化現象. 目前實驗已經完成了第一個部分並且大部分的轉植植株的A1-2mRNA量都非常的稀少.接下了則是要進行測試這些轉植植株在老化過程中是否有延遲老化的現象.

 

 

Leaf senescence is considered to be a highly regulated process. During senescence, chloroplast proteins degrade into nutrients that are exported. Previous research showed that CND41 played a role in the senescence process in tobacco. CND41 is a chloroplast DNA binding protein that also contains an A1 aspartic protease domain. In Arabidopsis, three CND41 orthologs have been found, A1-1, A1-2, and A1-3. A1-2 and A1-3 are considered to potentially play a role in senescence as their expression is upregulated in older leaves.However, individual a1-2 and a1-3 knockout Arabidopsis mutants did not show any obvious delays in senescence. The reason might be because the A1-2 and A1-3 genes can compensate for each other due to redundancy. Therefore, in order to verify the function of A1-2 and A1-3 genes, we plan to knock down A1-2 expression in an a1-3 knockout mutant and observe if there is any delay of leaf senescence in the a1-2KD/a1-3KO double mutant. It is difficult to obtain an a1-2/a1-3 double knockout line by crossing the a1-2 KO and a1-3 KO mutant because these are adjacent genes. Thus, we are using RNAi to obtain the double mutant. The first step is to design and construct the RNAi vector and produce transgenic lines while the second part is to analyze the senescence phenotypes of double mutants.  RNAi constructs have been obtained, transgenic lines have been selected, and our initial analysis demonstrates silencing of A1-2 gene expression in nearly all transgenic lines.  These lines are currently being grown for analysis of senescence phenotypes.  (This work funded by NSF 0415108.)