The regulation of proline metabolism related genes in Nicotiana benthamiana under stresses

本論文探討菸草(Nicotiana benthamiana)在不同重金屬逆境影響，脯胺酸代謝的調控機制。脯胺酸合成的榖胺酸(glutamate)路徑與鳥胺酸(ornithine)路徑代謝的四個酵素，pyrroline-5-carboxylate synthetase (P5CS)、pyrroline-5-carboxylate reductase (P5CR)、ornithine aminotransferase (OAT)、proline dehydrogenase (PDH)。本試驗以菸草葉圓片作為材料，進行重金屬銅、鎘、鋅、鉻、離層酸等處理。期能了解重金屬與離層酸單獨對脯胺酸含量，與其對脯胺酸代謝酵素基因表現量的影響。結果顯示，離層酸能誘導菸草葉圓片脯胺酸增加，但離層酸處理所誘導的脯胺酸增加程度僅及乾旱處理的十分之一。以即時聚合酶鏈反應(Real-time Polymerase Chain Reaction)偵測脯胺酸三種脯胺酸生成酵素基因P5CS、P5CR、OAT與一種脯胺酸代謝酵素基因PDH的結果顯示，離層酸處理增加P5CS基因表現約80倍，P5CR、OAT、PDH基因表現僅約兩倍。缺水處理時OAT、P5CS、P5CR的基因表現分別增加1.5、5.7、3.4倍，PDH基因表現則受到抑制，為控制組之2/3。隨著重金屬處理濃度的上升，菸草葉圓片脯胺酸含量先有顯著的上升，然後逐漸下降。銅處理則未見脯胺酸含量上升。低濃度重金屬處理使OAT、P5CS、P5CR、PDH基因表現略為升高。高濃度重金屬處理使脯胺酸含量降低，但OAT、P5CS、P5CR基因表現依然升高，而PDH基因表現則有大量的上升。據此推論，高濃度重金屬處理降低脯胺酸含量，可能與PDH基因表現上升有關。結論，菸草全株於重金屬逆境下脯胺酸的大量累積可能是由於缺水所誘導，且可能透過大量表現P5CS、P5CR基因。當過多重金屬進入植體時，脯胺酸下降，則可能是透過大量表現PDH基因。The aim of this thesis are to investigate the regulation of proline metabolism in Nicotiana benthamiana under heavy metal stresses. Two pathways of proline synthesis: one via glutamate , the other via ornithine were involved in this thesis. The profile of gene expression of four proline metabolism related enzymes, pyrroline-5- carboxylate synthetase (P5CS)、pyrroline-5-carboxylate reductase (P5CR)、ornithine aminotransferase (OAT) and proline dehydrogenase (PDH) were described. The accumulation of proline in plant under heavy metal stress might due to three factors: (1) the damage of root that cause water deficit in the shoot, (2) the increase of abscisic acid level in the root causes proline accumulation as it transport to shoot, (3) heavy metals themselves induce proline accumulated directly. In order to evaluate the impact of each single factor on proline accumulation under heavy metal stress, the leaf discs of Nicotiana benthamiana were floated on the solution of various treatments, such as abscisic acid, heavy metals and proline synthesis precursors. The experiment were conducted in two parts: the physiological assay and the gene expression of proline metabolism related enzymes. Four heavy metal, CuSO4, CdCl2, ZnCl2 and K2Cr2O7 were chosen for heavy metal treatments. The result showed that, abscisic acid could induce proline accumulation in leaf discs, but the level was only one tenth of the level by water deficit treatment. With the analysis of real-time polymerase chain reaction (RT-PCR) on four proline metabolism related enzymes, the result of the abscisic acid treatment showed that P5CS gene expression increased up to 80 fold mean while PDH, OAT、P5CR gene expression were only up regulated about 2 fold. In water deficit treatment, the expression of OAT, P5CS and P5CR gene was up regulated significantly, but PDH gene was reduced. In heavy metal treatment, proline content increase gradually with the increase of heavy metal concentration. Meanwhile, the expression of OAT、P5CS、P5CR and PDH genes were up regulated. As the heavy metal concentration up to 0.5 mM proline content begin to decrease, whereas though the expression of OAT、P5CS、P5CR genes were up regulated, PDH gene up was regulated massively. Thus, it is likely that the decrease of proline content in higher concentration of heavy metal treatment might due to the up regulation of PDH gene expression. In summary, the proline accumulation on the whole plant of Nicotiana benthamiana in response to heavy metal stress mainly due to water deficit and via the up regulation of the expression of P5CS and P5CR genes. Consequently, when the heavy metal contents in plant increased up to certain level, proline content decreases, the inducement of the expression of PDH gene seems to dominate the regulation of proline level in plant.摘要----------------------------------------------------- I Abstract----------------------------------------------- III 目錄----------------------------------------------------- V 圖目錄------------------------------------------------- VII 表目錄--------------------------------------------------- X 前言------------------------------------------------------1 第一章、前人研究------------------------------------------2 一、脯胺酸與逆境的關係------------------------------------2 二、離層酸與逆境的關係----------------------------------- 4 三、脯胺酸累積之機制------------------------------------- 5 四、植物復育法--------------------------------------------7 五、過量銅對植物的生理之影響------------------------------8 六、過量鎘對植物的生理之影響------------------------------9 七、過量鋅對植物的生理之影響-----------------------------10 八、過量鉻對植物的生理之影響-----------------------------11 第二章、材料方法 一、試驗材料---------------------------------------------13 二、逆境與藥劑處理---------------------------------------13 (1)重金屬處理--------------------------------------------13 (2)鳥胺酸(ornithine)處理---------------------------------14 (3)離層酸(abscisic acid)處理-----------------------------14 (4)脯胺酸(proline)處理-----------------------------------15 (5)Polyethylene Glycol(PEG)處理--------------------------15 (6)乾旱、回水處理----------------------------------------15 三、脯胺酸之測定-----------------------------------------16 四、Malondialdehyde(MDA)之測定---------------------------16 五、菸草葉圓片重金屬含量測定-----------------------------17 六、離層酸含量測定---------------------------------------17 七、總量RNA(total RNA)之萃取-----------------------------19 八、去氧核糖核酸分解酶(DNase I)處理----------------------20 九、反轉錄聚合酶反應(reverse transcription polymerase reaction)------------------------------------------- 20 十、P5CR、P5CS、OAT、PDH基因引子對設計------------------ 21 十一、Real-time PCR--------------------------------------23 (1) primer濃度測定---------------------- ----------------23 (2) 標準曲線---------------------------------------------23 (3) 實測-------------------------------------------------30 第三章、結果與討論 一、重金屬對菸草葉圓片脯胺酸的影響-----------------------31 (1) 重金屬對菸草葉圓片脯胺酸的影響-----------------------31 (2) 脯胺酸在重金屬逆境下的功能---------------------------31 (3) 脯胺酸累積對菸草葉圓片氧化逆境的影響---------------- 32 二、離層酸對菸草葉圓片中脯胺酸的影響---------------------45 三、重金屬對菸草葉圓片離層酸的影響-----------------------52 四、重金屬對脯胺酸合成代謝酵素基因表現量的影響---------- 52 五、離層酸對脯胺酸合成代謝酵素基因表現量的影響---------- 60 六、缺水處理對脯胺酸合成代謝酵素基因表現量的影響-------- 61 第四章、綜合討論-----------------------------------------63 第五章、結論---------------------------------------------72 參考文獻-------------------------------------------------7