利用病毒誘導基因沈寂探討逆境誘導下菸草脯胺酸合成酵素的角色

Abstract

植物於逆境下，常發現有脯胺酸累積的現象。Δ1-pyrroline-5-carboxylate synthetase （P5CS）及ornithine-δ-aminotransferase （OAT）是已知合成脯胺酸的兩種酵素，目前對逆境下兩酵素所扮演的角色尚未確認。本論文的目的在探討逆境下，脯胺酸的合成是透過何種酵素，以及此二酵素可能扮演的角色。本論文利用菸草嵌紋病毒（Tobacco mosaic virus, TMV）載體誘導基因沈寂（virus-induced gene silencing, VIGS）降低十週大菸草體內P5CS（TMV-antiP5CS）或 OAT 表現（TMV-antiOAT）或同時降低 P5CS 及 OAT 表現（TMV-antiP5CS + antiOAT），然後取病毒接種葉以上第一葉及第二葉的葉圓片，分別處理29％ PEG （polyethylene glycol）、100 μM 離層酸 （abscisic acid, ABA）或、50 μM 氯化鎘，代表重金屬逆境下會誘導脯胺酸累積的三個因子。PEG處理下，接種TMV-antiP5CS葉圓片脯胺酸累積量減少80-89％，而接種TMV-antiOAT葉圓片脯胺酸量僅減少5-15％，鎘與ABA處理也有類似的結果。推論P5CS為重金屬逆境下使脯胺酸累積的主要酵素。再則進一步利用葉綠素與MDA含量變化，觀察逆境下抑制P5CS或OAT對生理的影響，結果顯示滲透逆境下接種TMV-antiP5CS葉圓片之葉綠素分解比接種TMV-GFP葉圓片快，而接種TMV-antiOAT葉圓片之葉綠素分解卻比接種TMV-GFP葉圓片緩慢。各處理下MDA含量則無明顯差異。由於OAT的反應物為鳥胺酸（ornithine），鳥胺酸亦可用於多元胺（polyamine）之合成，而多元胺有保護植物的功能。因此測定各處理葉圓片的多元胺含量，確認葉綠素分解是否受多元胺含量影響。結果顯示滲透逆境下抑制OAT 確實會增加多元胺含量。綜合上述論點歸結要點如下：P5CS為滲透逆境、ABA處與重金屬逆境理下使脯胺酸累積的主要酵素，而OAT僅參與滲透逆境誘導之脯胺酸累積。滲透逆境下抑制P5CS路徑會加速葉綠素分解。滲透逆境下抑制 OAT 路徑則會延緩葉綠素分解，其原因可能與多元胺含量增加有關。

Proline accumulation is wide spread phenomenon in stressed plants. Proline could be synthesized with Δ1-pyrroline-5-carboxylate synthetase (P5CS) from glutamate and with ornithine-δ-aminotransferase (OAT) from ornithine in plants. It dose don not know well about the role of P5CS and OAT in stressed plants. The aim of this thesis is to investigate which enzyme is more important for proline synthesis, and what is the possible function of these enzymes in response to abiotic stress. We used Tobacco mosaic virus (TMV) as vector with the insertion of proline metabolized related gene fragments to decrease the expression of P5CS (TMV-antiP5CS) , OAT (TMV-antiOAT), or both of P5CS and OAT (TMV-antiP5CS＋antiOAT) in 10 weeks old tobacco via the mechanism of VIGS (virus-induced gene silencing). The tobacco leaf discs taking form first and second leaf above the inoculated leaf were treated with 29％PEG (polyethylene glycol), 100 μM abscisic acid (ABA), or 50 μM CdCl2 to mimic three abiotic stresses. Under PEG treatment, the proline level of tobacco leaf discs inoculated with TMV-P5CS or TMV-OAT decreased about 80-90％ and 5-15％ respectively. The changes of proline level under ABA or Cd treatment were similar to PEG treatment. It is suggests that P5CS could be the major enzyme for proline accumulation under heavy metal stress. In order to investigate the possible function of P5CS and OAT expression in stressed plant, the chlorophyll and MDA content of leaf discs treated with VIGS were analyzed. The degradation of chlorophyll in TMV-antiP5CS inoculated tobacco leaf discs was faster than in TMV-GFP under PEG treatment. But the degradation in TMV-antiOAT was slower than in TMV-GFP. There was no difference in MDA content among all VIGS treated plant under stresses. It is noticed that the content of polyamine increased in TMV-antiOAT inoculated tobacco leaf discs under osmotic stress. In summary, P5CS is the major enzyme of proline accumulation under osmotic stress, ABA or heavy metal stress. OAT participates the proline accumulation only in osmotic stress. Under osmotic stress, inhibiting P5CS pathway can improve the rate of chlorophyll degradation. But inhibiting OAT pathway can delay the rate of chlorophyll degradation that migh be related to the increase of polyamine content in tobacco leaf discs.