以蛋白質體學探討熱休克處理打破番木瓜種子休眠機制

Proteomic Analysis of Dormancy Breakage of Papaya (Carica papaya L.) Seeds Pre-treated with Heat Shock Treatment

楊開雲(Kai-Yun Yang)

指導教授 ： 吳俊達

共同指導教授 ： 張英峯(Ing-Feng Chang)

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.00293

摘要

番木瓜 ( Carica papaya L. )為臺灣重要的經濟果樹之一，其繁殖方式有實生苗、嫁接、扦插及組織培養，但目前其種苗生產方式仍以實生苗為主。然而，番木瓜種子因本身休眠性的因素，造成在發芽上有發芽時間冗長與發芽不整齊的問題，雖已有多位學者提出促進發芽的方法，但其促進效果卻會因品種及種子休眠程度不同而有所改變。眾多研究中發現，一般使用植物荷爾蒙-激勃素 ( gibberellins )打破種子休眠，熱處理亦可促進番木瓜種子的萌芽。然而，有關熱處理是如何打破番木瓜種子的休眠的機制目前仍所知闕如。
本論文以蛋白質體學 ( Proteomics ) 為研究方法，試圖分析熱處理誘導番木瓜種子打破休眠的可能機制。然而，在眾多文獻中尚未有番木瓜種子二維電泳樣品製備的相關方法，故本論文試驗首先以Tris-HCl作為基礎之萃取液，將浸潤5天番木瓜種子均質萃取後，直接以10% TCA/acetone ( TCA-A ) 沉澱，與經酚 （ phenol ） 抽取再以0.1 M ammonium acetate-methanol ( P-AA-M ) 沉澱，比較兩種樣品製備方法對蛋白質電泳圖譜解析之差異。試驗結果發現P-AA-M法較TCA-A法可增加一維SDS-PAGE圖譜之蛋白質條帶數至少五條，且其中較明顯的兩條帶經LC-MS/MS鑑定後以「功能未知」的蛋白質所占之比例最高 ( 34% )，其次則為與「能量代謝」 ( 20% )、細胞週期 ( 14% ) 相關之蛋白質。此外，在二維電泳圖譜方面，P-AA-M法可改善TCA-A樣品於二維電泳圖譜條紋 ( streaking ) 與拖尾 ( smearing ) 之次級代謝物干擾問題，提升蛋白質點間的分離效果與圖譜解析度及增加蛋白質點的數目。
將經26℃黑暗環境浸潤5天再以36℃熱水浴處理5小時熱處理後的番木瓜種子透過上述之P-AA-M萃取方法進行二維電泳後發現，熱處理可使鈣離子訊息傳導中的CBL及CDPK、小分子熱休克蛋白之17.5 sHSP、能量代謝相關之G3PDH及ATP synthase epsilon chain與轉錄作用相關之GR-RBP等蛋白質的表現，並較只浸潤無熱水浴處理的種子為高出3倍以上。推測熱處理可誘導番木瓜種子經過一連串的訊息傳導與能量代謝使其休眠弱化後，開始進入生長發育的階段，並在進入此階段前開始累積能量而使特定的轉錄因子開始表現，進而提高胚內的生長勢，最後使胚根向外推擠的力量大於胚乳與種殼的物理障礙，使種子得以順利發芽。
熱處理打破番木瓜種子的休眠機制從試驗結果得知與GAs生合成之間具有相當重要關連性存在。然而除了GAs之外，BR亦為參與種子發芽過程的重要荷爾蒙之一。然而以36℃熱水浴處理5小時後熟番木瓜種子置於0.01~5 μM之BR生合成抑制劑，Brassinazole，中進行培養後卻發現所有濃度皆無法抑制發芽。故推測熱處理誘導番木瓜種子發芽的過程中BR應該不是最主要的因子，而可能由其他植物荷爾蒙與環境因子共同調控。
熱處理雖然可有效打破番木瓜種子的休眠，然而對於不同休眠程度的番木瓜種子其效應卻會有所不同。本試驗將浸潤5天後的種子分別以34、36、38℃之水浴處理後發現熱處理對新鮮製備的番木瓜種子發芽效果不彰，然而若將種子進行2~8週之後熟作用則可增加熱處理之效果，其中又以後熟8週的效果最好。另一方面，將熱處理後的種子以不同乾燥速率乾燥後經貯藏2週發現，以36℃乾燥6小時之快速乾燥最能保留熱處理之效應約80%，而若以較低的溫度進行慢速乾燥則較無法保留熱處理之效果。

關鍵字

蛋白質體學 ； 熱休克 ； 番木瓜 ； 種子 ； 休眠

並列摘要

Papaya (Carica papaya L.) is one of the important economical fruit crops in Taiwan, and it can be propagated by seeds, grafting, cutting and tissue culture. However, papaya is propagated mainly by seeds. Nevertheless, freshly harvested papaya seeds germinate poorly and unevenly. Even though numerous researchers had reported many kinds of treatments to improve the germination, results were different due to cultivars and environmental factors. Gibberellins and heat treatments have been reported to promote the germination of papaya seeds. However, the mechanism of how heat treatment breaks the dormancy of papaya seeds is not clear. Therefore, it is an important topic to understand how heat treatment induces the germination of papaya seeds.
In this research, I use the method of proteomic analysis to investigate the possible mechanism of heat treatment on the dormancy breakage of papaya seeds. Unfortunately, there are no related protocols have been reported for protein preparation from papaya seed for proteomics analysis. Therefore, in this study I established the optimized protocol of protein extraction for sample preparation of two-dimensional electrophoresis. The protein extraction mixture for 5 day-imbibed papaya seeds was a Tris-HCl base buffer. The extracted solution was then precipitated by 10% TCA/Acetone (TCA-A) or extracted by phenol followed by 0.1M ammonium acetate-methanol precipitation (P-AA-M). The results showed that the method of P-AA-M could increase at least 5 bands than TCA-A in SDS-PAGE analysis. Two of the bands were assessed via LC-MS/MS and the proteins identified included “unknown proteins”, with the highest ratio (34%), “energy metabolic proteins”, the minor category (20%), and “cell cycle proteins”( 14%). P-AA-M could reduce the problems of streaking and smearing in 2D gel analysis, enhance the efficiency of protein separation, and therefore, increase the number of protein spots identified.
Application of 36℃ hot water immersion for 5 hours to 5 day-imbibed papaya seeds would induce the expression of proteins related to calcium signal transduction (CBL and CDPK), small heat shock protein (17.5 sHSP), energy metabolism (G3PDH and ATP synthase epsilon chain), and transcription (GR-RBP). It is assumed that after the heat treatment, the papaya seeds would be released from dormancy by a series of physiological events including signal transduction and energy metabolism, increase of the embryo growth potential, overcome the physical constraint of testa, and then germination.
According to the results, the possible mechanism of heat treatment on dormancy breakage of papaya seeds would be through GA, but not brassinoids, biosynthesis, since brassinzole, a brassinoids synthesis inhibitor, was unable to inhibit the germination of the seeds treated with the hot heat immersion
Even though heat treatment could induce the germination of papaya seeds, the effects would be various depending on degree of the dormancy. Hot water immersion treatment with 34, 36, or 38℃ could enhance the germination of after-ripened seeds. The longer the after-ripening time applied, the better the efficacy of dormancy releasing. Moreover, the germination capability of papaya seeds acquired from heat treatment could be preserved up to 80% if the seeds were prepared by fast drying method (36℃ forced air for 6 hours), but could not be preserved by slow drying method.

並列關鍵字

proteomics ； heat shock ； papaya ； seed ； dormancy

參考文獻

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