綠豆第一族低分子量熱休克蛋白質與低溫耐性之探討

Abstract

綠豆?不耐低溫性植物，將其白化幼苗放在低溫4℃處理12小時後，便會有延緩生長的現象；而在處理2天後，進行回溫時則會有相當明顯的傷害出現並且死亡。 將白化幼苗以40℃進行短暫熱休克前處理30分鐘，便可明顯降低其在低溫下的傷害且能夠在回溫後繼續生長，而隨著熱休克時間的增加至2小時，其在低溫逆境下可耐到長達12天以上的時間。 綠豆白化幼苗在高溫40℃處理2小時後，經35S-methionine放射性元素標定、雙向電泳的分析與熱休克蛋白質抗體反應，發現有一群高、中、低分子量熱休克蛋白質被誘導出來，而由抗體所辨識的一群19-21kD第一族低分子量熱休克蛋白質會隨熱休克時間不同而生成量不同；在致死溫度（4℃）與冷馴化溫度（10℃）處理下這群19-21kD蛋白質並不被誘導生成。此蛋白質的量在常溫（28℃）下可維持約48小時，至72小時則完全消失；在4℃低溫下可維持超過12天。除了正面證明誘導的熱休克蛋白質愈多，其幼苗在低溫下的忍受力（生長勢）愈強外，亦反面證明此低分子量熱休克蛋白質消失時，幼苗便會喪失其低溫耐性。 以抗大腸桿菌60kD與抗酵母菌60kD熱休克蛋白質抗體檢測反應，發現在綠豆幼苗處理過程中，並沒有與抗體性質相似的中分子量熱休克蛋白質被誘導出來。 藉由熱休克處理來增加低溫忍受性的研究在過去已有報導，但均未直接針對低分子量熱休克蛋白質的角色進行探討，是以本研究已確立了綠豆第一族低分子量熱休克蛋白質在低溫下的生理特性，並證明其與綠豆幼苗耐冷性間的相關性。

The growth of etiolated mungbean seedlings (Vigna radiata L. cv. Tainan No.5), a chilling-sensitive crop, was significantly inhibited by low temperature (4℃) treatment of 12 hr, and a significant injury appeared 2 days after low temperature treatment followed by 1 day recovery at 28℃. Pretreatment of 40℃ could improve chilling tolerance of mungbean seedlings, and a set of isotope labeled HSPs was separated by 1-D gel electrophoresis. A group of 19-21kD class I low-molecular-weight HSPs was recognized by westerrn blotting using rabbit anti-soybean class I low-molecular-weight heat-shock protein antibody. This group of 19-21kD HSPs was only induced by high temperature at 40℃ and reached the maximum level at 2 hr. The half life of these 19-21kD HSPs were about 2 days at 28℃, but the amount of proteins maintained at high level at 4℃ for 12 days. The more of the 19-21kD HSPs, the more chilling tolerance was observed. When these 19-21kD HSPs were degraded completely, the plant has lost its chilling tolerance. Using E. coli and yeast 60kD HSPs antibodies for western blot analysis, no HSPs ranging in 80kD to 40kD in mungbean seedlings were detected. The roles of each group of HSPs are still yet to be clearfied. Furthermore, the chilling tolerance observed in our mungbean system could be achieved by multiple HSPs but not only a single HSPs.