胺基酸類似物誘導大豆第一族低分子量熱休克蛋白質生合成之生理生化分析及其與耐熱性的探討

Abstract

大豆幼苗以高溫或胺基酸類似物azetidine（proline之類似物，以下簡稱Aze）處理，均會誘導第一族低分子量熱休克蛋白質（class I low-molecular-weight heat shock proteins，簡稱class I LMW HSPs）的生合成。但是以10 mM Aze處理24小時後的大豆幼苗，以大豆第一族低分子量熱休克蛋白質抗體進行免疫轉印分析（immunoblotting）的結果顯示class I LMW HSPs的累積量雖然能達到與40℃處理2小時相當，但在氯化四唑染色法（TTC reduction test）所進行的細胞活性測定中，卻顯示不能相對地提高幼苗在45℃致死溫度的忍受力。 本實驗發現將大豆幼苗先以10 mM Aze處理6小時，就足以在往後24小時累積與40℃處理2小時相當量之class I LMW HSPs，但只有10 mM Aze處理後接著以10 mM Pro處理的大豆幼苗對45℃處理2小時具有耐熱性，而接著以其他不同種類胺基酸（如Pro、Gly、Phe、Cys及Gln）處理的幼苗則否。 在次細胞分離（Subcellular fractionation）及免疫轉印分析（Immunoblotting）的結果中顯示，Aze處理後再以10 mM Pro處理24小時的大豆幼苗，約40％的class I LMW HSPs分佈於去核糖體上清液（post-ribosomal supernatant，簡稱PRS）中，而以其他胺基酸繼續處理之幼苗的class I LMW HSPs，僅約10％位於PRS。在熱保護（thermoprotection）實驗中，顯示出純化自10 mM Aze處理6小時後再以10 mM Pro處理24小時之大豆幼苗的class I LMW HSP complex，能降低PRS中蛋白質因受熱而變性沉澱的情形。 由蛋白質胺基酸成份分析（Amino acid composition analysis）的結果顯示，Aze不會存在class I LMW HSP中（pI 6.2，MW 17.3）至於其與大豆幼苗耐熱性的關係，將在本論文中討論。

Class I low-molecular-weight heat shock proteins (LMW HSPs) which are likely to promote thermotolerance in soybean seedlings are induced by heat shock treatment as well as azetidine-2-carboxylic acid (Aze), a proline analog. The amount of class I LMW HSPs induced by 10 mM Aze for 24 hours was equivalent to that induced by 40℃ for 2 hours. However, these class I LMW HSPs induced by Aze treatment had no effect on thermotolerance of seedlings. When soybean seedlings, treated with 10 mM Aze for 6 hours, were then incubated with several different amino acids for 24 hours, respectively, all seedlings of each treatment accumulated certain amount of class I LMW HSPs. However, only the seedlings incubated with proline subsequently acquired thermotolerance. In these seedlings, about 40% of class I LMW HSPs were found in post-ribosomal supernatant (PRS) fraction of total protein. On the other hand, only around 10% of class I LMW HSPs induced by subsequent incubated with other amino acids (e.g., Gly, Phe, Cys and Gln) were found in PRS. Class I LMW HSP complex from the soybean seedlings treated with Aze and subsequently with proline reduced denaturation of PRS of soybean seedlings at high temperature. Analysis of amino acid composition indicated that Aze was not incorporated into class I LMW HSP (pI 6.2, MW 17.3), but was found in protein which was not inducible during heat shock.