大豆低分子量熱休克蛋白質抗體之製備及其定量

Abstract

本實驗利用雙向電泳分析來純化大豆的15-18 KD熱休克蛋白質（heat shock proteins, HSPs）。並且以15-18KD HSPs其中的一種蛋白質（HSP1, pI6.2 MW17.3KD）做?抗原，製備得對15-18KD這一?的HSPs具有專一性的抗體。籍由免疫轉印分析（Immunoblot）的技術配合Densitometer，以純化得到已知量的HSPs做?標準（standard）來定量各種情形所誘導產生的15-18KD HSPs，探討這?蛋白質累積的量與所提供的耐熱性之間的關係。 此外，利用抗體與抗原之間的專一性，證實了這?植物所特有的HSPs在不同的植物之間可以產生交叉反應（crossreaction）。而大豆本身的15-18KD HSPs由氨基酸組成分析及免疫轉印分析都可以證實他們彼此之間有著極高度的相似性。而且整?蛋白質的行?，以及對各種誘導因數所產生的反應都具有一致性，一被誘導合成則每一種蛋白質都同時產生，一起合成，一起增加，顯示這?蛋白質可能是同一multigene family的產物。 本實驗同時發現大豆種子在吸水發芽16?20小時之後，在受到熱休克誘導下才會有大量的15-18KD HSPs的合成。在正常溫度下已有少量的低分子量HSPs存在大豆種子胚（embryo）中，而隨著種子的發芽成長，這?蛋白質亦逐漸地消失。大豆白化幼苗的低分子量HSPs除了與植物耐熱性的形成有直接的關係之外，可能還有一些其他的生理功能值得進一步深入探討。

Heat shock proteins (HSPs) from soybean seedlings were found enriched in 65-100% ammonium sulfate fraction. This HSPs enriched fraction was used for obtaining one of the low-molecular-weight (LMW) HSPs, p16.2 with MW 17.3 KD by 2-D PAGE. This protein spot was used as an antigen for raising a polyclonal antibody by injecting into New Zealand white rabbit. We found this antibody crossreacted with 13 polypeptides of 15-18 KD LMW soybean specific HSPs and also to some of the LMW HSPs from mung bean, rice, pea, maize and tobacco by immunoblot analyses. When the growth temperature of two-day-old soybean seedlings was shifted to 40℃ for 4h, the level of 15-18 KD HSPs accumulated to the highest level. All 13 polypeptides of HSPs in the range of 15-18KD were enhanced simultaneously. A brief 10-minute exposure to 45℃ followed by incubation at 28℃ also resulted in the synthesis of 15-18KD HSPs. Arsenite treatment also induced the synthesis of 15-18KD HSPs which were indistinguishable from the heat shock treatment but accumulated to much lower level. We estimated that the accumulation of 15-18KD HSPs to 0.6-0.76 ug/100 ug total protein was nessary for the seedling to acquire thermo-tolerance. During 6h to 12h of post-imbibition, a detectable amount of LMW HSPs was found regardless of heat shock temperature. But the levels of these LMW HSPs were much lower and were found even under non-heat shock condition (28℃). After 16-20h of germination, soybean seedlings exhibited a normal response with the synthesis and accumulation of large quantity of LMW HSPs. At the optimum temperature 42.5℃ rather than 40℃ in 1-day-old soybean seedlings synthesized the highest amount of 15-18KD HSPs. This was different from 2-day-old or 3-day-old seedlings which synthesized 15-18 KD HSPs to the highest level at 40℃.