大豆種子中三種group Ⅳ的LEA蛋白質對人工微脂粒在乾燥下之保護作用

Abstract

本實驗利用人工微脂粒乾燥滲漏實驗，分析大豆種子中三種屬於group Ⅳ的LEA蛋白質，GmPM16、GmPM21和GmPM01，在乾燥時對人工微脂粒(liposome)的保護效果。探討LEA蛋白酸鹼度之不同、微脂粒所帶電荷之不同、蛋白與磷脂質質量比之改變與Tm等如何影響受到乾旱逆境後的膜系。在pH=8.0的環境下，三種LEA蛋白質對三種帶不同電荷的liposome均有程度不一的保護效果。當膜外蛋白質與lipid比例較低（質量比為25:1）時，GmPM16和GmPM28的確對帶相反電荷的膜保護效果最好，而GmPM01則無顯著差異。當膜外蛋白質與lipid比例較高（質量比為100:1）時，由保護效果的不同，發現這三種group Ⅳ之LEA蛋白質可大致分為兩群—GmPM16對三種膜的保護效果均較GmPM01與GmPM21好。將保護效果最好的GmPM16與三種不同的雙醣（sucrose、raffinose、stachyose）混合，在膜外測試保護效果的差異。在帶負電的膜系中，糖類單獨存在時的保護效果最好，其與GmPm16各半的組合，在保護物質與磷脂質質量比為25/1時均較GmPM16單獨存在為佳。在帶正電的膜系中，當膜外物質：磷脂質比例為25/1時糖之效果最佳，當質量比提高後，糖與蛋白質各半的組合其保護效果最好。在不帶電的膜中，GmPM16與三種糖的分別組合其保護效果均較只有糖存在時的顯著為低。在三種不同的膜中，當膜外的蛋白質（GmPM16）與lipid比例較低（質量比為25:1）時，內外均有GmPM16蛋白質會使保護效果顯著大幅提高，尤以帶負電膜系的結果最顯著。在Tm 值的結果方面，對不帶電的膜來說，保護效果與使Tm下降的程度具有正相關。保護效果愈好的，會使乾燥復膜系的Tm愈低。對帶正電和負電的膜來說，保護效果與使Tm下降的程度沒有關係。這代表對帶有電荷的膜而言，保護效果主要由電荷之間的作用力決定。本研究證實group Ⅳ之LEA蛋白，尤其是GmPM16，確實能在乾燥過程中對不同表面電荷之膜系有保護的功能。這些生物體外的實驗證據可以幫助我們推測這些group Ⅳ的LEA蛋白質在生物體內的功能，以及蛋白質保護乾燥膜系的機制。

We have examined whether purified group Ⅳ LEA proteins in soybean seeds can maintain the structure of artificial liposome under desiccation in this study. We also studied the relationships between protective effect and phase transition temperature (Tm) of desiccated membrane. All LEA proteins (GmPM01, GmPM16 and GmPM28) were proved to be able to protect various dehydrated liposomes. GmPMI6 was more effective in terms of membrane protection than other group IV LEA proteins. The occurrence of GmPM16 on both side of liposome had better effect on structural integrity of dehydrated vesicles than only GmPM16 existing externally. Three oligosaccharides of soybean seed, sucrose, stachyose and raffinose, were more effective than GmPM16 to maintain neutral liposome membrane integrity after desiccation and rehydration. The level of protection observed under the combinations of sugar and protein was close to that observed under only GmPM16 present. The combinations of stachyose and GmPM16 maintained the negative membrane as well as stachyose, and better than only GmPM16. The different combinations of three oligosaccharides and GmPM 16 were more effective than either only sugar or protein to maintain the positive liposome above 50 mg protective material / mg of phospholipid. The degree of depression of Tm is correlated with the protective effect of the neutral liposome under dehydration. However, Tm was not correlated with protective effect in the positive and negative liposomes. It may be because that the charge of protein and the vitrification of sugar are also important to maintain the integrity of vesicle under dehydration. In vitro study might help us to hypothesize the mechanism of these LEA proteins in membrane protection in vivo.