Studies on physiological roles of galactolipase in the chloroplasts and cloning of a cDNA encoding galactolipase from Phaseolus vulgaris L.

本研究旨在研究半乳糖脂酶在胁迫诱导的脂质代谢中的生理作用、半乳糖脂酶的免疫学特性，并尝试从菜豆叶片中分离半乳糖脂酶的cDNA。主要研究结果如下：1.从半乳糖脂到三酰甘油的代谢变化是叶绿体中半乳糖脂酶活性增强的结果。脂质的变化不仅发生在菠菜中，而且也发生在SO_2处理、水分胁迫和原生质体分离的叶片中.在菜豆和红花菜豆叶中至少有两种类型的半乳糖脂酶，膜结合型和可溶型，并已报道制备了抗这种酶的兔抗血清。该抗血清不仅完全抑制叶绿体组分中的半乳糖脂酶活性，而且也完全抑制可溶性半乳糖脂酶活性。可溶性蛋白和可溶性蛋白的Western blot均显示单一染色带，表明两个组分中均不存在免疫学上不同的同工酶.从菜豆幼叶中提取poly（A）^+ RNA，经oligo（dT）-cellulose柱纯化，构建了菜豆cDNA文库。用半乳糖脂酶的抗血清对文库进行早期筛选是不成功的。在解决了上述几个问题并成功地构建了文库后，我们继续分离半乳糖脂酶的cDNA克隆。

In this research project, we studied physiological roles of galactolipase in the stress-induced lipid metabolism, immunological properties of galactolipase, and in addition, we tried to isolate a cDNA encoding galactolipase from kidney bean leaves. The results obtained are as follows :1. The metabolic changes from galactolipids to triacylglycerols are results of the enhancement of galactolipase activity in chloroplasts. The lipid changes was shown to occur not only in spinach but also occurred in leaves subjected to SO_2 treatment, water deficiency, and protoplast isolation.2. At least two types of galactolipase, membrane-bound in chloroplasts and soluble, have been reported in kidney bean and runner bean leaves and prepared rabbit antiserum against this enzyme. The antiserum completely inhibited galactolipase activities not only in the chloroplast fractions but also in the soluble ones. Western blot of both solubilized chloroplast proteins and soluble proteins showed single stained band, suggesting the absence of immunologically different isozymes in both fractions.3. A cDNA library was constructed from poly (A) ^+ RNA which was isolated from younger leaves of kidney bean and purified by oligo (dT)-cellulose column. Early screening of the library using antiserum against galactolipase was unsuccessful. After the solution of the sevsral problems and the reconstruction of the library, we now continue to isolate a cDNA clone encoding galactolipase.