Department of Fruit Crops, University of Florida, Gainesville, Florida 32611.
Plant Physiol. 1991 Apr;95(4):1259-64. doi: 10.1104/pp.95.4.1259.
A method is presented for preservation of isolated intact chloroplasts and isolated thylakoids for use in chloroplast protein import and thylakoid protein integration studies. Chloroplasts of pea (Pisum sativum) were preserved by storage in liquid nitrogen in the presence of a cryoprotective agent. Dimethyl sulfoxide was the most effective of several cryoprotectants examined. Approximately 65 to 70% of chloroplasts stored in liquid nitrogen in the presence of dimethyl sulfoxide remained intact upon thawing and were fully functional for the import of precursor proteins. Imported proteins were correctly localized within these chloroplasts, a process that for two of the proteins tested involved transport into the thylakoids. Lysate obtained from preserved chloroplasts was functional for protein integration assays. Preserved chloroplasts retained import and localization capability for up to 6 months of storage. Thylakoids were preserved by a modification of a method previously described (Farkas DL, Malkin S [1979] Plant Physiol 64: 942-947) for preservation of photosynthetic competence. Preserved thylakoids were nearly as active for protein integration studies as freshly prepared thylakoids. The ability to store chloroplasts and subfractions for extended periods will facilitate investigations of plastid protein biogenesis.
本文介绍了一种用于保存完整的叶绿体和类囊体的方法，可用于叶绿体蛋白导入和类囊体蛋白整合研究。通过在有冷冻保护剂的条件下保存在液氮中，豌豆（Pisum sativum）的叶绿体得以保存。几种冷冻保护剂中，二甲基亚砜的效果最佳。在二甲基亚砜存在的条件下保存在液氮中的叶绿体解冻后约有 65%至 70%保持完整，完全能够用于前体蛋白的导入。导入的蛋白在这些叶绿体中正确定位，其中两种蛋白的测试结果表明，这些蛋白参与了向类囊体的运输。从保存的叶绿体中提取的裂解物可用于蛋白整合测定。保存的叶绿体在长达 6 个月的储存期内保持导入和定位能力。类囊体的保存方法是对先前描述的一种方法（Farkas DL，Malkin S [1979] Plant Physiol 64：942-947）的改进，以保持光合作用能力。保存的类囊体在蛋白整合研究中的活性与新鲜制备的类囊体几乎相同。这种能够长期储存叶绿体和亚组分的能力将有助于研究质体蛋白生物发生。
