小球藻Rubisco在叶绿体中的免疫金标定位

应用免疫技术对Rubisco在中国小球藻(Chlorellaspp.640909)叶绿体中进行了分子定位及Native-PAGE电泳、SDS-PAGE电泳及其Westen印迹分析,并对小球藻淀粉核(Pyrenoid)超微结构进行了观察.结果显示Native-PAGE电泳图谱主要为一条主带,Westen印迹反应证明该条带即为Rubisco酶,SDS-PAGE电泳及其Western印迹图谱显示Rubisco大亚基分子量大约为55kD.中国小球藻淀粉核为椭圆形,被淀粉鞘所包围,中央有一条由2个类囊体组成的纵向通道,并在蛋白核内段处稍膨胀.淀粉核与叶绿体基质存在多处联系.免疫分子定位显示Rubisco大亚基和全酶分子主要分布于叶绿体的淀粉核上,且Rubisco在淀粉鞘部位也有少量分布,极少部分分布在叶绿体基质中,表明叶绿体淀粉核与光合作用关系密切.Rubisco聚集于淀粉核可能有利于藻类对CO2固定.

Imrmunogold localization of ribulose-1,5-bisphosphate carborylsae/oxygenase in chloroplasts of Chlorella

Ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco)is a first key enzyme in the Calvin Circle of plant cell photosynthesis. This paper mainly studied gold immunolocalizaion of Rubisco of Chlorella spp. 640909, and the Native-PAGE and,SDS-PAGE and Western blotting analysis, as well as the observation to pyrenoid ultra structure. The Native-PAGE result showed a main band, evidenced as the Rubisco band by the Western blot with the antibody against the Rubisco from C. prototothe-coides , The special immunoacton of Rubisco from Chlorella spp. 640909 and the antibody to large subunit of Rubisco from C. prothecoides showed the large subunit proteins of Rubisco in the two species of Chlorella shared the high homology. The SDS-PAGE and Western blotting maps showed the molecule weight of the large subunit of Rubisco of Chlorella spp. 640909 was about 55KD. The shape of pyrenoid ultra structure of the electronic microscope was oblong, and was embedded in starch sheath, with 2 swelling thylakoids through out a center portrait channel of the pyrenoid. There were some connections between pyrenoid and the chloroplast stroma. The distribution of the large subunits and the whole Rubisco in the chloroplast of Chrolella spp. 640909 was studied by immunoelectron microscopy by embedded sections with antibody to large subunit and whole enzyme followed by second antibody, goad anti-rabbit immunoglobulin G conjugated to 10nm gold particles(Sigma production). The result showed the antibodies against large subunit and whole enzyme heavily labeled the pyrenoid, as well as starch sheath region, whereas the thylakoid region of the plastid was lightly labeled. And the whole Rubisco antibody labeled the pyrenoid surface more heavily than the large subunit antibody did. It is demonstrated the pyrenoid and starch sheath have the photosynthesis function. Rubisco concentrating in pyrenoid and starch sheath is valuable to fix CO2 for photosynthesis in algae.