大叶藻(Zostera marina L.)PSⅠ和PSⅡ复合物的分离鉴定

ISOLATION AND IDENTIFICATION OF PSⅠAND PSⅡ COMPLEXES FROM EELGRASS ZOSTERA MARINA L.

采用蔗糖密度梯度离心法分离纯化大叶藻类囊体膜 ,经 1 0 %SDS增溶后 ,用蔗糖密度梯度超速离心分离其色素蛋白质复合物。经稳态光谱分析、DCIP光还原活性测定及P680 、P70 0 差示光谱检测结果表明 ,2 0 %蔗糖层的CP3和 40 %蔗糖层 (上 )的CP4为PSⅡ复合物 ,具有光化学活性 ;40 %蔗糖层 (下 )的CP5为PSⅠ复合物 ,其P70 0 特征吸收峰位于 695nm处。CP3和CP4的DCIP光还原活性 :CP3为 34.2 7微电子当量 /(mgchl·h) ,CP4为 7.2 9微电子当量 /(mgchl·h)。

Eelgrass Zostera marina L. is one of a few higher plants that live in seawater at depth of 4—5 meter. It has complete root-stem-leaf structure that higher plants possess, and its whole life history (blooming, pollinating, fruiting etc.), takes place entirely in the sea. In this living environment, Z. marina L. has gained a number of special physiological characteristics, and plays an important role in marine ecology. In addition, it is also important for its place in evolution of photosynthetic organisms.It is widely believed that the most of higher plants that live on the land were originated in the ocean at the beginning of plant evolution. However, Z. marina L. and other seagrasses originated on the land. This suggested that, since photosynthetic organisms evolved from the sea to the land, some of them returned to the sea and gradually adapted to marine environment. The study of the photosynthetic characteristics of Z. marina L. is, therefore, of great interest. In this study, Z. marina L. sample was first disaggregated by supersonication (400W for 5 minutes) and isolated by sucrose density gradient centrifugation for 4 hours. Then two main bands located on the 60% and 50% of the sucrose densities were collected respectively. The absorption, fluorescence emission and excitation spectra of the two bands were exactly the same, indicating that the two bands were the thylakoid membrane although their sedimentation rates were different. After that, the isolated thylakoid membrane was solubilized in SDS solution, which contains 0.3mol/L Tris-HCl, 10% glycerol, 1% SDS, pH 8.0, for 10 minutes, and then the pigment-protein-complexes were isolated by sucrose density gradient centrifugation for 15 hours. After centrifugation, six bands clearly appeared at different sucrose densities. The complexes, except for the scraps of the thylakoid membrane between 60% and 50% of the sucrose densities, were named CP1, CP2, CP3, CP4, CP5 corresponding to the different sucrose densities: 10%, 15%, 20%, 40% (upper) and 40% (lower). The absorption, fluorescence emission and excitation spectra were determined in order to study the spectral characteristics of these pigment-protein-complexes. The DCIP photoreduction activity of each complex was measured to identify PSⅡ particles. The data showed that CP3 with the DCIP photoreduction activity of 34.27 μE/(mg chl·h) and CP4 with that of 7.29μE/(mg chl·h) were PSⅡcomplexes, which was further confirmed by P 680 differential spectrum. P 700 differential spectrum determination confirmed that CP5 has an absorption peak at wavelength 695nm, suggesting that CP5 was PSⅠcomplex. In comparison with the P 700 absorption of continental higher plants, there is a 5nm blue shift. All results indicated that PSⅡ with photoreduction activity and PSⅠcomplexes can be successfully isolated by sucrose density gradient centrifugation at 20% and 40% (lower) of sucrose densities, respectively.