杨树胞外蛋白的提取分离及2-D电泳体系的建立

Extraction and 2-D Electrophoresis Analysis of Extracellular Proteins from NL895 Poplar Leaves and Stems

该研究以美洲黑杨杂种优良无性系NL895杨（Populus deltoides×Populus euramericana cv.）组培苗叶片和茎段为研究对象,对NL895杨叶片细胞壁蛋白（cell wall proteins,CWPs）和茎段质外体蛋白（apoplastic proteins,APPs）的提取、分离和双向电泳等技术进行了系统研究。结果表明：10g以上叶片、超声波破碎10min、CaCl2法提取的细胞壁蛋白效果较好,经G-6-PDH酶活性检测,提取的细胞壁蛋白胞质污染率较低;真空渗透法提取的茎段质外体蛋白胞质污染率较前者高,但在允许范围之内。TCA/丙酮沉淀法纯化提取的细胞壁蛋白、pH 4-7的24cm胶条、上样量为500μg的双向电泳体系,其蛋白电泳图谱中的斑点多而清晰,斑点数达550多个,是叶片细胞壁蛋白电泳分析较适合的体系;pH 3-10胶条对茎段质外体蛋白的电泳分离效果较好。该研究初步建立了杨树胞外蛋白的提取、分离及2-DE电泳体系,为木本植物胞外蛋白的研究奠定了基础。

Plant cell apoplast plays a major role in defensing abiotic stresses such as drought,salinity,and it is composed of cell wall matrix and extracellular spaces. The extraeellular proteins（ECPs） are one of main composition in apoplast. Poplar is the pattern of species in tree genomies and proteomies research. In this paper, the methods of extraction, purification and two-dimensional electrophoresis about ECPs were studied by use of leaf and stem segments as sample from NL895 poplar（Populus deltoides X Populus euramericana cv.,NL895）. The results were that the effect of cell wall proteins（CWPs） from leaf segments（~10 g） which was extracted for 10 min on ultrasonic processor was better than other methods. By measuring G- 6PDH enzyme activity,it was found the rate of cell cytoplasmic contamination was only 2.9% in the CWPs from leaf segments,and was 14. 1% in the apoplastic proteins from stem sections. The optimum system for ECPs analysis was leaf segments which purified by method of acetone/precipitation,CaC12,24 cm pH 4--7 IPG strips,and loading protein sample of 500 μg. More than 550 protein spots with clear background were observed in 2-D electrophoresisgel. This work preliminarily established the optimum system including extraction,separation, purification and 2-DE electrophoresis technology for poplar ECPs, and this protocol could be beneficial to develop high-level ECPs studies in poplar.