转脂蛋白CaMBP10的胶体金标记及对白菜中CaMBP10结合蛋白的鉴定

Colloidal Gold Labeling of Lipid Transfer Protein CaMBP10 and Identification of its Binding Protein in Cabbage

植物转脂蛋白(LTP)是一类广泛存在于高等植物中的空间结构高度保守的碱性小分子蛋白,其确切功能和调节机制至今仍不清楚.本室从白菜中分离的钙调素结合蛋白10(CaMBP10),经序列分析被鉴定为植物转脂蛋白家族成员.近期研究结果表明,CaMBP10参与了植物的生物与非生物胁迫反应.为了深入探讨CaMBP10的抗性机制,确定植物中与其相互作用的蛋白质,本文拟建立胶体金标记CaMBP10的方法,通过凝胶覆盖分析,检测植物样品中的CaMBP10结合蛋白.为此,对标记反应的最适条件进行了优化,确定最佳条件为:交联剂戊二醛用量为0.034%,交联反应pH值为7.0,交联反应时间为40 min,胶体金颗粒度为10 nm,胶体金溶液的pH为7.0.本文确定建立了植物样品中CaMBP10结合蛋白的分析与鉴定方法.

Plant lipid transfer proteins （LTPs） are ubiquitously distributed throughout the plant kingdom. LTPs are a class of low molecular weight, basic proteins with sconserved three-dimensional structures. The biological functions of LTP and its in vivo regulation remained unclear. A CaM binding protein10 kD （CaMBP10） was isolated from Chinese cabbage and was identified as a member of plant LTPs family. It was found that CaMBP10 was involved in the plant responses of biotic and abiotic stresses. To identify the proteins interacted with CaMBP10 in plants, a CaMBP10 labeling method with colloidal gold was developed and used in gel overlay assay with cabbage extracts. The optimum reaction conditions for CaMBP10 labeling were screened by an orthogonal design and determined as in 0. 034% glutaraldehyde, pH 7. 0 for 30 rain with 10 nm colloidal gold particle, and pH 7. 0 for colloidal gold solution.