《植物生理学报》 2011, 47(3): 256-262

苹果蔗糖载体蛋白MdSUT1多克隆抗体的制备及MdSUT1蛋白表达特异性分析

彭昌操, 肖文芳, 李文燕, 赵小兰*

华南农业大学林学院, 广东省森林植物种质创新与利用重点实验室, 热带亚热带林业生物技术实验室, 广州510642

通信作者：赵小兰；E-mail: xiaolanpeng@scau.edu.cn；Tel: 020-85280259

摘　要：

高等植物光合同化产物蔗糖的质外体运输主要是靠蔗糖载体蛋白来完成的。MdSUT1是从苹果果实中克隆的蔗糖载 体家族基因, 本文将MdSUT1构建到酵母表达载体pMETαB中, 重组质粒转化毕赤酵母PMAD16经0.5%甲醇诱导后获得Md- SUT1表达。纯化的MdSUT1异源表达蛋白免疫Balb/C小鼠制备多克隆抗体, 抗体特异性分析结果显示该抗体对酵母表达 和苹果中的MdSUT1识别具有较高的特异性。免疫共沉淀实验结果也证明抗体能够应用于苹果果实中MdSUT1的功能分析。

关键词：苹果; 蔗糖载体; 多克隆抗体; 蛋白质互作

收稿：2010-10-15   修定：2011-01-27

资助：国家自然科学基金(30771759和30972388)、广东省自然科 学基金团队项目(9351064201000002)、广东省自然科学基 金重点项目(7118121)。

Polyclonal Antibody Preparing and Specific Expression Analyzing of Sucrose Transporter MdSUT1 in Apple

PENG Chang-Cao, XIAO Wen-Fang, LI Wen-Yan, ZHAO Xiao-Lan*

Bio-technology Laboratory of Tropical and Subtropical Forestry, Guangdong Key Laboratory for Innovative Development and Utilization of Forest Plant Germplasm, College of Forestry, South China Agricultural University, Guangzhou 510642, China

Corresponding author: ZHAO Xiao-Lan; E-mail: xiaolanpeng@scau.edu.cn; Tel: 020-85280259

Abstract:

Apoplast transport of sucrose is mainly mediated by sucrose transporters in high plants. MdSUT1 is a member of sucrose transporter (SUT) family isolated from apple fruit. In this study, the cDNA of MdSUT1was subcloned into pMETαB and the recombinant plasmid was used to transform the Pichia methanolica PMAD16. The fusion proteins induced by 0.5% methanol were used for standard immunization protocols in Balb/C mouse to prepare MdSUT1 polyclonal antibody. The affinity-purified antisera were evaluated by immunoblotting and shown to recognize MdSUT1 in vivo and in vitro specifically. Co-immunoprecipitation assay also confirmed that the prepared antisera were specific and reliable enough to be used to analyze the function of MdSUT1 in apple fruit.

Key words: apple (Malus domestica cv. Starkrimson); sucrose transporter; polyclonal antibody; protein-protein interaction