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

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

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。

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 MdSUT1 was 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 MdSUT 1 in apple fruit.