oleosin-MT融合蛋白在拟南芥中的表达及鉴定  

Expression and identification of oleosin-MT fusion protein in Arabidopsis

作　　者：官丽莉[1,2] 陈昱[1,2] 朱栋[1,2] 韩怡来 崔琪[1,2] 李海燕[1,2] 李校堃[1] 姜潮[1] 

机构地区：[1]吉林农业大学生物反应器与药物开发教育部工程研究中心,吉林长春130118 [2]吉林农业大学生命科学学院,吉林长春130118

出　　处：《西北农林科技大学学报（自然科学版）》2015年第10期155-161,共7页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家高技术研究发展计划(863)项目(2011AA100606);国家自然科学基金项目(31201237;31101172);吉林省科技厅中青年领军人才及优秀创新团队项目(20111815);教育部博士点基金青年教师基金项目(20122223120002);吉林省科技型中小企业创新基金项目(13C26212201223)

摘　　要：【目的】获得转金属硫蛋白(MT)基因的拟南芥植株,为利用植物生物反应器规模化生产MT奠定基础。【方法】利用融合PCR方法,扩增拟南芥MT基因与油体蛋白oleosin基因的融合基因,将融合基因克隆至表达载体pOP上,构建重组质粒pOP-oleosin-MT。采用冻融法将质粒pOP-oleosin-MT转入根癌农杆菌EHA105中,通过农杆菌介导法转化拟南芥,用PCR检测并筛选转基因拟南芥阳性植株,采用SDS-PAGE法检测oleosin-MT融合蛋白的表达,用邻苯三酚自氧化法和结晶紫法测定融合蛋白的体外抗氧化活性。【结果】成功构建了植物油体特异表达载体pOP-oleosin-MT,融合蛋白oleosin-MT在拟南芥种子中成功表达,其分子质量为26ku;总蛋白质量浓度达到50μg/μL时,超氧阴离子的清除率最高,为58.5%;总蛋白质量浓度为10μg/μL时,羟自由基清除率可达69.7%。【结论】成功获得转MT基因的拟南芥株系,并证明融合蛋白oleosin-MT在拟南芥中具有良好的体外抗氧化活性。【Objective】This study obtained MTgene transgenic Arabidopsis thaliana plants to improve the plant bioreactor technology and reduce the cost of separation and purification of therapeutic proteins.【Method】Molecular biology method（PCR）was used to amplify MTand oleosinfusion gene.Then,oleosin-MT gene was cloned into the expression vector pOP to construct recombinant plasmid pOP-oleosin-MT and the freeze-thaw method was used to transfer plasmid pOP-oleosin-MT into Agrobacterium tumefaciens EHA105.It was transformed into Arabidopsisby Agrobacterium-mediated.Positive transgenic plants were determined by PCR analysis and SDS-PAGE.Antioxidant activity analysis of MT protein was also detected by pyrogallol autoxidation method and crystal violet method.【Result】Plant binary expression vector pOPOleosin-MT was successfully constructed.SDS-PAGE showed that the fusion protein expressed in Arabidopsis seeds with a molecular weight of 26 ku.In vitro antioxidant activity assay showed that the superox-ide anion free radical clearance rate was the highest of 58.5% when MT protein concentration was 50μg/μL.Total protein concentration was 10μg/μL and hydroxyl radical scavenging rate was 69.7%,indicating that transgenic Arabidopsis had high antioxidant activity.【Conclusion】Arabidopsis lines with MT gene were successfully obtained,and it was proved that recombinant protein had good antioxidant activity.

关 键 词：金属硫蛋白 拟南芥 油体蛋白 基因表达 

分 类 号：Q943.2[生物学—植物学]