摘要
本研究克隆了甘蓝型油菜线粒体功能未知基因ORF117,构建了带有线粒体转运信号肽序列(TP)的植物过表达载体35S∷TP-ORF117、35S∷TP-EGFP和35S∷TP-ORF117-EGFP,转化野生型拟南芥并进行抗除草剂筛选,共获得纯合的转基因拟南芥株系62个。qPCR表明,ORF117在转基因材料中得到高效过表达。用转35S∷TP-EGFP、35S∷TPORF117-EGFP拟南芥制备原生质体,经线粒体特异染料染色,在激光共聚焦显微镜下做线粒体、GFP共定位检测,GFP蛋白和ORF117-EGFP融合蛋白被准确定位到了线粒体。
In this study,the gene of ORF117(open reading frame of rapeseed mitochondria)was cloned.Three plant expression vectors 35S∷TP-ORF117,35S∷TP-EGFP and 35S∷TP-ORF117-EGFP were constructed and transformed into the wild type of Arabidopsis thaliana,and 62 transgenic lines were obtained.qPCR analysis testified that ORF117 was overexpressed in the transgenic plants.The protoplasts isolated from35S∷TP-EGFP,35S∷TPORF117-EGFPtransgenic plants were stained by mitochondrion-specific dye.Positioning observations of GFP and mitochondrion were carried out under the laser scanning confocal microscope.Results demonstrated that protein of GFP and ORF117-GFP were expressed accurately in the mitochondria.Our study laid a solid foundation to the functional research of ORF117,and explored a new way to study the unknown mitochondrion genes.
出处
《氨基酸和生物资源》
CAS
2015年第2期43-51,共9页
Amino Acids & Biotic Resources
基金
国家863计划重点项目(2013AA102602)
关键词
甘蓝型油菜
线粒体
转运肽
载体构建
Brassica napus L.
mitochondrion
transit peptide
vector construction
