甘蓝型油菜线粒体功能未知基因 ORF 117的载体构建与转化

本研究克隆了甘蓝型油菜线粒体功能未知基因 ORF 117，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，ORF 117在转基因材料中得到高效过表达。用转35S ∷TP-EGFP 、35S ∷TP-ORF117-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。

Construction and Transformation of Plant Expression Vector Carrying an Unkown Gene (ORF117)from Mitochondria (Brassica napus L.)

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 from 35S ∷TP-EGFP ,35S ∷TP-ORF117-EGFP transgenic plants were stained by mitochondrion-specific dye.Positioning observations of GFP and mi-tochondrion 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.