目的 克隆地黄多药及有毒化合物外排（multidrug and toxic compound extrusion，MATE）转运蛋白基因RgMATE6，并进行亚细胞定位与时空表达分析，为深入研究其在地黄转运次生代谢产物中的分子功能奠定基础。方法 利用地黄转录组数据库候选基因序列，利用实时荧光PCR （RT-PCR）克隆RgMATE6基因；利用生物信息学软件对其编码蛋白的结构、理化性质、同源性和进化树进行分析；通过与绿色荧光蛋白（GFP）融合表达进行亚细胞定位；利用实时荧光定量PCR （quantitative real-time PCR，qRT-PCR）的方法测定该基因的时空表达模式。结果 克隆获得1892 bp的RgMATE6基因序列，RgMATE6基因编码524个氨基酸、具有2个典型的MatE结构域和12个跨膜结构；RgMATE6蛋白与越橘的VcMATE6蛋白亲缘关系最近；RgMATE6-GFP载体瞬时表达的结果发现RgMATE6蛋白定位在液泡膜上；qRT-PCR分析发现RgMATE6基因在地黄不同时期的根中表达最高，尤其是块根膨大前期。结论 RgMATE6蛋白定位在液泡膜上，在地黄块根膨大期的根中表达较高，初步表明RgMATE6可能参与地黄体内次生代谢产物向液泡转运的过程。

Objective To clone a multidrug and toxic compound extrusion gene (RgMATE6) in Rehmannia glutinosa, determine its subcellular localization and analyze its spatio-temporal expression pattern for further exploring its molecular function of secondary metabolite transports. Methods RgMATE6 gene, which was obtained from R. glutinosa transcriptome data, was cloned by reverse transcription-polymerase chain reaction (RT-PCR) method. The structures, physicochemical properties, homology and phylogeny of RgMATE6 protein were analyzed by bioinformatic software. RgMATE6 was localized via the fusion expression system of its green fluorescence protein (GFP). The expression pattern of RgMATE6 gene was detected by quantitative real-time PCR (qRT-PCR) technology. Results The cloned RgMATE6 gene sequence was 1892 bp in length. It encoded 524 amino acids, and its protein had two typical MatE domains and 12 transmembrane structures. Homologous and phylogenetic analysis revealed that RgMATE6 had a closed homology with VcMATE6 from Vaccinium corymbosum. The transient expression of RgMATE6-GFP vector found that RgMATE6 localized in vacuolar membrane. The qRT-PCR analysis showed that the expression levels of RgMATE6 gene were higher in roots than in other tissues at various stages (especially tuberous expansion earlier stage).Conclusion The RgMATE6 protein localized in vacuolar membrane, and the gene expression was higher in R. glutinosa roots relative to stems and leaves at tuberous expansion earlier stages. The results preliminarily suggested that RgMATE6 may involve in the vacuolar transport process of secondary metabolite in R. glutinosa.

R286.12

国家自然科学基金资助项目（81973417）；河南省重点研发与推广专项（182102310606）；国家重点研发计划项目（2017YFC1700705）；河南工业大学科学研究基金项目（2017RCJH05）