目的 克隆地黄Rehmannia glutinosa毛蕊花糖苷合酶基因（RgAcS1），分析其亚细胞定位和表达模式。方法 在地黄的转录组数据库中通过注释和比对，获得地黄RgAcS1的cDNA序列，利用聚合酶链式反应（PCR）方法进行分子克隆。构建绿色荧光蛋白（GFP）融合表达载体，以农杆菌瞬时表达法观测RgAcS1的亚细胞定位。利用实时荧光定量PCR（qRT-PCR）检测RgAcS1基因在地黄块根不同部位的表达模式。结果 获得地黄1个莽草酸-O-羟基肉桂酰基转移酶的全长编码序列，cDNA长度为1 659 bp，包含1个1 296 bp的开放阅读框，编码431个氨基酸残基，蛋白质相对分子质量为475 900，具有莽草酸-O-羟基肉桂酰基转移酶的典型结构域，命名为RgAcS1。亚细胞定位结果显示RgAcS1主要分布在细胞质中，在细胞核中也有分布。qRT-PCR分析表明，RgAcS1在地黄的周皮和根毛中表达量较高，在木质部和韧皮部表达量较低。RgAcS1基因在地黄品种北京1号、QH1和85-5中非菊花心中表达量均高于菊花心中的表达量，且差异达极显著水平。结论 获得地黄RgAcS1的cDNA序列，明确了RgAcS1的亚细胞定位和时空表达模式，为进一步研究RgAcS1基因在毛蕊花糖苷合成过程中的作用奠定基础。

Objective To clone the acteoside synthase gene (RgAcS1) from Rehmannia glutinosa, and analyze its subcellular localization and expression pattern. Methods The cDNA sequence of RgAcS1 was identified based on the annotation of the transcriptome data of R. glutinosa, and the RgAcS1 gene was cloned by polymerase chain reaction (PCR). Constructing the GFP fusion expression vector and observing the subcellular localization of RgAcS1 mediated by Agrobacterium tumefaciens. The expression pattern of RgAcS1 in different parts of tuberous root of R. glutinosa was detected by real-time fluorescence quantitative PCR (qRT-PCR). Results A full-length coding sequence of a shikimate-O-hydroxy cinnamoyl transferase from R. glutinosa was obtained and named RgAcS1. The length of the RgAcS1 cDNA was 1659 bp, including an open reading frame (ORF) of 1 296 bp, encoding 431 amino acid residues, the molecular weight of the protein was 475 900, and it has a typical domain of shikimic acid-O-hydroxy cinnamoyl transferase. The result of subcellular localization showed that RgAcS1 was mainly distributed in cytoplasm and also in nucleus. The qRT-PCR analysis showed that the expression levels of RgAcS1 were higher in the periderm and root hair of R. glutinosa tuberous root, but lower in the xylem and phloem. The expression levels of RgAcS1 were higher in non-radial striation than that in radial striation of BJ1, QH1 and 85-5. Conclusion In this study, we obtained the cDNA sequence of RgAcS1, and analyzed the subcellular location and expression patterns of RgAcS1, which will lay foundations for further study on roles of RgAcS1 gene in the synthesis of acteoside in R. glutinosa.

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国家自然科学基金资助项目（81872950）；国家自然科学基金资助项目（81473299）；国家重点研发计划项目（2017YFC1700705）