袁媛 刘予涵 李建昆 万超 张月 陈清西 伍炳华

摘  要：基于雙瓣茉莉[Jasminum sambac （L.） Aiton]的花、叶转录组，筛选了一个在花朵中高表达且与香气释放规律对应的P450基因进行克隆和表达模式分析。利用cDNA末端快速扩增（RACE）技术获得该基因的5?cDNA序列，与转录组中序列进行拼接并验证，显示该基因是一个含1545 bp的开放阅读框、编码514个氨基酸残基的蛋白。此P450蛋白具有特征性的亚铁血红素结合结构域和半胱氨酸残基，与油橄榄、小叶咖啡等其他植物CYP71A氨基酸序列同源性较高，将其命名为JsCYP71A。遗传进化分析显示，该蛋白与拟南芥P450家族中CYP82亚族CYP82G亲缘关系最近。CYP71A和CYP82G多个成员与萜类香气代谢相关，推测JsCYP71A蛋白有相似功能。JsCYP71A在茉莉花白花苞里的表达量最高，在茎中的表达量较低，在叶片中几乎不表达，茉莉花开放过程中，JsCYP71A在夜间11点和1点表达量高，白天表达量低，与萜类香气物质释放呈正相关。JsCYP71A定位于细胞质中。为获得异源表达蛋白，构建真核表达载体，在酵母BY4742株系中表达JsCYP71A，经Western Blot检测发现株系能成功表达大小约60 kDa的蛋白，为后续JsCYP71A酶学功能分析奠定基础。本研究结果为分析JsCYP71A在茉莉花萜类香气合成代谢中的作用提供参考。

关键词：茉莉花;P450;基因克隆;亚细胞定位;真核表达

中图分类号：S685.16      文献标识码：A

Cloning and Eukaryotic Expression of JsCYP71A from Jasminum sambac

YUAN Yuan， LIU Yuhan， LI Jiankun， WAN Chao， ZHANG Yue， CHEN Qingxi， WU Binghua

College of Horticulture， Fujian Agriculture and Forestry University， Fuzhou， Fujian 350002， China

Abstract： Based on the flower and leaf transcriptome of Jasminum sambac， a P450 gene with high expression in flowers and corresponding to the pattern of volatile fragrance release was screened for cloning and expression pattern analysis. The 5?cDNA sequence of this gene was obtained by RACE technology， which was aligned with the sequence in the transcriptome and verified by PCR. JsCYP71A contained an open reading frame at 1545 bp and encoded a protein with 514 amino acid residues. JsCYP71A had characteristic heme binding domains and cysteine residues. JsCYP71A had a high homology with the amino acid sequence of CYP71A in olive， coffee and other plants， therefore this gene was named as JsCYP71A. Phylogenetic analysis showed that JsCYP71A was closely related to CYP82G in the family of Arabidopsis thaliana P450. Multiple members of CYP71A and CYP82G subfamily were related to the aroma metabolism of terpenoids， so it is speculated that JsCYP71A has similar functions. The expression level of JsCYP71A was the highest in the white bud of jasmine， lower in the stem， and very low in the leaf. During the flowering process of jasmine， the expression level of JsCYP71A was high at 11 oclock and 1 oclock at night and low at daytime， which was positively correlated with the release of flower aroma. JsCYP71A was located in the cytoplasm of the cells. Eukaryotic expression vector of JsCYP71A was constructed and expressed in yeast BY4742 strain， and Western Blot showed that a protein about 60 kDa was successfully expressed. The result would lay a foundation for the subsequent enzymatic function analysis of JsCYP71A and for the future analysis of the role of JsCYP71A in the metabolism of terpenoids of jasmine.

Keywords： Jasminum sambac L.; P450; gene cloning; subcellular localization; eukaryotic expression

DOI： 10.3969/j.issn.1000-2561.2021.07.005

植物细胞色素P450是一种加单氧氧化酶，可切割进入底物活性位点并与血红素铁结合的氧分子的双键断裂，将其中的一个氧原子添加到底物的氧化位点，使底物发生氧化[1-2]。植物P450蛋白能结合和催化分子体……