目的 从布渣叶中克隆黄酮碳苷合成途径关键酶黄烷酮-2-羟化酶（flavanone 2-hydroxylase，F2H）基因，构建原核表达载体，对其进行生物信息学和表达模式分析。方法 根据布渣叶转录组数据中注释为F2H的Unigene设计特异性引物，利用PCR法扩增MpF2H基因的开放阅读框（open reading frame，ORF），基于在线工具对cDNA序列进行生物信息学分析。同时，依据F2H序列，设计特异引物，采用RT-qPCR方法对其在芽、叶、枝、花、果等组织中的表达进行测定。结果 MpF2H基因ORF全长为1 557 bp（Genbank登录号KY652921），编码518个氨基酸；相对分子质量54 500，理论等电点5.49，含有信号肽，不含跨膜域，可能定位于叶绿体。同时，MpF2H基因在不同组织中均有表达，其中在叶中表达量最高，在枝和花中表达量最低。结论 MpF2H基因在不同组织中表达量差异较大。克隆了MpF2H基因cDNA，构建了其原核表达载体pET30a-MpF2H，为MpF2H基因的原核表达和功能验证奠定了基础。

Objective To obtain the key enzyme gene involved in flavone C-glycosides biosynthesis pathway, a flavanone 2-hydroxylase (F2H) gene was cloned from Microcos paniculata, and its bioinformatics analysis and gene expression pattern were also performed. Methods The specific primers were designed according to Unigene in F2H annotated in the transcriptome data of M. paniculata. The open reading frame (ORF) of MpF2H gene was amplified by PCR. Then the PCR product was purified and ligated to pET30a, and finally a prokaryotic expression vector pET30a-MpF2H was constructed. The bioinformation of F2H gene cDNA sequences was analyzed by some online tools. Using RT-qPCR with suitable primers, the quantitative expression analysis of MpF2H gene in different tissues, namely, buds, leaves, twigs, flowers and fruits was carried out. Results The length of MpF2H gene ORF was 1 557 bp (GenBank accession number KY652921), which encoded a protein with 518 amino acid residues, relative molecular weight of 54 500, theory isoelectric point of 5.49. In which was no transmembrane domain. It was hypothesized that this protein located in chloroplast. MpF2H gene was expressed in different tissues, with the highest expression in leaves and the lowest expression in twigs and flowers. Conclusion The expression of MpF2H gene varied widely in different tissues. The MpF2H gene was cloned from M. paniculata based on pET30a-MpF2H expression vector. This study will provide the fundamental information for the further preparation and functional research of MpF2H protein in flavone C-glycosides biosynthesis pathway.

国家自然科学基金资助项目（31300273）；广东省科技计划项目（2015A030302082）