目的 为了研究独行菜黄酮类化合物生物合成途径的关键基因，从独行菜中克隆了黄烷酮-3-羟化酶（flavanone-3-hydroxylase，F3H）基因，命名为LaF3H，并进行序列分析、原核表达和纯化。方法 根据独行菜转录组数据中LaF3H基因序列设计特异性引物，克隆LaF3H基因的cDNA序列，构建pET-32a-LaF3H原核表达载体并在大肠杆菌中诱导表达LaF3H重组蛋白。结果 LaF3H基因开放阅读框（ORF）长1 080 bp，编码359个氨基酸，其蛋白质相对分子质量为40 320。序列分析结果表明LaF3H具有F3H蛋白的5个保守基序，系统进化分析结果显示LaF3H蛋白与拟南芥等十字花科植物F3H蛋白同源性较高。通过构建原核表达载体pET-32a-LaF3H，在大肠杆菌BL21（DE3）菌株中成功表达LaF3H重组蛋白，利用Ni²⁺亲和色谱得到了纯化的LaF3H重组蛋白。结论 克隆了LaF3H基因，获得LaF3H纯化蛋白，为下一步制备LaF3H蛋白抗体、酶学活性检测，研究LaF3H基因在独行菜黄酮类化合物生物合成途径中的功能奠定基础。

Objective In order to study the key genes involved in flavonoid biosynthesis pathway, the flavanone-3-hydroxylase (F3H) gene was isolated from Lepidium apetalum, which is named as LaF3H. Meanwhile, the sequence analysis, prokaryotic expression, and purification were also performed. Methods Specific primers were designed according to LaF3H gene sequences in the transcriptome data of L. apetalum, and the cDNA sequence of LaF3H gene was isolated from L. apetalum. By construction the prokaryotic expression vector pET-32a-LaF3H, the recombinant LaF3H protein was expressed in Escherichia coli BL21 (DE3) cells under IPTG induction. Results The open reading frame (ORF) of LaF3H was 1 080 bp, which encoded a protein of 359 amino acid residues, with a predicted molecular mass of 40 320. Sequence analysis showed that LaF3H contains five conserved motifs of F3H protein. The phylogenetic analysis indicated that LaF3H protein showed the highest homology with F3H protein from cruciferous plants (such as AtF3H from Arabidopsis thaliana). The prokaryotic expression vector pET-32a-LaF3H was constructed and the recombinant LaF3H protein was successfully expressed in E. coli BL21 (DE3) cells. Furthermore, the recombinant LaF3H protein was purified through Ni²⁺ affinity chromatography. Conclusion The LaF3H gene was isolated from L. apetalum and the recombinant LaF3H protein was obtained. The results of this study provided the foundation for the further preparation of LaF3H antibody and detection of LaF3H enzyme activity, and were helpful for functional characterization of LaF3H gene involved in flavonoid biosynthesis pathway of L. apetalum.

国家重点基础研究发展计划"973"项目（2013CB531802）；中央引导地方科技发展专项（河南道地大宗药材种质评价及集约化种植与示范）；河南省科技攻关计划（162102310468）