目的 从滇龙胆叶片中克隆单萜合成关键酶环烯醚萜氧化酶（GrIDO）基因及其启动子序列，并进行序列分析。方法 根据滇龙胆转录组GrIDO基因序列设计基因特异性引物，使用RT-PCR方法克隆GrIDO基因的开放阅读框（open reading frame，ORF）序列，基于在线软件对GrIDO基因序列进行生物信息学分析。同时，根据GrIDO基因ORF序列，设计特异性引物，采用PCR法对该基因启动子序列进行扩增，并进行序列分析。结果 GrIDO基因（GenBank登录号KP722034）ORF全长1 557 bp，编码518个氨基酸；GrIDO蛋白相对分子质量58 920，理论等电点8.40，属于细胞色素P450超家族成员，可能定位于叶绿体；无信号肽，为亲水不稳定蛋白，主要由α-螺旋（51.07%）和环（42.69%）构成；GrIDO蛋白与长春花CrIDO蛋白具有较高的相似性（85.83%），且亲缘关系较近。GrIDO基因启动子（GenBank登录号KT428570）长720 bp，具有TATA-box和CAAT-box，还具有参与脱落酸和茉莉酸甲酯应答的顺式作用元件、光应答元件和MYB结合位点。结论 GrIDO基因表达受多种因素调控。克隆了GrIDO基因ORF及其启动子，为GrIDO基因的功能验证奠定基础。

Objective To obtain the key enzyme gene involving in the monoterpenoid biosynthesis pathway, an iridoid oxidase gene (GrIDO) and its promoter were cloned from the leaves of Gentiana rigescens, and its bioinformatics analysis were also performed. Methods The gene specific primers were designed according to the GrIDO gene of transcriptome in G. rigescens. The open reading frame (ORF) of GrIDO gene was cloned by RT-PCR method. The bioinformation of GrIDO gene was analyzed by online softwares. Meanwhile, the gene specific primers were designed according to the cloned GrIDO gene, and the promoter of GrIDO gene was amplified by PCR method. Its sequence analysis was also performed. Results The length of GrIDO gene ORF (GenBank accession number KP722034) was 1 557 bp, which encoded a protein with 518 amino acids. Its relative molecular weight was 58 920 with the theoretical isoelectric point of 8.40. GrIDO was the member of cytochrome P450 superfamily and may localize in chloroplast. GrIDO was a hydrophilic stable protein without signal peptide and composed of mainly α-helix (51.07%) and loops (42.69%). GrIDO protein had a high similarity with CrIDO of Catharanthus roseus (85.83%) and their genetic relationship was close. The cloned GrIDO promoter had a length of 720 bp (GenBank accession number KT428570), which possessed cis-regulatory elements TATA-box and CAAT-box, and had cis-acting elements involved in the abscisic acid and MeJA responsiveness, part of a light responsive element and MYB transcription factor binding site. Conclusion The expression of GrIDO gene were regulated by multifactors. The GrIDO gene and its promoter were cloned from G. rigescens. This study will lay foundations for the functional research of GrIDO gene in monoterpenoid biosynthesis pathway.

云南省教育厅重点项目（2015Z171）；云南省大学生创新项目（201511390006）；云南省自然科学基金重点项目（2017FA049）