目的对怀地黄3-酮酯酰CoA-硫解酶(Rehmannia glutinosa f.hueichingensis 3-ketoacyl CoA-thiolase,RghKAT)cDNA全长基因进行克隆及分析,为怀地黄分子育种提供候选基因和理论依据。方法根据其他植物ARGOS基因序列的保守结构域设计简并引物,采用RT-PCR和RACE技术,获得RghKAT cDNA全长序列;通过生物信息学技术对其核苷酸序列和氨基酸序列进行比对;利用实时荧光定量PCR技术检测了其在2个时期、10个组织的表达。结果 RghKAT基因全长1 713 bp,包含了1 395 bp的开放阅读框(ORF),编码464个氨基酸;同源比对和系统进化分析表明,RghKAT的核苷酸序列与葡萄、番茄、毛果杨、拟南芥和小麦的KAT核苷酸序列同源性分别达84%、82%、82%、79%、73%;RghKAT编码的氨基酸序列与矮牵牛、葡萄、黄瓜、拟南芥和小麦的KAT氨基酸同源性分别为88%、88%、86%、87%、78%;各物种KAT酶进化树符合物种进化规律;理化性质表明该蛋白为略成碱性的稳定蛋白质,蛋白质二级结构主要由α-螺旋、不规则卷曲、β-折叠和β-转角构成;在N端存在一个由70个氨基酸残基组成的信号肽;RghKAT蛋白三维结构具有硫解酶典型的特征序列;表达谱分析表明,RghKATmRNA在各时期、各组织中均有表达,盛花期花瓣中表达最强,而在幼苗期叶中表达量最低。结论成功克隆了RghKAT cDNA全长序列,具有KAT基因的结构特性及其产物硫解酶典型的特征序列,其在盛花期花瓣中表达量最高。 Objective To clone and analyze the full-length cDNA of Rehmannia glutinosa CoA-thiolase (RghKAT) from Rehmannia glutinosa, and to provide a candidate gene and theoretical basis for the breeding of Rehmannia glutinosa. Methods Degenerate primers were designed based on the conserved domains of other ARGOS gene sequences. The full-length cDNA sequence of RghKAT was obtained by RT-PCR and RACE. The nucleotide and amino acid sequences of RghKAT gene were aligned by bioinformatics. Real-time fluorescence quantitative PCR was used to detect the expression of 10 tissues in two stages. RESULTS: The RghKAT gene was 1 713 bp in length and contained 1 395 bp of open reading frame (ORF) encoding 464 amino acids. Homologous alignment and phylogenetic analysis showed that the nucleotide sequence of RghKAT was closely related to that of grapes, tomatoes, Yang, Arabidopsis and wheat KAT nucleotide sequence homology 84%, 82%, 82%, 79%, 73%; RghKAT encoded amino acid sequence and petunia, grape, cucumber, Arabidopsis The KAT amino acid homology with wheat was 88%, 88%, 86%, 87% and 78%, respectively. The phylogenetic tree of KAT showed that the phylogenetic tree was consistent with species evolution. The physical and chemical properties showed that the protein was a slightly alkaline stable protein, The secondary structure of protein consists of α-helix, irregular curl, β-sheet and β-turn. There is a signal peptide consisting of 70 amino acid residues at the N-terminus. The three-dimensional structure of RghKAT protein has typical characteristics of thiolase Sequence analysis showed that RghKAT mRNA was expressed in all tissues and tissues at all stages, with the expression of RghKAT mRNA being the highest in the full-flowering petals and the lowest in the leaves of seedlings. Conclusion The full-length cDNA sequence of RghKAT has been cloned successfully. It has the structural characteristics of KAT gene and its typical characteristic sequence of thiolase. The expression of RghKAT gene is the highest in full-flowering petal.