怀地黄3-酮酯酰CoA-硫解酶基因的克隆、序列特征和时空表达分析

Gene cloning, features of sequence, and analysis on temporal and spatial expression of Rehmannia glutinosa f. hueichingensis 3-ketoacyl CoA-thiolase

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 f. hueichingensis 3-ketoacyl CoA-thiolase （RghKAT） gene and to provide the candidate genes and theoretical basis for the molecular breeding of R. glutinosa f. hueichingensis. Methods The full-length cDNA sequence of RghKAT gene was amplified by quantitative RT-PCR and RACE techniques with degenerated primers being designed based on the conserved domain of ARGOS base sequences from other plants. The nucleotide and amino acid sequences were compared by bioinformatics technology. The temporal and spatial expression levels in 10 tissues of regenerated plantlets at two stages were detected by quantitative RT-PCR. Results The full-length of RghKAT gene was 1 713 bp, including an open reading frame （1 395 bp） encoding a 464-amino acid protein. Homology comparison and phylogenetic analysis revealed that RghKAT shared the high nucleotide sequence identity to those of Vitis vinefera （84%）, Solanum lycopersicum （82%）, Populus trichocarpa （82%）, Arabidopsis thaliana （79%）, and Triticum aestivum （73%）, respectively. Meanwhile, the amino acid sequence coded by RghKAT gene shared the high identity to those of Petunia x hybrida （88%）, V. vinefera （88%）, Cucumis satiuus （86%）, A. thaliana（87%）, and T. aestivum （78%）. The phylogenetic tree of RghKAT was consistent with the evolutionary relationship among the species. The physicochemical properties of RghKAT indicated that it was a slightly alkaline and stable protein, and the secondary structure was made of a-helixes, random coil, β-sheets, and B-turns. There was a putative signal peptide composed of 70 amino acid residues in the N-terminus, and the three-dimensional structure of RghKAT protein showed a typical characteristic sequence of thiolases. The quantitative RT-PCR assay demonstrated that the RghKAT mRNA could be detected in all 10 tissues examined at seedling and blooming stages with the highest expression level in the petals at blooming stage and the lowest one in the young leaves at seedling stage. Conclusion The full-length eDNA of RghKAT is cloned successfully, with thestructral properties ofKAT gene and the typical characteristic sequence of product thiolases, which has the highest expression level in the petals of blooming stage.