黄花菜生物钟基因HcLHY的克隆及时空表达分析

Cloning and expression pattern analysis of circadian clock gene HcLHY in Hemerocallis citrina cv.’DatongHuanghua’

为解析黄花菜LHY基因的开花时间生物学功能和调控机理,本试验以大同黄花(Hemerocallis citrina cv.‘DatongHuanghua’)为材料,通过转录组数据库检索和PCR技术克隆了黄花菜生物钟基因HcLHY,分析了该基因的生物信息学、系统发育和时空表达特性。结果表明:该基因cDNA全长共计1929 bp,编码642个氨基酸,蛋白相对分子量为69470.67 D,理论等电点(pI)为5.82。生物信息学预测其为亲水蛋白,不具有跨膜结构域;其为转录因子,定位于细胞核中,不具备信号肽,该基因所编码蛋白的N端具有典型的Myb DNAbinding保守结构域;系统发育分析显示,HcLHY蛋白与芦笋AoLHY(XP020245416.1)亲缘关系最近,同时发现不同植物的LHY蛋白保守结构域也有较高的相似性,在被子植物生物钟系统中的功能高度保守;时空表达特性分析HcLHY基因在开花前-12 h表达量最高,且在花瓣中表达量最高,显著高于叶片、雌蕊、雄蕊、花萼及根。此结果为以黄花菜为生物钟模式植物的分子生物学研究提供了借鉴,为进一步通过遗传和外源影响开花时间调控黄花菜采收提供理论依据。

To analyze the biological function and regulation mechanism of flowering time of LHY gene in Hemerocallis citrina,we used Hemerocallis citrina cv.‘DatongHuanghua’as the material,and the circadian clock gene HcLHY was cloned by transcriptome database search and PCR technology for analyzing the bioinformatics,phylogeny and spatiotemporal expression characteristics of this gene.The results show that the total length of the gene cDNA is 1929 bp,encoding 642 amino acids,the relative molecular weight of the protein is 69470.67 D,and the theoretical isoelectric point(pI)is 5.82.Bioinformatics predicts that it is a hydrophilic protein and does not have a transmembrane domain;it is a transcription factor that is localized in the nucleus and does not have a signal peptide;the N-terminus of the protein encoded by this gene has a typical Myb DNA-binding conserved domain.Phylogenetic analysis shows that HcLHY and AoLHY(XP020245416.1)are closely related.At the same time,it is found that the conserved domains of LHY proteins in different plants also have high similarities,and their functions are highly conserved in the angiosperm biological clock system.The expression of HcLHY gene is the highest at-12 h before flowering and the highest in petals,which is significantly higher than those of leaves,pistil,stamen,calyx and root.This article provides a reference for molecular biology research of daylily as a biological clock model plant,and also the theoretical basis for regulating the flowering time and harvest of daylily by genetic and exogenous influence.