油茶转录因子基因CoSOC1-like的克隆和表达分析

Molecular Cloning and Expression Analysis of Transcription Factor Gene CoSOC1-like in Camellia oleifera

[目的]克隆油茶的SOC1同源基因(CoSOC1-like),分析其序列特征和表达模式及其蛋白进化。[方法]以3年生油茶嫩叶为材料提取RNA,利用RT-PCR和RACE方法克隆油茶的CoSOC1-like基因,用生物信息学工具分析其序列特征,用荧光定量PCR分析其表达模式,用基因瞬时表达法分析其蛋白的亚细胞定位。[结果]油茶CoSOC1-like基因的CDS全长为654 bp,推测蛋白质由127个氨基酸组成,蛋白分子量为24.958 kD,等电点(pI)为6.8,基因库的登录号为MT036382。CoSOC1-like具有植物Ⅱ型MADSbox基因的蛋白结构,且C末端含有MOTIF结构域,是一个MADS-box家族转录因子。CoSOC1-like蛋白有31个磷酸化位点,建立在二级结构基础上的CoSOC1-like蛋白的三级结构具有明显活性部位;CoSOC1-like基因瞬时表达分析表明,油茶CoSOC1-like蛋白定位于细胞核,符合转录因子的细胞核定位特征。系统进化分析表明:油茶CoSOC1-like与茶树SOC1-like蛋白聚类在同一个进化支上。荧光定量PCR分析表明:CoSOC1-like基因存在于油茶所有的器官中,尤其在花芽中的相对表达量最多。[结论]CoSOC1-like基因在油茶的花芽分化中起重要作用,也可能参与根、茎、叶和种子等器官的生长发育。本结果为进一步研究油茶成花的分子机制奠定了基础。

[Objective]To clone the homologous gene of SOC1 from Camellia oleifera(CoSOC1-like)and to analyze its sequence structure,expression pattern and protein evolution.[Method]Total RNA was extracted from the young leaves of three-years-old C.oleifera,and the CoSOC1-like gene was cloned by using RT-PCR technology and RACE(rapid amplification of cDNA ends)technology.The sequence and expression pattern of CoSOC1-like were analyzed using the bioinformatic tools and fluorescence quantitative PCR,respectively.The subcellular localization and evolution of CoSOC1-like protein were analyzed using the method of gene transient expression and MEGA7 software,respectively.[Result]The full length cDNA of CoSOC1-like contained 654 bases,encoding 217 amino acids,and the relative molecular weight was 24.958 kD and the isoelectric point was 6.8.The Genbank accession number of CoSOC1-like is MT036382.The CoSOC1-like protein had the structure of MADS-box family transcription factors of plant typeⅡ,and there was a SOC1 MOTIF in C-domain.The CoSOC1-like protein had 31 phosphorylative loci in its amino acid sequence,and its tertiary structure based on secondary structure had obvious active sites,and the transient expression of CoSOC1-like gene showed the CoSOC1-like protein located in nuclear,which was consistent with the nuclear localization characteristics of transcription factors.The phylogenetic analysis showed that the CoSOC1-like protein was clustered in the same evolutionary branch with SOC1-like protein of Camellia sinensis.Fluorescence quantitative PCR analysis showed that the CoSOC1-like gene could be detected in all organs,and there was a maximal relative expression level in flower buds of C.oleifera.[Conclusion]The CoSOC1-like gene may play an important role in the flower bud differentiation,and participate in the growth and development of other organs such as root,stem,leaf and seed of C.oleifera.