百子莲CONSTANS同源基因的克隆及表达分析

Molecular cloning and expression pattern analysis of a CONSTANS homolog from Agapanthus praecox ssp. orientalis

根据前期百子莲转录组测序分析的结果，获得了1个与光周期调控开花途径关键基因CONSTANS（CO）同源性较高的核心片段。采用cDNA末端快速扩增（RACE）方法得到了百子莲CO基因eDNA全长序列，命名为却COL，GenBank登录号为KF683287。序列分析表明，百子莲ApCOL基因eDNA全长1648bp，5’非编码区（5’UTR）和3’非编码区（3’UTR）分别为126和355bp，开放阅读框（ORF，127～1293bp）1167bp，编码388个氨基酸。ApCOL具有CO蛋白典型的结构域：氨基末端有2个B-box结构域，羧基末端有1个CCT保守结构域。氨基酸同源性比对发现，ApCOL与葡萄（XP_002274384．2）、可可（EOX99181．1）和大豆（NP001241023．1）等植物的CO蛋白有很高的相似性，同源性均在50％以上。系统进化树分析表明，ApCOL与小麦CO（EMS51329．1）聚类关系最近。实时荧光定量qRT-PCR结果表明：叶片中apCOL的表达量在花芽分化3个时期均高于花芽中的表达量，且叶片中的最高表达量和花芽中最低表达量均出现在花芽诱导期；在整个初花期内，各器官中却COL表达量由高到低依次是花梗、叶片、幼果、子房、花瓣、茎和花葶，花梗中表达量分别为叶片和茎中的2．68和114．3倍；不同光周期处理的叶片中ApCOL基因表达模式相近，均在暗处理时期呈现高表达。说明该基因的表达具有明显的时空差异性和生物钟调节特性，推测却COL在百子莲成花过程中以及花发育过程中发挥着一定的作用。

A core fragment highly homologous with the key gene of photoperiodic regulatory blossoming pathway, CONSTANS （CO） was obtained on the basis of preliminary Agapanthus praecox ssp. orientalis transcriptome sequencing results. The full length sequence of cDNA, the CO gene of A. praecox ssp. orientalis was gained by the RACE method, which was named as ApCOL with the GenBank accession number of KF683287. The results of sequence analysis showed that the full length of cDNA was 1 648 bp, which was composed of one open reading frame of 1 167 bp, 388 encoded amino acids, uncoded region （UTR） 5＇ and 3＇ with the length of 126 and 355 bp, respectively. The ApCOL had the typical domain of CO proteins： two B-box domains were boned at both of the hydroxy ends and a conserved domain CCT was located at the amino end. The results of homology comparison indicated that the ApCOL was highly homologous with the CO proteins of some plants such as Vitis vinifera （ XP_002274384.2 ） , Theobroma cacao （EOX99181. 1） and Glyeine max （NP_001241023.1）, with the homology of 50% or more. On the basis of phylogenetic analysis, it was demonstrated that ApCOL had the closest genetic relationship with clustering Triticum urartu （EMS51329.1）. The real-time quantitative PCR results suggested that the expression of ApCOL in leaves was obviously higher than that in blossom bud in the whole period of flower bud differentiation, and the highest and lowest expression of ApCOL in leaves and blossom bud were both happened in the period of flower bud induction ; in the beginning-flower stage, the expression of ApCOL in the various organs from high to low was pedicel, leaf, young fruit, ovary, petal, stem and scape, the ApCOL expression in the pedicel was 2.68 and 114.3 times of that in leaf and stem ; during different light cycle treatment, ApCOL gene expression patterns were similar, and were higly expressed during the dark treatment in A. praecox ssp. orientalis leaves. The results show that the ApCOL expression has obvious time and space difference and circadian regulation characteristics, and may play a crucial role in the processes of flowering and flower development.