甜荞FeFUL2基因的克隆与表达分析

Cloning and expression analysis of FeFUL2 gene from buckwheat(Fagopyrum esculentum)

关键词:  荞麦,花发育,FRUITFULL,MADS-box,基因表达

Key words:  buckwheat, flower development, FRUITFULL, MADS-box, gene expression

摘要:

为了探索甜荞FUL同源基因参与花与籽粒发育调控的分子机制,该文采用同源克隆的方法从甜荞(Fagopyrumesculentum)长花柱和长雄蕊突变体(lpls)中克隆到1个长837bp的FeFUL2基因(GenBank登录号为MG779493.1),其包含长690bp的完整开放阅读框,编码1个由229个氨基酸残基组成的MADS-box转录因子。通过对FeFUL2进行分子系统发生、同源蛋白比对与转录因子结构分析,结果显示FeFUL2与核心真双子叶植物AP1/FUL亚家族转录因子中的euFUL进化系聚于1个进化分支,属甜荞euFUL型MADS-box转录因子,且包含1个57个氨基酸残基长的高度保守的MADS结构域、1个69个氨基酸残基长的次级保守的K结构域,其C末端转录激活区在序列长度和氨基酸残基组成上与其他euFUL型转录因子差异较大,但仍含有2个euFUL型转录因子特有的保守基元:FULmotif和paleoAP1motif。用qPCR检测基因表达的组织特异性显示:FeFUL2基因在甜荞lpls突变体的根、茎、叶、花被片、雄蕊、雌蕊和发育4d的幼果中均有表达,但其在花被片中表达量极显著高于该基因在其他器官中的表达量(LSD,P<0.01)。综合转录因子的结构与基因的表达模式推测,FeFUL2基因与其他euFUL型基因的功能可能存在一定差异,其在花发育过程中可能主要参与甜荞花被片的发育调控。

Abstract:

In order to uncover the molecular mechanisms of FRUITFULL(FUL)homologous genes involving in regulating flower and fruit development in buckwheat, an 837 bp of FeFUL2 cDNA containing a 690 bp full ORF(Open Reading Frame)encoding 229 amino acids(GenBank Accession Number MG779493.1)was isolated from a Fagopyrum esculentum mutant line with long pistil and long stamen(lpls)through homologous cloning. Moreover, the FeFUL2 cDNA contains a 30 bp 5'UTR(untranslated region, UTR)and a 117 bp 3'UTR including poly-A. The results showed that the buckwheat FeFUL2 was classified into the core eudicot euFUL lineages of AP1/FUL subfamily MADS-box transcription factors through phylogenetic, protein alignment and sequence analyses. In addition, FeFUL2 was classified transcription contained a highly conservation MADS-box domain(1-57)with 57 amino acids(aa), a secondary conserved K domain(91-159)with 69 aa, as well as two conserved motifs: FUL motif and paleo AP1 motif lying variable C terminal region. The highly conserved MADS domain was responsible for DNA binding, dimerization and nuclear localization of MADS-domain transcription factors. The secondary conserved K domain was involved in the formation of amphipathic helices and responsible for protein dimerization and multimeric complex formation protein-protein interactions. Finally, the C domain was important for transcriptional activation and multimeric complex formation. Moreover, the C terminal region of FeFUL2 was variable in sequence and length comparing with other euFUL-like transcriptors, which suggested that FeFUL2 may play different roles regulating flower and fruit development with FUL-like homologs from other species. qPCR revealed that FeFUL2 expression was detectable in all tissues including root, stem, leaf, tepal, stamen, gynoecium and 4-day-old juvenile fruit. However, FeFUL2 expression level in tepal was significantly higher than those in other organs(LSD, P<0.01). In addition, FeFUL2 expression level in stamen, gynoecium and 4-day-old juvenile fruit displayed no significant differences(LSD,P>0.05), but FeFUL2 expression level in stem and leaf was significantly higher than root(LSD,P<0.05). However, FeFUL2 expression level in stem and leaf showed no significant differences(LSD,P>0.05). Above all, our data suggest that the function of FeFUL2 may show a difference with other euFUL-like gene, and FeFUL2 play a major role involving in perianth development.