张良波 王旋 钱成旭 刘志雄

摘 要： 為了探索甜荞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（Fagopyrum esculentum）长花柱和长雄蕊突变体（lpls）中克隆到1个长837 bp 的FeFUL2基因（GenBank登录号为MG779493.1），其包含长690 bp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：FUL motif和paleo AP1 motif。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4 d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。

关键词： 荞麦， 花发育， FRUITFULL， MADS-box， 基因表达

中图分类号： Q943.2

文献标识码： A

文章编号： 1000-3142（2021）04-0591-07

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.

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

甜荞（Fagopyrum esculentum）是蓼科（Polygonaceae）荞麦属食用和保健兼用的粮食作物，其籽粒含有丰富的赖氨酸、膳食纤维、芦丁和抗氧化活性物等，具有很高的营养和保健功效（Quinet et al.， 2004; Li et al.， 2017）;作为典型的二型花柱作物，甜荞自然群体中thrum型（短花柱长雄蕊）和pin型（长花柱短雄蕊）花植株按1∶1分离，自花或相同花型植株间授粉不亲和，仅异型花植株间相互授粉才能正常结实，产量低，不利于开展杂交育种工作，成为制约这一重要经济作物推广应用的瓶颈（Li et al.， 2017）。……