春兰AGAMOUS同源基因的克隆及表达分析

Cloning and Expression Analysis of AGAMOUS Homologous Genes from Cymbidium goeringii

为深入探索春兰(Cymbidium goeringii)中C类MADS-box基因参与春兰花器官发育调控的分子机理,用同源克隆的方法从春兰花芽中分离出两个C类MADS-box基因CygoAG1与CygoAG2。在分析其序列结构的基础上,分别检测2者在春兰不同花器官中的表达差异。序列结构分析表明:CygoAG1序列全长为831bp,包含702 bp完整开放阅读框(open reading frame, ORF),编码233个氨基酸和1个终止密码子;CygoAG2基因cDNA序列全长996 bp,包含705 bp完整开放阅读框,编码234个氨基酸和1个终止密码子,2个基因编码的转录因子均包含保守的MADS、K-box结构域和AG基序。分子系统发生与进化树重建结果显示:春兰CygoAG1与CygoAG2与拟南芥MADS-box基因家族中AG (AGAMOUS)基因的同源性最高。实时荧光定量分析结果表明,春兰CygoAG1基因只在花粉团、合蕊柱和子房中表达,在其它花器官及营养器官叶片中不表达,其在子房中的表达量最高。CygoAG2基因在春兰的花器官如萼片、花瓣、唇瓣、花粉团、合蕊柱和子房中均有表达,但在营养器官叶片中不表达,其在合蕊柱(雌雄蕊合生结构)中的表达量最高。CygoAG1和CygoAG2基因在春兰花器官中的表达模式呈现一定的差异。

In order to explore the molecular mechanism of the Cymbidium goeringii C-type MADS-box genes involving in floral development,two C-type MADS-box genes CygoAG1 and CygoAG2 were cloned from Cymbidium goeringii floral buds by homologous cloning.Sequence analysis showed that CygoAG1 was 831 bp in length,including 702 bp open reading frame(ORF),encoding 233 amino acids and one stop codon.The cDNA sequence of CygoAG2 gene has a total length of 996 bp,including 705 bp complete open reading frame,encoding 234 amino acids and one termination codon.The transcription factors encoded by the two contain conserved MADS,K-box domains and AG motifs.Protein sequence alignment and phylogenetic analyses showed that CygoAG1 and CygoAG2 display highest similar with the AG(AGAMOUS)MADS-box protein in Arabidopsis thaliana.The relative expression levels of CygoAG1 and CygoAG2 genes in young leaves,sepals,petals,labellum,anther,gynostemium and ovary of Cymbidium goeringii were detected by q PCR.It was found that CygoAG1 gene was only expressed in Cymbidium goeringii anther,gynostemium and ovary,but was not expressed in other floral organs and leaves.Moreover,the relative expression levels of CygoAG1 in the ovary was the highest,CygoAG2 genes in Cymbidium goeringii organs such as sepals,petals,labellum,pollen,gynostemiu and ovary were expressed,but not in the leaves,its gynostemium(female stamens connation structure)amount of expression in the highest.The expression patterns of CygoAG1 and CygoAG2 show obvious difference.