蝴蝶兰花发育基因PhSTK的克隆及在突变体中的表达分析

Cloning of the floral organ identity gene PhSTK from Phalaenopsis and its expression analysis in floral organ mutants

采用RT-PCR及RACE技术从蝴蝶兰蕊柱中克隆了一个D类MADS-box基因PhSTK (GenBank登录号为MW574998),通过实时荧光定量PCR (qRT-PCR)方法分析了该基因在蝴蝶兰不同组织及突变体中的表达水平。结果表明,该基因cDNA全长为1 051 bp,具有705 bp的开放阅读框(ORF),可编码234个氨基酸,在其C端具有AG I、AG II和MD基序;系统进化树分析显示该基因编码蛋白与蝴蝶兰属的PeMADS7和PhalAG2蛋白亲缘关系最近;组织表达分析表明,PhSTK基因主要在蕊柱、子房和胚珠中表达,并随着子房发育其表达水平逐渐升高,在胚珠中表达水平最高。在不同突变体中,PhSTK基因在退化雄蕊瓣化突变体的蕊柱中表达水平显著降低,在其子房中的表达水平显著升高;在侧萼唇瓣化和侧瓣唇瓣化突变体的蕊柱和子房中,其表达水平均显著降低;在侧瓣雄化突变体中,该基因的表达水平在蕊柱和子房中均显著升高;在侧瓣退化突变体的子房中其表达水平显著降低。由以上结果可知,PhSTK基因主要调控蝴蝶兰胚珠的发育,并维持蕊柱和子房的形态建成。D类基因可能与其他MADS-box基因互作参与花器官形态的发育调控。

A Class D MADS-box gene PhSTK(GenBank accession number:MW574998) was cloned from the column of Phalaenopsis using RT-PCR and RACE technique,and the expression levels of Ph STK in different tissues and floral organ mutants of Phalaenopsis were analyzed by qRT-PCR.The results showed that the full-length cDNA of Ph STK was 1 051 bp and contained a 705 bp open reading frame encoded 234amino acids.Multiple protein sequence alignment revealed that PhSTK protein contained the AG I,AG II and MD motifs in C-terminal.The phylogenetic tree analysis indicated PhSTK protein was closely related to PeMADS7 and PhalAG2 proteins from Phalaenopsis.The tissue-specific expression analysis showed that Ph STK was mainly expressed in the column,ovary and ovule,and its expression level increased gradually during ovary maturation,with the highest in the ovule.The expression pattern of PhSTK of the floral homeotic mutants was further investigated.The expression level of PhSTK was reduced significantly in the column of the petalled-staminode mutant,while its expression level was increased significantly in the ovary.In both lip-like sepals and lip-like petals mutant,PhSTK expression was significantly decreased in the column and ovary.The expression of PhSTK was significantly higher in the column and ovary of the stamen-like petals mutant,and decreased significantly in the ovary of the petals degeneration mutant.These results suggest that Ph STK may play a role in the development of the ovule and the maintenance of the morphogenesis of column and ovary in Phalaenopsis.The class D gene may exert its function through interacting with other MADS genes in the regulation of floral organ development.