1个兰花MADA-box基因的克隆与表达分析

Cloning and expression analysis of a MADS-box gene from orichid

为阐释兰科植物成花的分子调控机制,根据MADS-box基因保守序列设计简并引物,用RACE方法从蝴蝶兰花葶中克隆到1个MADS-box家族基因.序列和系统进化树分析表明,该基因与其他植物的MADS-box基因具有很高的同源性,属于AP1/FUL-like亚家族,命名为DtpsMADS2,GeneBank登录号为JQ065098.实时荧光定量PCR检测结果显示,DtpsMADS2在营养器官和生殖器官均有表达,表达量以盛花期花葶中最高;在根中,DtpsMADS2随着苗期、抽葶期和盛花期表达量逐渐降低,而花后期又升高;在叶中,DtpsMADS2在苗期表达量较高,随后在抽葶期下降,又随着盛花期和花后期表达量升高,DtpsMADS2在花葶中以抽葶期表达量最低,盛花期大幅度升高,花后期有所下降;而在花器官各轮结构中,DtpsMADS2只有少量表达.由此推断,DtpsMADS2的表达与开花进程呈正相关,主要参与开花调控,而对花器官的发育没有明显的决定和调控作用.

To elucidate the molecular mechanisms of floral development in the monocotyledonous species Phalaenopsis hybrid, we isolated eDNA of a MADS-box genes from this plant by the method of 3 ＇ and 5＇ rapid amplification of eDNA ends using degenerate primers designed according to the MADS- box protein family conserved region from scape at flowering stage. Sequence and phylogenetie analysis indicated a high degree of predicted protein sequence identity with those from other species. The gene belongs to the class of AP1/FUL-like group of MADS-box genes and was termed DtpsMADS2 （accession No. JQ065098）. Real-time quantitative PCR （qRT-PCR） results showed that DtpsMADS2 was expressed in both vegetative and reproductive organs, and the highest expression was observed in scape at full-blossom stage; In root, the expression of DtpsMADS2 decreased gradually along with seedling stage, scape forming stage and full-stage, and increased at post-flowering stage; In leaf, DtpsMADS2 had higher level of expression at seedling stage and the level decreased at scape forming stage, and then increased with full-blossom stage and post-flowering stage; In scape, DtpsMADS2 was expressed in the lowest level at scape forming stage, and increased greatly at full-blossom stage, then decreased at post-flowering stage ; In each wheel of flower organs, the expression of DtpsMADS2 showed the lower level. The results suggested that the expression of DtpsMADS2 was positive to flowering process, Dtps- MADS2 played a role mainly in regulating the flowering process, and played no significant decisive and regulatory role in floral morphogenesis.