铁皮石斛胚胎发生相关基因DoEMB8的克隆及表达分析

Cloning and expression analysis of embryogenesis-associated protein EMB8(DoEMB8) in Dendrobium officinale Kimura et Migo

采用RACE和巢式PCR技术克隆出铁皮石斛胚胎发生相关基因DoEMB8(Embryogenesis-associated protein EMB8)序列。序列分析表明该基因全长2077 bp,包含一个1824 bp的CDS,编码608个氨基酸残基。分子进化分析显示,DoEMB8与海枣(Phoenix dactylifera)、油棕(Elaeis guineensis)、小果野芭蕉(Musa acuminata subsp.malaccensis)等单子叶植物的亲缘关系较近。跨膜区分析显示DoEMB8蛋白含有两个跨膜螺旋结构,亚细胞定位显示该基因可能存在于线粒体。RT-q PCR分析表明,DoEMB8在原球茎不同发育时期表达量较为恒定,在PLB(Protocorm like body)中的表达量较原球茎不同时期要高,在组织表达分析中,种子中的表达量最高,较根中的表达量上升了3.16倍,表明该基因在胚胎发育时期有着持续稳定且较高的表达,可能对植物胚胎发育起着重要作用。

The embryogenesis-associated protein EMB8 （DoEMB8） in Dendrobium officinale Kimura et Migo was achieved by RACE and nest PCR technologies.The full length of DoEMB8 was 2077 bp, contained an open reading frame （ORF） of 1824 bp that encoded 608 amino acid residues.Phylogenetic analysis showed that DoEMB8 had the closest relationship with Phoenix dactylifera, Elaeis guineensis, Musa acuminata subsp.malaccensis and some other monocotyledon.DoEMB8 had 2 transmembrane helix structures and probably existed in mitochondrion analyzed by TMHMM and TargetP, respectively.RT-qPCR analysis showed that the expression level of DoEMB8 was relatively constant in different development stages of protocorm, while it was higher in PLB（Protocorm like body） compared to that in protocorm.In different tissues, the expression level of DoEMB8 was highest in seed and was 3.16 times higher than that in root, indicating that DoEMB8 had relatively constant and higher expression level in embryonic development and may play an important role in plant embryonic development.