兜兰DEFICIENS(DEF)-和GLOBOSA(GLO)-like基因的克隆及表达分析

Cloning and Expression Analysis of DEFICIENS(DEF)-and GLOBOSA(GLO)-like Genes From Paphiopedilum

以‘同色兜兰’品种为材料,采用RT-PCR和RACE技术获得了DEFICIENS(DEF)-和GLOBOSA(GLO)-like基因的cDNA全长,命名为PcDEF和PcGLO,并用半定量RT-PCR和实时PCR研究了PcDEF和PcGLO在花芽发育过程和不同组织部位的表达特性。结果表明,PcDEF和PcGLO的全长cDNA分别为1 039bp和934bp,分别编码224和210个氨基酸;蛋白比对表明,PcDEF和PcGLO蛋白都具有典型MADS-box蛋白的MADS和K结构域;蛋白同源性分析显示,PcDEF和PcGLO与已登录的其它兰科植物的DEF/AP3和GLO/PI蛋白的相似性分别在75%～96%和87%～98%;系统进化树分析表明,PcDEF和PcGLO分别属于B类MADS-box蛋白家族的AP3和PI亚家族。表达分析显示,PcDEF和PcGLO在花芽发育中均有表达,PcDEF在成熟花、唇瓣和花瓣中的表达量高,在蕊柱、萼片、苞叶和根中次之,在花茎和叶中较低,在子房中几乎不表达;PcGLO在各组织中均有不同丰度的表达。

The full-length cDNA sequences of DEFICIENS（DEF） and GLOBOSA（GLO）-like genes were cloned from Paphiopedilum concolor Pfitz. by reverse transcriptase-polymerase chain reaction （RT-PCR） and rapid amplification of cDNA ends （RACE）. They were named PcDEF and PcGLO, respectively. Ex- pression transcript levels of PcDEF and PcGLO in different flower bud developmental stages and different tissues were analyzed by semi-quantitative RT-PCR and real-time PCR. The results revealed that the full- length cDNA sequences of PcDEF and PcGLO were 1 039 bp and 934 bp,respectively,and encoded 224 and 210 amino acids, respectively. Protein alignment analysis indicated that both PcDEF and PcGLO proteins had typical structuredomains of MADS-box protein, containing a MADS domain and a K domain. Protein sequence identity search showed that PcDEF shared 75% -96% similarities with other known DEF/APg- like MADS-box proteins of Orchidaceae and PcGLO shared 87% - 98% similarities with other known GLO/PI-like MADS-box proteins of Orchidaceae. Phylogenetic analysis clearly showed that PcDEF and Pc- GLO proteins were classed into AP3 and PI subgroups of B group of MADS-box proteins＇ family,respec-tively. Expression analyses revealed that PcDEF and PcGLO were detected in all six flower bud develop- mental stages,but showed different expression patterns in tissues examined. Compared with the transcripts of PcDEF in columns, sepals, bracts and roots, higher levels in mature flowers,lips and petals, lower levels in flower stems and leaves,and only low transcript levels were detected in ovaries. PcGLO was expressed in all tested tissues,including leaves, roots, flower stems, ovaries, bracts, sepals, petals, lips and columns, but at different abundance.