铁皮石斛泛素结合酶基因DoUBC24的克隆及表达分析  

Cloning and Expression Analysis of Ubiquitin-Conjugating Enzyme 24 in Dendrobium officinale Kimura et Migo

作　　者：安红强[1] 范静[1] 梁易[1] 裴薇[1] 王万军[1] 

机构地区：[1]西南交通大学生命科学与工程学院,四川成都610031

出　　处：《生物技术通讯》2016年第5期643-648,共6页Letters in Biotechnology

基　　金：国家自然科学基金(31371232)

摘　　要：目的:克隆铁皮石斛泛素结合酶基因DoUBC24,预测其蛋白结构特征和进化关系,分析其时空差异表达情况,并推测其在植物生长发育中的功能。方法:提取铁皮石斛全苗总RNA,采用cDNA末端快速扩增(RACE)和巢式PCR技术克隆Do UBC24基因;用生物信息手段分析基因序列结构特征,并预测其编码蛋白及其理化性质和亚细胞定位;用MEGA6.0构建系统发育树,分析物种进化关系;用qRT-PCR检测DoUBC24在原球茎不同发育时期及不同组织中的相对表达量。结果:克隆得到基因全长为3811 bp,包含2880 bp的CDS,编码959个氨基酸残基,编码产物含有一个保守的UBCc结构域;分子进化分析显示,DoUBC24同海枣、油棕、小果野芭蕉、玉米、短花药野生稻、二穗短柄草和大麦等单子叶植物的亲缘关系较近;qRT-PCR分析显示,DoUBC24在铁皮石斛叶原基维管系统形成、原球茎退化时期表达显著下降,而在椭球形原球茎、根端分生组织和类原球茎中表达量较高,在组织中的表达量水平依次为花>茎>种子>根>叶。结论:铁皮石斛泛素结合酶DoUBC24基因在铁皮石斛原球茎不同发育时期及不同组织中均有显著的差异表达,可能对原球茎发育及植物组织的形态建成具有重要调控作用。Objective： To clone the ubiquitin-conjugating enzyme 24 gene（DoUBC24） in Dendrobium officinale Kimura et Migo and predict the structure and phylogeny of its protein, then infer its possible role in plant growthand development according to the spatio-temporal difference of relative expression level. Methods： DoUBC24 wasamplified by rapid-amplification of c DNA ends（RACE） and nest PCR technologies from total RNA, which was ex-tracted from the plantlets. The sequence structure, physicochemical property and subcellular localization were anal-ysed by bioinformatic tools. The phylogenetic tree of DoUBC24 was constructed by MEGA6.0 in order to analyzethe evolutionary relationship of species. Relative expression level of DoUBC24 in different development stages ofprotocorm and different tissues were tested by q RT-PCR. Results： The full length of DoUBC24 was 3811 bp, con-tained an open reading frame（ORF） of 2880 bp that encoded 959 amino acid residues, whose product contains aconserved UBCc domain. Phylogenetic analysis showed that the DoUBC24 has the closest relationship with monocot-yledons, such as Phoenix dactylifera, Elaeis guineensis, Musa acuminata subsp. Malaccensis, Zea mays, Oryza brachyantha, Brachypodium distachyon and Hordeum vulgare subsp. vulgare. q RT-PCR analysis showed that the rela-tive expression levels of DoUBC24 decreased significantly in leaf primordium and vascular system, degeneration ofprotocorm, while increased in spheroidicity protocorm, root apical meristem and protocorm-like body. In differenttissues, the rank of relative expression levels was flower〉stem〉seed〉root〉leaf. Conclusion： The relative expressionlevels of DoUBC24 were significantly varied in different developmental stages of protocorm and different tissues,which indicated that the DoUBC24 plays an important role in the development of protocorm and the morphogicalestablishment of tissues in plant.

关 键 词：铁皮石斛 泛素结合酶 CDNA末端快速扩增 QRT-PCR 

分 类 号：Q943.2[生物学—植物学]