铁皮石斛泛素结合酶DoUBC9的克隆鉴定与表达分析

目的:本研究旨在探讨泛素结合酶UBC9在铁皮石斛原球茎发育过程中的分子机制,方法:利用生物信息学手段,从已获得的铁皮石斛基因组中鉴定出UBC9基因,命名为DoUBC9,并利用RACE技术,克隆出DoUBC9 3’端,通过与基因组序列拼接,获得完整序列。结果:本研究首次从铁皮石斛基因组中运用生物信息学的方法,鉴定出DoUBC9,并通过RACE技术克隆后拼接,得到完整的DoUBC9,基因全长为7176bp,CDS长为447bp,共编码148个氨基酸。亚细胞定位预测结果表明,DoUBC9大概率分布于分泌囊泡和质膜上。序列比对和进化树分析表明,铁皮石斛Do UBC9和其他物种中的UBC9拥有高度保守的UBCc结构域,进化关系高度保守。qRT-PCR分析表明,在不同组织部位中,DoUBC9在愈伤组织中的表达量最高、叶中表达量最小;在原球茎发育过程中,在P1时期具有最高表达量,其他各时期表达量较小,表明该基因可能促进了植物体的细胞快速分裂与生长。结论:通过分子建模方式,首次构建出DoUBC9蛋白的基本模型,并通过不同的评价方式确定了以人泛素结合酶E2 H结构(2Z5D)为模板的为最优模型。

Clonal Identification and Expression Analysis of Ubiquitin-conjugating enzyme DoUBC9 in Dendrobium officinale Kimura et Migo

ABSTRACT Objective: To study the molecular mechanism of the ubiquitin-conjugating enzyme UBC9 in the development of protocorms of officinale. Methods: UBC9 was identified from Dendrobium officinale Kimura et Migo genome by bioinformatics, named DoUBC9. The cloning and splicing of RACE was used to obtain the complete DoUBC9, and the 3'' end of DoUBC9 gene was cloned and then spliced with the genomic sequence to obtain the complete sequence. Results: The full length of the gene was 7,176 bp and the CDS was 447 bp, encoding a total of 148 amino acids. Subcellular localization prediction results show that DoUBC9 has a high probability of being distributed on secretory vesicles and plasma membrane. Sequence alignment and phylogenetic tree analysis indicated that UBC9 from officinale and other species had highly conserved UBCc domains, and the evolutionary relationship was highly conserved. qRT-PCR analysis showed that DoUBC9 had the highest expression in callus and the lowest expression in leaves in different tissues. During protocorm development, it had the highest expression in P1 and less expression in other periods, indicating that the gene may promote rapid division and growth of cells in plants. Conclusion: The basic model of DoUBC9 protein was constructed for the first time by molecular modeling. The optimal model of human ubiquitin-binding enzyme E2 H structure (2Z5D) was determined by different evaluation methods.