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[摘要]
目的 克隆珍稀濒危兰科药用铁皮石斛F-box蛋白基因即S期激酶相关蛋白(S phase kinase-associated protein)基因DoSKP2A,并进行生物信息学和表达特征分析。方法 采用RACE技术获基因全长;利用生物信息软件预测基因编码蛋白的理化性质、结构域和亚细胞定位等分子特性;用DNASTAR 7.0和MEGA 7.0分别进行氨基酸多序列比对和进化树构建;借助定量PCR检测基因表达模式。结果 克隆到DoSKP2A(GenBank注册号KU160472),cDNA全长1 507 bp,开放阅读框(ORF)长1 101 bp,编码蛋白含366个氨基酸,相对分子质量39 590,等电点7.9。DoSKP2A蛋白不含跨膜域和信号肽,包含F-box核心结构域(26~88)、亮氨酸重复序列LRR(202~226)和多个保守基序;DoSKP2A与植物SKP2As蛋白一致性为64.6%~72.4%,所在分支隶属于SKP2As分子进化的单子叶植物分支,与小兰屿蝴蝶兰SKP2A亲缘关系近;DoSKP2A基因转录本在石斛3种器官中为组成型表达,根中相对表达量较高,为叶中的6.16倍,茎中次之,叶中表达量很低。结论 获得一个全新的铁皮石斛F-box蛋白基因DoSKP2A全长、生物信息及其表达特征,为研究其在铁皮石斛生长发育、信号传导和抗逆境胁迫的分子机制提供基础。
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[Abstract]
Objective To clone an F-box protein namely S phase kinase-associated protein gene DoSKP2A with full length cDNA, in a rare endangered medicinal orchid species Dendrobium officinale, followed by bioinformatics analysis and expression pattern analysis. Methods RACE technology were used for gene identification. Characteristics of physiochemical properties, conserved domains and subcellular localization of the protein were determined using a series of bioinformatics tools. The analyses of multiple alignment and phylogenetic tree were performed using DNASTAR 7.0 and MEGA 7.0, respectively. Real time quantitative PCR was used for gene expression analysis. Results DoSKP2A gene was cloned (GenBank accession KU160472). The full length cDNA of DoSKP2A was 1 507 bp in length, and ORF was 1 101 bp, encoding a 366-aa protein with a molecular weight of 39 590 and an isoelectric point of 7.9. The deduced DoSKP2A protein, without transmembrane or signal peptide residues, contained an F-box core domain (26—88), a leucine-rich repeat (202—226), and multiple conserved motifs. DoSKP2A had high identities (64.6%—72.4%) with SKP2As proteins from various plants. DoSKP2A belonged to the monocotyledons subgroup of the SKP2As evolutionary tree. DoSKP2A gene was differentially expressed in the three included organs. The transcripts were more abundant in the roots, with 6.16 fold, then the stems and the lowest in the leaves. Conclusion The novel full-length F-box protein gene DoSKP2A was obtained, along with bioinformatics and expression characteristics, which provided molecular basis for the growth and development, signal transduction, and stress resistance of D. officinale.
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[基金项目]
陕西中医药大学“秦药”品质评价及资源开发创新团队项目;公益性行业(中医药)科研专项经费项目(201507002-1-08);陕西中医药大学新进博士科研启动经费(104080001);陕西省高校青年杰出人才支持计划项目;咸阳市中青年科技领军人才项目