刺五加Actin基因cDNA全长序列的克隆与生物信息学分析

Clone of Full-length cDNA Sequence of Actin Gene of Eleutherococcus Senticosus and Bioinformatics Analysis

目的:克隆刺五加Actin基因的全长cDNA序列,并对其进行生物信息学分析。方法:以刺五加的叶片为材料,提取总RNA,逆转录为cDNA,根据初步克隆到的刺五加Actin基因保守序列设计引物,利用套式PCR进行3'和5'RACE扩增,得到Actin基因的3'和5'端cDNA序列片段,拼接获得cDNA全长。将该序列进行BLAST比对、相似度分析,并对刺五加Actin1蛋白的二级结构和三级结构进行预测。结果:刺五加的Actin基因全长1 507 bp,命名为ESActin1,注册号KC469585。该基因开放阅读框(ORF)长1 134 bp,编码377个氨基酸,5'端非编码区长140 bp,3'端非编码区长233 bp。ESActin1与GenBank中注册的其它植物肌动蛋白核苷酸序列的相似性在75%以上,蛋白质序列的相似性在94%以上。结论:首次报道了刺五加Actin基因的全长cDNA序列,为刺五加的分子生物学研究奠定基础。

This study was aimed to clone the full-length cDNA sequence of actin gene of Eleutherococcus sentico- sus. And bioinformaties analysis was used. The total RNA was isolated from leaves of E. senticosus, and cDNA was synthesized by reverse transcription of total RNA. Primers were designed according to the conserved se- quence that had been cloned of Actin of E. senticosus. Then, the 3＇and 5＇ cDNA fragments were cloned by nested PCR. The full-length gene was obtained by gene splicing method. Sequencing results were compared and treated with similarity analysis by blast analysis in the GenBank. Protein secondary structure and tertiary struc- ture of Actin of E. senticosus was predicted by online software. The results showed that the full-length cDNA of Actin of E. senticosus is 1507 bp, which named EsActinl, GenBank accession No. KC469585. The conserved sequence, which contained a 1134 bp open reading frame that encoding a 377 amino acid residues, a 5＇-UTRof 140 bp and a 3＇-UTR of 233 bp. Homologous alignment showed that it shared over 75% nucleotide se- quence similarity and over 94% amino acid sequences similarity with Actins in other plants. It was concluded that this study first isolated and reported the full-length cDNA sequence of actin gene of E. senticosus, and laid a foundation for the molecular bioloav research of E. senticosus .