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为了在百合功能基因表达研究中选择一个理想内参基因,依据岷江百合cDNA文库所获得的百合肌动蛋白(Actin)基因的EST序列,采用RACE技术进行该基因cDNA全长克隆,并利用实时荧光定量PCR分析其在不同组织中的表达模式,获得百合肌动蛋白基因cDNA全长序列(GenBank登录号:JX826390),命名为lilyActin。该基因cDNA全长1367bp,其中,5′非编码区91bp,3′非编码区233bp,开放读码框1134bp,编码377个氨基酸。序列比对发现,该基因与其它15种植物肌动蛋白核苷酸序列的相似性均在80%以上,氨基酸序列的相似性达98%。进化分析显示,百合肌动蛋白与郁金香肌动蛋白的亲缘关系最近。实时荧光定量PCR结果显示,该基因在百合的花蕾、叶片和鳞片组织中恒定表达,表明相对于其他物种的内参基因,lilyActin更适宜作为百合属植物的内参基因。
In order to select an ideal reference gene for lily functional gene expression study, full-length cDNA of Actin gene was obtained from the cDNA library of Minjiang River Lily by RACE technique. The full-length cDNA was cloned by real-time fluorescence quantitative PCR analysis of its expression patterns in different tissues to obtain lily actin gene cDNA full-length sequence (GenBank accession number: JX826390), named lilyActin. The full-length cDNA of this gene is 1367bp, of which 91bp in 5 ’non-coding region and 233bp in 3’ non-coding region. The open reading frame is 1134bp and encodes 377 amino acids. Sequence alignment showed that the similarity of this gene with other 15 plant actin nucleotide sequences was over 80% and the amino acid sequence similarity was 98%. Phylogenetic analysis showed that Lili actin is the most closely related to tulip actin. Real-time PCR results showed that the gene was expressed consistently in buds, leaves and scales of lily, indicating that lilyActin is more suitable as a reference gene for Lilium than other species.