百合肌动蛋白基因lilyActin的克隆与表达分析

Cloning and Expression Analysis of Actin Gene(lilyActin)from Lily

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 this study,the objective is to provide a reference gene for gene expression studies of Lilium. Based on the actin gene EST sequence of the cDNA library of Lilium regale Wilson,a full-length cDNA sequence was cloned from Lilium regale Wilson through rapid amplification of cDNA ends（RACE） method and the gene expression characters were analyzed by the real time PCR. The full-length cDNA sequence（GenBank：JX826390）designated as lilyActin from Lilium regale Wilson was 1 367 bp in length, contains a 1 134 bp open reading frame（ORF）encoding a 377 amino acid proteins,with 91 bp in the 5′ UTR and 233 bp in the 3′ UTR. Homologous alignment shows that it shares over 80% nucleotide sequence similarity and over 98% amino acid sequence similarity with actins in other plants. The phylogenetic tree reconstructed on the base of amino acid sequences suggests that the relationship of actin between Lilium regale and Tulipa gesneriana is the most intimate. Real time PCR analysis revealed that lilyActin was constantly expressed in various organs of Lilium regale Wilson such as flowers,leaves,bulbs. The results showed that relative to other reference gene lilyActin can be more appropriate for Lilium.