卷丹LlAGO1基因的克隆及表达分析

Cloning and Expression Analysis of LlAGO1 in Lilium lancifolium

以卷丹(Lilium lancifolium)叶腋组织为研究材料,利用RT-PCR和RACE技术克隆得到AGO1基因的c DNA全长,命名为Ll AGO1。其全长4 014 bp,开放阅读框长3 687 bp,编码1 228个氨基酸残基,其编码蛋白分子量为135.36 k D,理论等电点(p I)为9.57。氨基酸序列分析表明Ll AGO1含有PAZ和Piwi两个AGO1典型的结构域;信号肽预测结果表明Ll AGO1蛋白不存在信号肽,为非分泌蛋白;亚细胞定位预测其主要定位于细胞核;与相关同源蛋白高度相似,且与芦笋AGO1a蛋白(XP_020260210.1)亲缘关系最近。q RT-PCR分析结果表明:Ll AGO1在卷丹叶腋、鳞片、根、叶等不同组织均有表达,其中在叶腋中的表达量最高,叶片和根中的表达较弱;腋生珠芽形成过程中,Ll AGO1仅在可形成珠芽的上部叶腋表达,且在珠芽形成时表达量最高,而在不形成珠芽的下部叶腋几乎不表达,推测Ll AGO1可能与卷丹珠芽的形成相关。

In this study,AGO1 gene was isolated from the leaf axil of Lilium lancifolium by using reverse transcription PCR（RT-PCR） and rapid-amplification of c DNA ends（RACE）techniques,and was named as Ll AGO1. The full length c DNA of Ll AGO1 was 4 014 bp,which contained a 3 687 bp complete open reading frame（ORF）and encoded 1 228 amino acid residues with a predicted molecular weight of 135.36 k D,bioelectric point value of 9.57. Amino acid sequence analysis showed that it contains two conserved domains named PAZ and Piwi. Signal peptide prediction indicated that Ll AGO1 without signal peptide and was a secreted protein. Ll AGO1 was predicted to locate in the nuclear. In the phylogenetic tree,Ll AGO1 has the closest evolutionary relationship with the homologous protein from Asparagus officinalis（XP020260210.1）. The q RT-PCR analysis showed that Ll AGO1 expressed in most of the tested tissues,but mainly occurred in leaf axil,and the lowest in leaf and root. During the process of axillary bulbil formation,Ll AGO1 gene only expressed in the upper leaf axils which were able to generate bulbils and the expression was the highest at S3 stage,but didn＇t express in the lower leaf axils which could not form bulbils,which implied that this gene might play an important role in regulating the formation of axillary bulbils.