番茄SlAGO7基因的克隆及表达分析

Cloning and Expression Analysis of SlAGO7 in Tomato

根据番茄基因组数据库中AGO7的EST序列信息,利用RACE和PCR技术从番茄花cDNA和叶基因组DNA中分别克隆SlAGO7的cDNA全长序列和基因组序列,用生物信息学方法对其基因序列特征进行分析;利用Real-time PCR分析SlAGO7在整个生长与发育过程中的时空表达模式。结果表明,SlAGO7的cNDA全长片段为3 238 bp(GenBank登录号:JX467714),含有3 003 bp的完整开放阅读框,编码1 000个氨基酸,共包含3个外显子和2个内含子。所编码的氨基酸序列含有两个高度保守的PAZ和PIWI结构域,具有典型的AGO类蛋白的结构特征;与已报道的AGO7蛋白序列有较高的同源性;SlAGO7基因属于AGO蛋白家族中ZIPPY/AGO7亚族。SlAGO7在营养组织叶中微弱表达,主要集中在根和茎表达;而在生殖器官花中强烈表达,表达丰度从花蕾到盛开的花呈递增趋势,但在果实不同发育时期中表达都非常微弱甚至不表达。由此推测,SlAGO7蛋白可能与番茄花器官的发育相关。这为进一步研究该基因通过ta-siRNA干扰途径来调控番茄花器官发育形成的分子机制奠定了基础。

Based on EST sequences from tomato genome database（SGN）,the full-length cDNA and genomic DNA sequence of SlAGO7 were isolated by using RACE and PCR.Bioinformatics methods were used to analyze the sequence characteristics of this gene and the Real-time PCR method was used to investigate the temporal and spatial expression pattern of SlAGO7 gene.These results showed that the cloned full-length cDNA of SlAGO7 was 3 238 bp in length（Genbank：JX467714）,containing a 3 003 bp open reading frame（ORF）which encoding 1 000 amino acids.In addition,the full length of genomic DNA contained three extrons and two introns.The deduced SlAGO7 protein with two conserved PAZ and PIWI domains had the typical characteristics of the AGO family.This deduced amino acid had a high identity with the known AGO7 protein.Phylogenetic analysis showed that SlAGO7 was classified into the ZIPPY/AGO7 subfamily of the AGO family.Real-time PCR analysis indicated that the expression of the SlAGO7 gene was detected in vegetative tissues such as roots and stems,but not in leaves.SlAGO7 gene was mainly expressed in flower organs（Buds and flowers）,while its mRNA was not detected in fruits.Therefore,SlAGO7 was probably involved in the development of flower organ in tomato.The results would provide a foundation for further dissection of the molecular and genetics mechanisms of SlAGO7 in the control of floral organ development by the ta-siRNA pathway.