草莓AP1同源基因的克隆、表达及启动子分析

Cloning,Expression and Promoter Analysis of AP1 Homologous Gene from Strawberry (Fragaria×ananassa)

【目的】从草莓(Fragaria×ananassa)中克隆APETALA1(AP1)同源基因,并分析其在不同组织、器官及不同花发育阶段的表达水平,探讨其在草莓植株成花进程中的作用。【方法】根据其它物种AP1同源基因的保守序列设计简并引物,以草莓幼叶和花芽为试材,克隆得到AP1的基因片段,在此基础上利用RACE的方法分离获得其cDNA全长。利用实时定量RT-PCR分析草莓不同组织、器官及不同花发育阶段中AP1同源基因的表达水平。利用染色体步移的方法分离启动子序列。【结果】从草莓品种‘花姬’中克隆出AP1同源基因的cDNA全长序列,命名为FaAP1;其CDS长度为735 bp,编码245个氨基酸,与玫瑰AP1-1的氨基酸序列同源性最高,达到92%,与拟南芥AtAP1的氨基酸序列同源性为64.00%。FaAP1编码的氨基酸全长序列符合MADS-box基因家族特征,包含MADS-box、I-间插域、K-box域和C-末端几个结构域,是MIKC类型的MADS-box基因家族的成员。实时定量RT-PCR结果表明,在不同组织、不同花器官及不同花发育阶段中FaAP1的表达量存在差异。其启动子除了具有TATA/CAAT-box外还包含一些特异作用元件。【结论】从草莓中分离出的FaAP1基因,在花分生组织形成和花器官发育中有一定的调控作用。

【Objective】The aim of this study was to isolate the AP1 homologous gene from strawberry（Fragaria×ananassa）,analyze its expression level in different tissues,organs and at different growth stages of flowers and elucidate the role of it in the floral development progress of strawberry【.Method】The fragment of an AP1 homologous gene was amplified from young leaves and flower buds of strawberry with the degenerate primers designed according to the conserved sequences of AP1 homologous genes in the other species.The full-length cDNA sequence of this gene was obtained with RACE technique.Real time RT-PCR was used to analyze the expression patterns of this gene in different tissues,organs and at different growth stages of flowering of strawberry.A promoter sequence was isolated with chromosome walking methods.【Result】 An AP1 homologous gene in strawberry was cloned from cultivar ‘Huaji’ and it was designated as FaAP1.The CDS length of FaAP1 is 735 bp and encodes a predicted protein of 245 amino acid residues.Its amino acid sequence shows a highest homology with the AP1-1 of Rosa,up to 92%,and also has a homology of 64.00% when compared with the AtAP1 of Arabidopsis thaliana.FaAP1 is a MIKC type MADS-box gene for its amino acid sequence contained the typical domains of MADS-box,I-region,K-box and C-terminal.The results of real time RT-PCR showed that the expression patterns of FaAP1 changed in different tissues,organs and flowers of different growth stages.In addition to the TATA/CAAT-box,its promoter also contained some specific regulatory elements【.Conclusion】FaAP1,an AP1 homologous gene in strawberry,was isolated and it may play an important role in the progress of flowering in strawberry.