基于EST数据库的葡萄APETALA2基因cDNA克隆及其表达分析

APETALA2(AP2)是拟南芥生长发育特别是花器官发育过程中重要的调控基因。利用生物信息学方法以拟南芥APETALA2基因cDNA序列作为模板,对葡萄EST数据库进行同源检索筛选,电子克隆出葡萄AP2基因(Vv-AP2)cDNA序列。以香悦葡萄花cDNA为模板,根据电子克隆的葡萄AP2基因cDNA序列设计特异引物,分别利用RACE技术和特异PCR技术获得该基因3’末端和5’末端,序列拼接后获得葡萄的APETALA2基因的cDNA全长。该cDNA的全长为2 208 bp,命名为Vv-AP2。Vv-AP2核苷酸序列有一个1 536 bp完整的开放读码框(ORF),其5’与3’末端非翻译区分别为268 bp和376 bp。其中,3’末端含有28 bp的Ploy+(A)。该基因在GenBank基因数据库的注册号为FJ809943。氨基酸推导结果表明该cDNA共编码了511个氨基酸,具备完全保守的核定位信号序列(KKSR)以及2个高度保守的重复序列即AP2结构域。分别采用半定量RT-PCR和SYBR Green I实时定量RT-PCR初步分析了Vv-AP2在葡萄叶、茎、花序、花等不同器官中的表达水平。其中,Vv-AP2在花序和花中的表达量明显高于叶和茎。

Cloning and expression analysis of APETALA2 gene from grapevine (Vitis vinifera)based on EST database

APETALA2(AP2)plays an important role in Arabidopsis development,especially in the development of flower or-gans.Based on the relative conservation of plant homologous genes,a full-length Vitis vinifera homologue of AP2(Vv-AP2) was bioinformatically cloned in this study.Accordingly,the 5’-and 3’-end sequences were obtained from cDNA of flower of Xiang yue grapevine cultivar by 3’RACE and specific PCR with two gene-specific primers designed against the Vv-AP2 se-quence,based on the full length cDNA of Vv-AP2(2 208 bp) which was spliced.This Vv-AP2 cDNA included an open reading frame(ORF)of 1 536 nucleotides,5’-untranslated region(UTR) of 268 bp,and 3'-UTR of 376 bp.The 3’-UTR contains a Poly +(A) of 28 bp.The sequence has been deposited in GenBank database with the accession number of FJ809943.The deduced amino acid sequence of Vv-AP2 includes 551 amino acids,contains a putative nuclear localization signal sequence(KKSR) and two highly conserved AP2 domains.The semi-quantitative RT-PCR and SYBR Green I Real-time qRT-PCR were employed to analyze the expression of Vv-AP2 in different organs of grapevine and the result showed that Vv-AP2 is expressed in much higher level in inflorescence and flower than that in leaf and stem.