基于EST库的枳APETALA2基因cDNA克隆及其表达分析

 根据物种间同源基因相对保守的特点，利用生物信息学方法以拟南芥APETALA2 cDNA序列作为模板，对柑橘EST数据库进行同源检索筛选，克隆了柑橘APETALA2基因的cDNA序列，并以枳[Poncirus trifoliata (L.) Raf.]花cDNA为模板，根据以上cDNA序列设计特异引物，利用RACE技术分别获得该基因的5'和3'末端，序列拼接后获得枳的APETALA2 cDNA全长。该cDNA全长为1 980 bp，命名为Pt-AP2。Pt-AP2核苷酸序列有一个1 539 bp完整的开放读码框(ORF)，5'末端起始密码子ATG其始于290 bp，3'末端非翻译区为152 bp，其中含有27 bp的Ploy⁺(A)。该基因已在GenBank基因数据库注册，注册号为EU883665。推导该cDNA编码512个氨基酸，与苹果、矮牵牛和拟南芥中相应序列同源性分别为59.1%，59.7%和63.8%。序列分析表明, Pt-AP2除了具备完全保守的核定位信号序列（KKSR）外，还具有两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方法分析Pt-AP2在枳叶、茎、根、花和果等不同器官中的表达水平，结果一致表明Pt-AP2在各个器官中的表达水平不同, 花中的表达量最高，果实中的表达量最低。

Cloning and Expression Analysis of APETLA2 Gene from Poncirus trifoliata Based on EST Database

Based on the relative conservation of plant homologous genes, a full full-length citrus homologue of APETALA2 was bioinformatically cloned by search of citrus EST database via Arabidopsis thaliana corresponding sequence. Accordingly, the 5'- and 3'-end sequences were obtained from cDNA of opening flower of Poncirus trifoliata (L.) Raf. by RACE with two gene-specific primers designed on the basis of the citrus sequence. The 1,980 bp complete cDNA, designated as Pt-AP2, contained an open reading frame (ORF) of 1,539 nucleotides and 289 bp of 5' -untranslated region (UTR) and a 152 bp 3'-UTR. The sequence has been deposited in GenBank database with the accession number of EU883665. The deduced amino acid sequence of Pt-AP2 (512 residues) showed 59.1%, 59.7%, 63.8% identity with those of Malus domestica, Arabidopsis thaliana, Petunia hybrida, respectively. Pt-AP2 amino acid sequence contained 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 Pt-AP2 in different organs, revealing similar expression profiles in leave, stem, root, flower, fruit, in which the flower and fruit exhibited the highest and the lowest expression, respectively.