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当前位置: 首页 > 文章 > 基于EST库的枳APETALA2基因cDNA克隆及其表达分析 园艺学报 2009,36 (6) 799-806
Position: Home > Articles > Cloning and Expression Analysis of APETALA2 Gene from Poncirus trifoliata Based on EST Database Acta Horticulturae Sinica 2009,36 (6) 799-806

基于EST库的枳APETALA2基因cDNA克隆及其表达分析

作  者：
宋长年;房经贵;王晨;上官凌飞;章镇
单  位：
南京农业大学园艺学院
关键词：
柑橘;枳;花器官发育;基因;表达分析
摘  要：
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[Ponci-rustri foliata(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1980bp,命名为Pt-AP2。Pt-AP2核苷酸序列有一个1539bp完整的开放读码框(ORF),5′末端起始密码子ATG其始于290bp,3′末端非翻译区为152bp,其中含有27bp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 GreenI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。
译  名：
Cloning and Expression Analysis of APETALA2 Gene from Poncirus trifoliata Based on EST Database
作  者：
SONG Chang-nian,FANG Jing-gui,WANG Chen,SHANGGUAN Ling-fei,and ZHANG Zhen(College of Horticulture,Nanjing Agricultural University,Nanjing 210095,China)
关键词：
Citrus; Poncirus trifoliata; flower; gene; expression
摘  要：
Based on the relative conservation of plant homologous genes,a 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 RT-PCR were employed to analyze the expression of Pt-AP2 in different organs,revealing similar expression profiles in leaf,stem,root,flower,fruit,in which the flower and fruit exhibited the highest and the lowest expression,respectively.

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