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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。
According to the relatively conservative homologous genes among species, the cDNA sequence of APETALA2 gene was cloned by homologous search and screening based on the APETALA2 cDNA sequence of Arabidopsis thaliana using bioinformatics method. The cDNA sequence of APETALA2 gene was cloned, Rosa was used as a template to design specific primers based on the above cDNA sequence. The 5 ’and 3’ ends of the gene were obtained by RACE technique. The full length cDNA of APETALA2 was obtained after splicing. The full-length cDNA was 1980bp and named Pt-AP2. The Pt-AP2 nucleotide sequence has a 1539bp complete open reading frame (ORF). The 5’-start codon ATG starts at 290bp and the 3’-terminal untranslated region is 152bp, which contains 27bp of Ploy + (A) . This gene has been registered in the GenBank database with the registration number EU883665. The cDNA was deduced to encode 512 amino acids. The homology was 59.1%, 59.7% and 63.8% respectively in apple, petunia and Arabidopsis. Sequence analysis showed that Pt-AP2 has two highly conserved repeats, ie AP2 domain, in addition to the fully conserved nuclear localization signal sequence (KKSR). The expression levels of Pt-AP2 in different organs of Trifoliate orange, stem, root, flower and fruit were analyzed by semi-quantitative RT-PCR and SYBR GreenI real-time RT-PCR. Different levels of expression, flowers in the highest expression, the lowest expression in the fruit.