一个新的枳NAC基因cDNA全长的克隆及其亚细胞定位分析

Cloning and Subcellular Localization Analysis of A New Gene NAC cDNA in Poncirus trifoliata

以拟南芥的NAC1 cDNA序列作为模板,对柑橘EST数据库进行同源检索筛选,利用生物信息学方法克隆了柑橘NAC1基因的cDNA序列。以枳(Poncirus trifoliata)花的cDNA为模板,根据以上cDNA序列设计特异性引物,利用5'RACE和3'RACE技术,分别获得了NAC1基因的5'和3'末端,序列拼接后获得枳的NAC1 cDNA全长,命名为Pt-NAC1。Pt-NAC1全长为1351 bp,含有1个1047 bp完整的开放读码框(ORF),5'末端起始密码子ATG起始于25 bp,3'末端非翻译区为280 bp。该cDNA推导编码348个氨基酸,与苹果、拟南芥、杨树中相应序列的同源性分别为64.8%、57.0%、61.3%。生物信息学分析结果表明:Pt-NAC1 cDNA序列中有miRNA164的识别位点,还有高度保守的NAC结构域。构建Pt-NAC1亚细胞定位载体35S-GW-GFP-FJ619349,用基因枪转化洋葱表皮细胞,亚细胞定位结果表明:Pt-NAC1均定位于细胞膜中。

Taking the NAC1 cDNA sequence in Arabidopsis thaliana as the template,the homologous gene in the EST database of citrus was searched and screened,and the cDNA sequence of NAC1 gene in citrus was cloned by bioinformatics method.Taking the cDNA sequence in the flower of Poncirus trifoliata（L.） Raf.as the template,the specific primers were designed according to the above cDNA sequence,and the 5′-end and 3′-end sequences of NAC1 gene were obtained by using 5′RACE and 3′ RACE techniques,respectively.Based on these,the corresponding full length cDNA of NAC1 in Poncirus trifoliata was acquired through sequence splicing.This complete cDNA,designated as Pt-NAC1,was 1351 bp in length,it contained a 1047 bp whole open reading frame（ORF）,its 5′-end included a putative translation start codon（ATG） at the position of 25 bp,and the length of its 3′-end untranslated region was 280 bp.The deduced amino acid sequence of Pt-NAC1 was 348 residues,which showed 64.8%,57.0%,61.3% identical levels with that of Malus×domestica,Arabidopsis thaliana,Petunia×hybrida,respectively.Bioinformatics analysis showed that the cDNA of Pt-NAC1 had the recognition site of microRNA164,and it also had highly conserved NAC domains.Recombinant plasmid 35S-GW-GFP-FJ619349 was transferred into onion＇s epidermal cells by using the particle bombardment method,and the subcellular localization analysis results indicated that Pt-NAC1 was all localized at the cell membrane.