利用生物信息学方法,以拟南芥TIR1和F-box cDNA序列为模板,对柑橘EST数据库进行同源检索,筛选出柑橘TIR1和F-box基因的cDNA序列,并以枳花cDNA为模板,根据以上cDNA序列设计5′末端和3′末端扩增的特异引物,利用5′RACE和3′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TIR1和F-box cDNA全长.分别命名Pt-TIR1和Pt-F-box,大小分别是2048,1695 bp,在GenBank的登录号分别是FJ502240和FJ502241,其分别编码569和468个氨基酸全长.生物信息学分析表明,Pt-TIR1和Pt-F-box的cDNA序列中分别有microRNA393和microRNA394的识别位点,其与其他植物的F-box一样有着高度保守的序列即F-box结构域.构建Pt-TIR1和Pt-F-box亚细胞定位载体35S-GW-GFP-FJ502237/FJ502238,基因枪转化洋葱表皮细胞,暗培养24 h后激光共聚焦显微镜下观察.亚细胞定位结果表明Pt-TIR1和Pt-F-box均定位于细胞核中.转录因子Pt-TIR1和Pt-F-box均具有核定位功能.%A bioinformatics strategy is applied to clone a full length cDNA of TIR1 and Pt-F-box gene from citurs by blasting search of EST database with homologous gene cDNA of Arabidopsis thaliana and identified. According to the cDNA of above sequence,the 5'-end and 3'-end sequence were obtained from cDNA library of opening flower of Poncirus trifoliata (L. ) Raf. Using two gene specific primers by 5'RACE and 3'RACE methods,respectively. The full length cDNA of TIR1 and F-box gene from Poncirus trifoliata was spliced based on 5'-end and 3'-end sequence. The complete cDNA,designated as Pt-TIR1 and Pt-F-box,were 2 048,1 695 bp,respectively. These sequences were deposited in GenBank database,accession numbers FJ502240 and FJ502241 ,with an open reading frame encoding 569 and 468 amino acids,respectively. T Bioinformatics analysis showed that the cDNA of Pt-TIRl and Pt-F-box have the recognition sites of microRNA393 and microRNA394. Pt-TIRl and Pt-F-box with other plant F-boxes have the same ami-no acid sequences that are highly conserved as the designed F-box domains. Recombinant plasmid 35S-GW-FJ502240/FJ502241-GFP was introduced into onion epidermal cells by the particle bombardment method with a PDS1000/He. Transformed cells were incubated for 24 h at 221 in the dark and green fluorescence was monitored under a laser scanning confocal microscope. Subcellular localization results showed that the Pt-TIRl and Pt-F-box were localized in the nucleus.
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