摘要： 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2 048,1 695 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。
关键词:
枳,
TIR1和F-box,
基因克隆,
亚细胞定位
Abstract: 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-TIR1 and Pt-F-box have the recognition sites of microRNA393 and microRNA394.Pt-TIR1 and Pt-F-box with other plant F-boxes have the same amino 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 22℃ in the dark and green fluorescence was monitored under a laser scanning confocal microscope.Subcellular localization results showed that the Pt-TIR1 and Pt-F-box were localized in the nucleus.
Key words:
Poncirus trifoliata,
TIR1 and F-box,
Cloning,
Subcellular localization
中图分类号:
张演义, 王化坤, 宋长年, 孙欣, 王西成. 枳2个F-box基因cDNA全长的克隆及其亚细胞定位分析[J]. 华北农学报, 2012, 27(5): 1-7. doi: 10.3969/j.issn.1000-7091.2012.05.001.
ZHANG Yan-yi, WANG Hua-kun, SONG Chang-nian, SUN Xin, WANG Xi-cheng. Cloning and Subcellular Localization Analysis of Two F-box Genes from Poncirus trifoliata[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2012, 27(5): 1-7. doi: 10.3969/j.issn.1000-7091.2012.05.001.