Abstract: In this study, an EST of NtZE1 gene that was differently expressed in zygote and egg cell of Nicotiana tabacum was obtained from cDNA subtraction library. The EST contained a 315 bp CDS sequence (Coding sequence) which could be cloned by RT-PCR (reverse transcription polymerase chain reaction). Bioinformatics analysis indicated that NtZE1 gene encoded 1074 amino acids, and NtZE1 was hydrophilic and instable, probably located in cell extracellular space, and had a signal peptide. By using genome walking technique, 2578 bp 5’ franking sequence (promoter and 5’ UTR) before ATG start codon was obtained. The 5’ franking sequence was connected into the EGFP nuclear localization vector, which was then injected into the leaf epidermal cells of N. benthamiana by transient expression technology. As obvious green fluorescence could be observed in the epidermal cells, the gene expression activity of the 5’ franking sequence was certified. This study had established a foundation for future research on NtZE1 gene function in tobacco zygote development.

Key words: Nicotiana tabacum, zygote, cloning, bioinformatics analysis, gene expression activity detection

摘要： 通过分析烟草Nicotiana tabacum长形合子和卵细胞的cDNA差减文库获得一条合子时期表达的基因NtZE1的EST序列。分析显示，EST序列中含有315 bp CDS序列(Coding sequence)，通过RT-PCR(reverse transcription polymerase chain reaction)成功克隆到该序列。生物信息学分析显示，NtZE1基因编码105个氨基酸残基；NtZE1蛋白为不稳定的亲水性蛋白，具有信号肽；蛋白二级结构多为α-螺旋，软件预测该蛋白可能定位于细胞外。利用Genome walking技术获得ATG起始密码子前共2578 bp的5’侧翼序列(启动子和5’UTR)，将该序列连入EGFP核定位载体中并通过瞬时表达技术使其在小叶烟草N. benthamiana叶表皮细胞中表达，有明显的绿色荧光，证实所获得的5’侧翼序列具有启动基因表达的活性。

关键词: 烟草, 合子, 基因克隆, 生物信息学分析, 基因表达活性检测