烟草合子时期表达基因NtZE1的克隆及结构分析

doi:10.3969/j.issn.1009-7791.2019.02.001

亚热带植物科学 ›› 2019, Vol. 48 ›› Issue (02): 103-108.DOI: 10.3969/j.issn.1009-7791.2019.02.001

• 植物生理生化与分子生物学 • 下一篇

烟草合子时期表达基因NtZE1的克隆及结构分析

罗岸，左紫怡，焦雄，刘夏

(长江大学生命科学学院，湖北荆州 434023)

收稿日期:2019-03-18 修回日期:2019-03-28 出版日期:2019-06-27 发布日期:2019-06-27
通讯作者: 罗岸
作者简介:罗岸，博士，从事植物生殖发育研究。
基金资助:
长江大学大学生创新创业训练计划(2017071)

Cloning and Structural analysis of Zygote Expressed Gene NtZE1 in Nicotiana tabacum

LUO An, ZUO Zi-yi, JIAO Xiong, LIU Xia

(College of Life Science, Yangtze University, Jingzhou 434023, Hubei China)

Received:2019-03-18 Revised:2019-03-28 Online:2019-06-27 Published:2019-06-27
Contact: LUO An

摘要/Abstract

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

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

中图分类号:

Q943.2

罗岸，左紫怡，焦雄，刘夏. 烟草合子时期表达基因NtZE1的克隆及结构分析[J]. 亚热带植物科学, 2019, 48(02): 103-108.

LUO An, ZUO Zi-yi, JIAO Xiong, LIU Xia. Cloning and Structural analysis of Zygote Expressed Gene NtZE1 in Nicotiana tabacum[J]. Subtropical Plant Science, 2019, 48(02): 103-108.

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烟草合子时期表达基因NtZE1的克隆及结构分析

Cloning and Structural analysis of Zygote Expressed Gene NtZE1 in Nicotiana tabacum

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