木薯锌指转录因子MeDi19-1基因克隆及表达分析

Cloning and expression analysis of zinc-finger transcription factor MeDi19-1 gene in cassava

  • 摘要: 【目的】克隆木薯锌指转录因子基因MeDi19-1,分析其编码蛋白特征、亚细胞定位、转录激活活性及在木薯不同组织中的表达水平,为探究MeDi19-1基因在木薯中的作用机制提供理论参考。【方法】从木薯KU50扩增MeDi19-1基因编码区(CDS)序列,利用生物信息学软件对其进行预测分析,并构建pNC-Green-SubC-MeDi19-1融合表达载体,通过农杆菌介导转染烟草表皮细胞,观察荧光信号以确定蛋白的亚细胞定位情况。利用酵母系统确定其转录激活活性,并基于木薯不同组织转录组数据分析其组织表达特性。【结果】MeDi19-1基因CDS序列(OL620080)长度为618bp,共编码205个氨基酸残基,蛋白分子量为22416.79 Da,理论等电点(pI)为6.10,属于不稳定疏水蛋白,含有Di19蛋白家族典型的锌指结构域zf-Di19。MeDi19-1蛋白的二级结构中含有无规则卷曲(53.66%)、α-螺旋(40.49%)、延伸链(4.39%)和β-转角(1.46%)。MeDi19-1蛋白氨基酸序列与橡胶树(Hevea brasiliensis)Di19蛋白氨基酸序列(XP_021655585.1)相似性最高,为83.50%。MeDi19-1基因启动子元件含有脱落酸(ABA)、赤霉素和茉莉酸等激素响应元件及胁迫响应元件和光响应元件。MeDi19-1蛋白亚细胞定位于细胞膜和细胞核中,具有转录激活活性,且活性区域在C端。MeDi19-1基因在叶、叶中脉和储藏根中相对表达量较高。【结论】MeDi19-1基因属于Di19基因家族成员,具有组织表达特异性,主要在叶、叶中脉和储藏根中发挥调控作用,其编码蛋白在木薯组织中作为转录因子参与调节多项生理活动。

     

    Abstract: 【Objective】In this study,a zinc finger transcription factor MeDi19-1 was cloned from cassava. Its coding protein characteristics,subcellular localization,transcriptional activation activity and expression levels in different tissues of cassava were analyzed. This research laid a foundation for further exploration of the function and mechanism of MeDi19-1 in cassava.【Method】The coding region sequence(CDS)of MeDi19-1 gene was amplified from cDNA of cassava KU50.The bioinformatics analysis predicted the physicochemical properties.The pNC-Green-SubC-MeDi19-1 fusion expression vector was also constructed. By Agrobacterium-mediated transfection of tobacco epidermal cells,and the fluorescence signal was observed to determine the protein subcellular localization.The yeast system was used to determine its transcriptional activation activity,and its tissue expression properties were analyzed based on transcriptomic data from different tissues in cassava.【Result】The CDS of MeDi19-1 gene(OL620080)was 618 bp in length and encoded 205 amino acids with molecular weight of 22416.79 Da and theoretical isoelectric point(pI)of 6.10. MeDi19-1 protein belonged to an unstable hydrophobin and contained the zinc finger domainzf-Di19 which was typical in Di19 protein family. The se-condary structure of the MeDi19-1 protein contained irregularly coils(53.66%),α -helix(40.49%),extended chain (4.39%)and β-turn(1.46%). The amino acid sequence of MeDi19-1 protein had the highest similarity(83.50%)to the amino acid sequence of Hevea brasiliensis Di19-6 protein(XP_021655585.1). The promoter of MeDi19-1 contained the cis-acting element involved in the response to abscisic acid(ABA),gibberellin,jasmonic acid,stress response element and light response element. MeDi19-1 protein was located in nucleus and cytomembrane. MeDi19-1 protein had transcriptional activation activity,and its active region was at the C-terminus. MeDi19-1 showed high expression levels in leaf,midveins and storage root.【Conclusion】The MeDi19-1 gene is a member of the Di19 gene family,has tissue expression specificity and may play a regulatory role in leaves,mid-veins and storage roots,and its encoded protein is involved in the regulation of multiple physiological activities as transcription factors in cassava tissues.

     

/

返回文章
返回