茅苍术AlWRKY2基因的克隆及表达分析

Cloning and expression analysis of Atractylodes lancea AlWRKY2 gene

WRKY转录因子对植物生长过程中的物质代谢以及应对各类胁迫起着重要作用。本研究根据茅苍术转录组数据库,克隆得到WRKY2基因序列,命名为AlWRKY2(GenBank登录号:OR343916)。利用软件对AlWRKY2基因进行生物信息学分析,显示该基因包含1个975 bp的开放阅读框,编码324个氨基酸,AlWRKY2蛋白理论等电点为6.62,理论分子质量为35426.90。蛋白质结构预测显示,AlWRKY2蛋白质二级结构主要由无规则卷曲构成。同源氨基酸序列分析表明茅苍术和软橡木树、杨梅、可可树、哥伦比亚锦葵等植物的WRKY2基因编码的氨基酸具有较高的同源性且都具有WRKY保守结构域。系统进化树分析表明茅苍术AlWRKY2氨基酸序列与菊科植物聚在同一分支,具有高度同源性;SDS-PAGE电泳结果显示AlWRKY2基因成功在大肠杆菌BL21(DE3)表达感受态中进行诱导表达,并在约50 kDa处表达出预期目的蛋白;含有AlWRKY2基因的重组菌在NaCl和甘露醇的胁迫条件下负调控菌落生长;利用qRT-PCR检测显示,在茅苍术中AlWRKY2的表达量在叶中最高,茎中最低,且在外源茉莉酸甲酯(MeJA)处理下提高了湖北茅苍术AlWRKY2基因表达量,并在24 h处表达量最高。亚细胞定位结果显示该基因位于细胞核中。本研究可为进一步研究茅苍术WRKY转录因子的功能提供参考。

WRKY transcription factor plays a important role in material metabolism and various types of stress response during plant growth.In this study,based on the transcriptome data of Atractylodes lancea,the WRKY2 gene sequence was cloned and was named AlWRKY2(GenBank accession number:OR343916).Bioinformatics analysis was performed by software,and the results revealed that AlWRKY2 open reading frame was 975 bp,contained 324 amino acids.The encoded protein’s theoretical isoelectric point was 6.62,and the molecular mass was 35426.90.The secondary structure of AlWRKY2 was predicted to be mainly composed of random coils.Homologous alignment revealed that the amino acids encod-ed by WRKY2 genes of A.lancea,Quercus suber,Morella rubra,Theobroma cacao,Herrania umbratica,shared high sequence identity and had a typical WRKYGQK domain.The phylogenetic analysis indicated that the AlWRKY2 amino acid sequence of A.lancea was clustered in the same branch with Asteraceae plants and had high homology.The results of SDS-PAGE electrophoresis showed that the AlWRKY2 gene was successfully induced and expressed in Escherichia coli BL21(DE3)competent cells.The expected target protein was successfully expressed as a soluble protein of about 50 kDa.The growth status of the recombinant strain containing AlWRKY2 gene under NaCl and mannitol stress was significantly lower than that of the control strain and may have a negative regulatory effect.In different tissues of A.lancea,the results of gene expression detected by qRT-PCR showed that the expression level of AlWRKY2 was the highest in leaves and the lowest in stems.The expression of AlWRKY2 gene in A.lancea was increased under exogenous methyl jasmonate(MeJA)treatment,and the expression level was the highest at 24 h.The nuclear localization analysis revealed that the AlWRKY2 gene resides within the nucleus.This study can provide a reference for further study on the functions of WRKY transcription factors in A.lancea.