摘要
根据北细辛全长转录组测序数据,选择在植物形态发生及应对逆境胁迫中发挥重要作用的WRKY家族基因,用在模式植物拟南芥中调控抗毒素camalexin合成的AtWRKY33比对同源基因,设计引物,以北细辛叶总RNA为模板,采用RT-PCR技术扩增得到AhWRKY33基因开放阅读框(ORF)片段,实时荧光定量PCR结果表明其在北细辛地上和地下部分有显著的表达差异,AhWRKY33在毒性成分马兜铃酸含量较根中高的叶片中有较高的表达量。经验证,北细辛WRKY33基因ORF全长1 686 bp,编码561个氨基酸。北细辛WRKY33有2个保守的WRKYGQK结构域,按经典的分类方法来说,属于Ⅰ组WRKY转录因子,与其他物种氨基酸具有一定的同源性。该研究成功构建植物真核表达载体PHG-AhWRKY33并转化拟南芥,经过PCR检测和潮霉素(Hyg)抗性平板的筛选获得了转基因拟南芥。发现加快了转基因拟南芥种子的萌发,提高了植株的抗逆性。这为继续深入分析WRKY转录因子在北细辛生长发育和次生代谢产物合成中的功能奠定了基础。
The WRKY family genes,which play an important role in plant morphogenesis and stress response,were selected based on the data of the full-length transcriptome of Asarum heterotropoides.Using AtWRKY33,which regulates the synthesis of the camalexin in the model plant Arabidopsis to compare homologous genes in A.heterotropoides,primers were designed to amplify the open reading frame(ORF)fragment of AhWRKY33 gene by RT-PCR using total RNA of A.heterotropoides leaves as template.Real-time PCR results showed that there was a significant difference between the aerial part and the underground part of A.heterotropoides,the toxic aristolochic acid content is highly expressed in the leaves higher than the root.After verification,the WRKY33 gene of A.heterotropoides is ORF long 1686 bp,encoding 561 amino acids.AhWRKY33 had two conserved WRKYGQK domains.According to the classical classification,it belongs to groupⅠWRKY transcription factor.A.heterotropoides WRKY33 had some homology with amino acids of other species.The study successfully constructed the plant eukaryotic expression vector PHG-AhWRKY33 and transformed Arabidopsis thaliana,the transgenic Arabidopsis was obtained by PCR detection and hygromycin resistant plate screening.It found that the germination of transgenic Arabidopsis seeds was accelerated and the stress resistance was increased.It laid a foundation for further analysis of WRKY transcription factor in the growth and development of A.heterotropoides and the synthesis of secondary metabolites.
作者
杨永成
王潇晗
王艳芳
刘秀岩
杨世海
YANG Yong-cheng;WANG Xiao-han;WANG Yan-fang;LIU Xiu-yan;YANG Shi-hai(Laboratory of Cultivation and Breeding of Medicinal Plants of National Administration of Traditional Chinese Medicine,College of Chinese Medicine Materials,Jilin Agricultural University,Changchun 130118,China;School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 102488,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2020年第13期3112-3119,共8页
China Journal of Chinese Materia Medica
基金
国家中医药管理局项目(ZYBZH-Y-JL-25)。
关键词
北细辛
WRKY基因
植物表达载体
遗传转化
Asarum heterotropoides
WRKY gene
plant lant expression vector
genetic transformation
