木薯干旱胁迫响应CC类谷氧还蛋白基因的筛选与体外表达分析

Screening and Expression Analysis in vitro of CC-type Glutaredoxin Genes of Cassava in Response to Drought Stress

CC类谷氧还蛋白(CC-type glutaredoxins)是高等植物中所特有的一类谷氧还蛋白,模式植物中的研究表明,CC类GRX可以与TGA转录因子互作,在植物器官发育及响应环境胁迫过程中起重要的调控作用。基因差异表达分析结果表明,木薯中有多个CC类GRX基因在叶片中的表达受干旱胁迫的诱导。为了进一步研究木薯中CC类GRX蛋白与TGA的互作,本研究选择了其中的Me GRX058和Me GRX785这两个基因作进一步体外蛋白表达分析。理化性质及二级结构预测分析表明Me GRX058和Me GRX785蛋白分子量均在11 k D左右,具有谷氧还蛋白以及CC类谷氧还蛋白典型的结构域。通过构建酵母表达载体及植物表达载体,利用HA-Tag和GFP分别对Me GRX058和Me GRX785蛋白进行标记,在酵母和转基因木薯中进行表达,随后对融合蛋白的表达进行了Western blot检测。结果表明Me GRX058和Me GRX785成功在酵母和转基因木薯中表达,为进一步筛选它们的互作蛋白、研究其结构与功能做铺垫。

CC-type Glutaredoxins （GRX） is a kind of glutaredoxin-like proteins which is unique in the higher plants. The research of the model plant showed that, CC-type GRX protein can interact with TGA transcription factors and play an important regulatory role in plant organ development and environmental stress response. The results of gene differential expression analysis showed that the expression of several CC-type GRX genes in leaves of cassava was induced by drought stress. In order to further study the interaction between CC-type GRX proteins and TGA in cassava, two genes, MeGRX058 and MeGRX785, were selected for further in vitro protein expression analysis in this study. The physicochemical properties and the secondary structure analysis showed that the MeGRX058 and MeGRX785 proteins were all around l l kD, with the typical domain of glutaredoxin and CC-type glutaredoxin. The expression of MeGRX058 and MeGRX785 proteins were identified by yeast expression vector and plant expression vector with HA-Tag and GFP-Tag respectively. The expressed proteins were expression in yeast and transgenic cassava, and the expression of fusion protein was detected by Westem blot. The results showed that MeGRX058 and MeGRX785 were successfully expressed in yeast and transgenic cassava, which laid a foundation for further screening their interaction proteins and studying their structure and function.