Regulation Of ZmMYBR31 And ZmMYBR43 On PEPC Gene Expression In Maize

As one of the annual gramineous herbaceous plants,maize?Zea mays L.?is an important raw material for food,feed,biological and industrial products,which plays an important role in the food security and economic stability worldwide.Photosynthesis is an important process for green plants to produce organic matter,therefore,enhancing C₄ photosynthesis has a huge effect on increasing corn yield.PEPC?phosphoenolpyruvate carboxylase?is a key enzyme in C₄ photosynthesis and plays a vital role in CO₂ fixation,so it is of great significance to study the regulation of PEPC gene expression.MYB-related transcription factors are a subfamily of the MYB transcription factor family.According to previous studies,they play an important role in seed germination and plant stress invasion.However,the regulation of MYB-related transcription factors on photosynthesis,especially on PEPC gene expression,is currently under-researched.In this study,the ChIP-seq data system and phylogenetic analysis were used to predict the MYB-related transcription factors ZmMYBR31 and ZmMYBR43 that regulate the expression of PEPC gene.ZmMYBRR31,43 were studied using dual-Luciferase,subcellular localization,yeast one-hybrid,and CRISPR/Cas9 gene editing technologies.And the regulation of PEPC gene expression lays the foundation for further research and understanding of C₄ photosynthetic mechanism.The following are the main research results:?1?According to the ChIP-seq results and Dual-Luciferase technology of ZmMYBR31,ZmMYBR31 can significantly activate the expression of PEPC gene by binding to the promoter region of PEPC gene.?2?A genetic analysis of ZmMYBR31 showed that ZmMYBR31 and ZmMYBR43 have high homology and similar expression patterns in leaves,so ZmMYBR43 may be a functionally redundant gene of ZmMYBR31.Through Dual-Luciferase technology,it was found that ZmMYBR43 can also significantly activate PEPC gene expression by binding to the promoter region of PEPC gene,and has similar functions to ZmMYBR31 in regulating PEPC expression.?3?The Split LUC technology was used to study the interaction between ZmMYBR31 and43 and verified by Dual-Luciferase technology.It was shown that there is an interaction between ZmMYBR31 and 43 in tobacco leaves,and the coexistence of ZmMYBR31 and 43can activate the expression of downstream reporter gene more than alone.?4?The subcellular localization of ZmMYBR31 and ZmMYBR43 was performed in maize protoplasts,and it was found that both ZmMYBR31 and ZmMYBR43 were localized in the nucleus.?5?The binding sites of ZmMYBR31,43 and PEPC promoter were studied by yeast one-hybrid technology,and the binding sites of ZmMYBR31,43 and PEPC promoter were reduced from 1014 bp to 40-50 bp.?6?Based on the genetic relationship analysis between Zea mays and Setaria viridis,the homologous genes Sevir.9G327500.1 of ZmMYBR31 and 43 were identified.A gene knockout mutant of Sevir.9G327500.1 in Setaria viridis was constructed using CRISPR/Cas9gene editing and Setaria viridis tissue culture and genetic transformation technology.The 73bp fragment was deleted from the homozygous mutant 1 of the T1 generation and the 59 bp fragment was deleted from the homozygous mutant 3.By observing the phenotype,no obvious phenotypes of the mutants were distinguished from the wild type,and further research is needed.