The Interaction Between MeCPK9 And MeDi19 System Regulate Drought Resistance Of Cassava Via ROS-scavenging

Cassava is a typical drought tolerant crop.The research of the drought tolerance mechanism of cassava is helpful to reveal the causes of the drought tolerance of cassava,enrich the theory of plant drought tolerance,and provide excellent genetic resources for crop molecular breeding.Drought induced ABA signaling plays an important role in the process of drought tolerance of cassava.However,the molecular mechanism of ABA signal regulating cassava drought tolerance is still not clear,especially the drought resistance function and action mode of key factors downstream of ABA signal are still not revealed.Calcium dependent protein kinase(CPK)is a typical kind of plant serine/threonine protein kinase.CPK is a key response factor in ABA signaling pathway and is widely involved in regulating ABA-mediated drought response through phosphorylation of target proteins.Based on the data of cassava whole genome sequencing,drought tolerance evaluation of three accessions of germplasms and transcriptome analysis,the research group selected Me CPK9 protein kinase,an important regulatory factor of ABA signaling pathway,as the research target.In this study,the interaction between Me CPK9 and Me Di19 and its functional and molecular mechanisms in drought stress and ABA signal response were investigated by yeast two-hybrid assay system,yeast one-hybrid assay system,in vivo coexpression and genetic transformation techniques.The main results are listed below:1.Based on the transcriptome data,the full-length c DNA of cassava Me CPK9 and Me Di19 genes were screened and cloned.Subcellular localization analysis showed that Me CPK9 located on the cell membrane and Me Di19 located on the cell membrane and nucleus.Medi19 protein was splited based on different domain and self-activation activity of each part was analyzed,the reselt revealed that Me Di19 had transcriptional activation activity,and its self-activation region was at the Cterminal.The interaction between Me CPK9 and the C-terminal region of Medi19 was further confirmed by Yeast two-hybrid experiment and Bi FC.2.MeCPK9 and MeDi19 were overexpressed in Col-0 Arabidopsis thaliana through Agrobacterium-mediated genetic transformation method to obtain transgenic lines.The drought resistance of Me CPK9 and Me Di19 was further confirmed by phenotypic analysis.Through physiological analysis,it was confirmed that Me CPK9 and Me Di19 could activate the up-expression of antioxidant enzymes POD and SOD.3.Based on previous transcriptome data,10 downstream antioxidant enzymes gene promoters were screened and cloned.Through yeast one-hybrid screening,it was found that transcription factor Me Di19 could interact with the promoter of Me POD10.Further,dual luciferase report system confirmed that Me Di19 could directly conbine the promoter and activate Me POD10 expression.In summary,the results of this study indicate that MeCPK9 interacts with MeDi19,and both have drought-resistant function.Me Di19 can directly bind and activate the Me POD10 gene promoter to improve drought tolerance of plants.