Effects Of Cytokinin Oxidase Gene ZmCKX6 On Maize Leaf Development

Cytokinin（CK,Cytokinin）is widely involved in the processes of cell division,leaf growth,stomatal opening and anti-aging.Cytokinin oxidase/Reductase（CKX,Cytokinin oxidase/dehydrogenase）can degrade cytokinin irreversibly and regulate plant endogenous cytokinin effectively.The role of CKX gene family in regulating leaf type in maize remains unclear.In previous study,a QTL controlling leaf width was obtained by combining association analysis and linkage analysis,and ZmCKX6 was confirmed as the key gene controlling maize leaf type by resequencing,haplotype analysis and expression analysis.In this study,the biological function of ZmCKX6 gene in maize leaf development was preliminarily analyzed by phenotypic analysis and transcriptome analysis of mutant line.Key findings include:（1）By exogenous application of cytokinin（6-BA）to maize plants,it was found that the increase of cytokinin content would significantly reduce the leaf width of maize plants.（2）Phylogenetic tree analysis and sequence consistency analysis of CKX gene family members revealed that ZmCKX6 protein contains FAD_binding₄ domain and Cytokin-bind domain,and its biological function may be similar to OsCKX3 gene.（3）In order to verify the gene function of ZmCKX6,we constructed a ZmCKX6 knockout mutant line.Phenotypic analysis of mutant KO showed that the leaf width of the mutant was significantly smaller than that of the wild type under different environments,and the leaf length and plant height were also significantly smaller than that of the wild type.The yield related traits of the ZmCKX6 mutant were analyzed.The results showed that the ear weight,ear length and kernal length of the mutant were significantly lower than those of the wild type.（4）The cytokinin oxidase activity cytokinin content and of the mutant and wild type were measured.It was found that The activity of cytokinin oxidase in mutant leaves was significantly lower than that of wild type.the cytokinin content of tZ,iP and iPA types in the mutant leaves was significantly higher than that of the wild type.（5）In order to analyze the possible molecular mechanism of ZmCKX6 regulating the development of maize leaves,transcriptomic sequencing was performed on the ear leaf of ZmCKX6 knockout mutant KO and wild type.A total of 1137 differentially expressed genes were identified by comparing the gene expression levels of wild type and mutant KO.Functional enrichment analysis was performed on down-regulated differential genes,which were mainly enriched in photosynthetic antenna protein,carotenoid biosynthesis,plant hormone signal transduction,zetin biosynthesis,starch and sucrose metabolism,and brassin steroid biosynthesis.GO enriched photosynthesisrelated pathways including photosystem I,photosystem II,chloroplast thylakoid membrane,photosynthetic membrane and other pathways.In conclusion,this study preliminarily suggested that ZmCKX6 may regulate the development of maize leaves by degrading the cytokinin content in maize plants.The results laid a foundation for revealing maize leaf development and maize plant type genetic improvement.