Cloning And Functional Identification Of ZmMCM Gene In Maize

Maize is one of the world’s major food crops and its increased production is of great importance to all countries.The endosperm of maize accounts for 80% of the weight of the kernel and its growth and development determines the yield of maize.It has been shown that endoreplication is associated with the rapid expansion of the endosperm during maturation.The endosperm of cereal grains undergoes a form of cell division during tissue development known as ’endoreplication’,by which the accumulation of nutrients and the morphological establishment of the kernel are rapidly completed,with the cycle occurring almost simultaneously with the rapid expansion of the kernel.In addition,several studies have shown that endoreplication plays an important role in the early development of the embryo,in cell differentiation and in response to abiotic stresses.Therefore,studying the function and regulation of endoreplication not only helps to enrich the knowledge of cell cycle regulation,but also provides a theoretical basis for breeding new high-yielding maize varieties by regulating the mode of occurrence of endoreplication.In this study,based on the preliminary association analysis,a ZmMCM gene,which regulates the assembly of the cell cycle replication initiation complex,was identified,its gene family was analysed on a bioinformatic basis,and the ZmMCM2 gene was cloned for transgenic functional verification and molecular interaction analysis.1.Bioinformatics analysis of the ZmMCM gene family: through database search,comparison and screening,a total of 17 MCM family members,named ZmMCM1-ZmMCM17 in order,were identified,all localized in the nucleus,with large differences in protein molecular weight;sequence similarity and high conservation exist among family members;tandem repeat sequences and replication fragments exist among some genes;phylogenetic analysis Phylogenetic analysis shows that maize ZmMCM genes are distributed in six subgroups,including I,II,III,IV,V and VI,and are involved not only in hormone regulation,stress regulation and growth regulation,but also in various regulatory and metabolic pathways.2.Specific expression analysis of ZmMCM gene under abiotic stress: The expression specificity of ZmMCM gene was analyzed by QRT-PCR.The results showed that ZmMCM gene could respond to abiotic stress such as ABA,drought,high salt,low temperature and high temperature,and ZmMCM gene might be involved in multiple signal transduction.3.ZmMCM2 gene overexpression analysis: Using the inflorescence immersion method,35S:ZmMCM2 was transferred into wild Arabidopsis plants,and the transgenic Arabidopsis showed dwarf plants,reduced rosette leaves and smaller seed size phenotypes.Nuclear DNA ploidy analysis and endoreplication analysis showed that the ZmMCM2 gene inhibited the endoreplication of Arabidopsis thaliana and affected the growth and development of Arabidopsis thaliana,which in turn led to phenotypic changes.4.ZmMCM2 gene expression mechanism analysis: subcellular localization showed that ZmMCM2 gene was expressed in the nucleus;in addition,the yeast library was screened for CDC73,a cell division cycle protein that interacts with MCM2,and through rotary verification,it was found that there was an interaction between MCM2 and CDC73,suggesting that MCM2 may regulate cell cycle-related protein activity by interacting with CDC73.This suggests that MCM2 may interact with CDC73 to regulate the activity of cell cycle-related proteins and thus regulate the transition between endoreplication and mitosis.