Cloning And Functional Analysis Of ZmEer1 Gene In Maize

Maize kernels are mainly composed of endosperm and embryos,which is the storage place of protein and starch.The development of kernels and endosperm determines the quality and yield of maize,which is also one of the hotspots of maize biology research in recent years.Opaque endosperm mutants are a kind of important germplasm resources,which are usually accompanied by changes in grain development and cell differentiation as well as higher essential amino acid levels.Therefore,studying opaque endosperm mutants is of great significance for understanding the mechanism of grain development and improvement of nutritional quality.In this study,we obtained a silty endosperm mutant named eer1-1 by EMS mutagenesis.Our specific research work on this material is as follows:(1)The analysis of the eer1-1 mutant showed that the endosperm was opaque,the embryo was abnormal,and the grain size was normal,but the 100-kernel weight was 20% lower than that of the wild type.Compared with wild type,the content of gliadin in 20 DAP kernels decreased,and the content of 27-k D γ-zein increased and the level of 19-k D α-zein decreased in mature kernels,but the content of non-zein did not change significantly.(2)Histological section observation of 16 DAP showed abnormal embryo development of eer1-1,abnormal cell morphology of Basal endosperm transfer layer(BETL)and Aleurone layer(AL)in endosperm.(3)We used the method of map-based cloning to locate the target gene at the physical distance of 370 kb on chromosome 2.There were 10 candidate genes in this range.Sequencing analysis revealed that a c-T base mutation occurred at 529 bp downstream of the start codon ATG in gene3,and the codon changed from CAA to UAA,leading to the premature termination of translation.We named gene3 as ZmEer1.(4)To further verify ZmEer1 gene,we obtained heterozygous eer1-2 mutants mutated by EMS in the background of B73.Allelic test results showed that phenotypic changes caused by ZmEer1 gene mutation were confirmed.(5)The content of gliadin in mature eer1-2 mutants was decreased,especially 19-k D α-zein,and the content of non-gliadin increased.Compared with the wild type,the amylose content of eer1-1 and eer1-2 mutants decreased and the total starch content increased..(6)Phylogenetic tree analysis showed that ZmEER1 and AtRKD5 belonged to the same branch of the evolutionary tree,indicating that ZmEER1 was a RKD type RWP-RK family protein.(7)In order to determine the location of ZmEER1,it was proved that ZmEER1 was located in the nucleus by comparing with empty GFP vector and using DAPI staining,while yeast transcription-activity experiment proved that ZmEER1 protein had no self-activation activity.(8)RNA-seq data showed that ZmEer1 caused silty grain phenotype and abnormal embryo development by affecting protein and starch biosynthesis.