Functional Analysis Of Transcription Factor ZmREE1 In Maize

Maize is the largest grain crop in China,with great production potential and high economic benefits.Its deeply processed products have various industrial uses.The main raw material for corn deep processing comes from corn endosperm starch,whose composition and structure determine the quality and use of maize.The synthesis of starch is a complex biological process involving the regulation of multiple transcription factors.Using maize kernel development mutants to screen and identify transcription factors related to starch synthesis has important theoretical and practical significance for clarifying the starch regulatory network and improving maize quality.In this study,a mutant ree1 with abnormal maize seed development was obtained by EMS mutagenesis of Maize inbred line W22,and preliminarily analyzed the formation mechanism of the mutant.The main results was obtained as follows:(1)The mature grains of the mutant ree1 were dysplasia,with the tip concave inward,and the endosperm starch was powdery.Compared with the wild type,the 100 grain weight,grain length,and grain thickness of the mutant decreased,while the grain width did not change significantly;(2)The total starch content of this mutant increased,while the amylose content and protein content decreased.The content of gliadin,which accounts for a large proportion of total protein content,also decreased compared to the wild type,but the lipid content significantly increased;The scanning electron microscopy results of starch structure showed that the starch particles in WT were compact in texture,complete in section,and irregularly polygonal,while in the ree1 mutant,the starch particles were exposed and loosely arranged in an oval shape;(3)Used Map-based cloning,the target gene was located within an interval of approximately 490 kb on chromosome 2.There are 12 candidate genes in this region.Sequencing analysis found that a single base mutation occurred in ORF 9,leading to the premature termination of the encoded amino acid,which affected the function of the gene,resulting in abnormal grain dysplasia,and the gene was named ZmREE1;Allelic testing was conducted using the hybrid ree2 mutant and ree1 mutant mutated by EMS under the B73 background,and the results demonstrated the phenotypic changes caused by the ZmREE1 gene mutation;(4)ZmREE1 belong to RWP-RK family protein,without signal peptide and transmembrane domain.The expression level is relatively high in the endosperm and reaches its maximum after 15 days of pollination;Subcellular localization of ZmREE1 using maize protoplasts showed that the gene was located in the nucleus;And the ZmREE1 protein has no self activation activity.(5)Preliminary analysis of transgenic plants overexpressing ZmREE1 showed an increase in grain size compared to the wild-type,with an increase in total starch content and amylose content;RNA-seq analysis showed that the differentially expressed genes of WT,ree1,and overexpression of ZmREE1 in maize are mainly enriched in the regulation of protein dimerization activity,which may be involved in the regulation of amylase activity;In addition,the expression level of endogenous genes related to starch synthesis in transgenic maize has changed,with the expression level of gene GBSSI related to amylose synthesis being higher than the wild type maize.It is speculated that ZmREE1 may be involved in regulating the synthesis process of maize amylose.