| Maize is one of the most important cereal crops in the world,and its domestication has spanned over 9000 years.Zein is the main storage protein in maize seeds,but the lack of essential amino acids,such as lysine and tryptophan,limits its nutritional quality.Although the mutation of 02 reduces zein content and increases lysine level,it also brings many negative effects,resulting in the reduction of important agronomic traits such as floury endosperm,weak seedling emergence and susceptibility.After continuous improvement and breeding by breeders,quality protein maize(QPM)containing o2 modifiers(mo2)was finally obtained.QPM has the characteristics of high nutrition and hard endosperm,but lacks germplasm resources.Therefore,it is of great significance to elucidate the regulation mechanism of 02 transcription factor and mo2 in seed development.In this study,polymorphisms and haplotypes were detected based on the six z2 genes in 60 maize and teosintes lines.Haplotypes were unevenly distributed,and abundant genetic diversity was found in teosintes.Polymorphism of z2?18 was the highest,and z2?15 was the lowest,indicating differential roles in the evolution of seeds.Indels showed clustered distribution,and most of them derived from teosintes.The indels not only led to tandem repeat polymorphisms,but also to frameshift mutations,which can also be used as null variants.In addition,neutral evolutionary tests,phylogenetic analyses,and population structures indicated that z2?10 and z2?50 had undergone natural selection.Indeed,natural selection imprint can also be found in z2?27 and z2?16 genes,whereas z2?18 and z2?15 genes tended to be under neutral evolution.These results suggested that the genetic diversity and evolution of a subset of sulfur-rich zeins could be under environmental adaptation during maize domestication.Zein genes belong to endosperm-specific expression genes,and in order to understand the regulation mechanism of them,the DNA methylation of z2 was analyzed.The results showed that high DNA methylation level in z2 promoter and gene body region inhibited gene expression,while moderate DNA methylation in gene body might be beneficial to gene expression.In addition,z2?10,z2?818 and z2?50 genes were regulated by the DNA methylation,while z2?15,z2?16and z2?27 may regulated by other genes or transcription factors,suggesting that the expression of z2 genes had different regulatory mechanisms.The O2 gene,an endosperm-specific transcription factor,is essential for maize seed development.Zein genes began to transcribe 5-10 days after pollination and reached its peak around 15 days.Compared with o2 mutant,most of zein genes had higher expression in wild type except for z2?16.However,the synthesis of non-zein protein was increased in o2 mutant,suggesting there was a complementary mechanism that can keep the total protein of seeds in a relatively stable state.In order to obtain the comprehensive O2 regulatory network,the regulatory genes of O2 were identified by genomic prediction,ChIP-Seq and RNA-Seq data.Finall,274 genes were discovered that were directly regulated by O2.Besides the promoter region,O2 gene can also bind to the exon,intron,TTS and intergenic region of the gene.The targets of O2 contained transcription factors and kinase-related genes,which can not only further regulate the expression of downstream genes,but also participate in a variety of stress responses.In addition to activate gene expression,O2 can also have inhibitory effect on its target genes.The differentially expressed proteins(DEP)between the wild type,o2 mutant and QPM were identified by label-free quantification(LFQ).Among them,the expression of 66 proteins were recovered in QPM that may play an important role in the formation of hard kernel.WGCNA and functional analyses showed that proteins involved in energy and protein metabolism,and protein modification in brown and blue modules were significantly associated with phenotypes such as zein content,seed hardness,pest resistance and germination ability.The acetylation and phosphorylation of storage protein might play a regulatory role in seed development and germination based on the protein modification results.And the combination of transcriptome and proteome indicated the resistance-related genes were differentially expressed in o2 mutant.However,in response to external stress,the plant need the synergistic action of a variety of resistant proteins,which led to the mutant being susceptible to disease and insect.Although the O2 mutation resulted in a large number of differentially expressed stress-response genes and ribosome-related genes at transcriptional level,most of them did not cause the same change in protein level,indicating the existence of the post-transcriptional regulation.This study revealed the environmental adaptability of cultivated maize in the process of domestication,and the regulation network of O2 was constructed based on the analyses of multi-omic data,which revealed the transcriptional and post-transcriptional regulation mechanism of O2 transcription factor,and enriched the research contents of complex QTL genes,excavating some key genes that may be related to mo2.This work provides a theoretical basis and important genetic resources for accelerating the process of maize high-yield and high-quality molecular breeding in China. |
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