Study On The Molecular Regulation Of Endosperm Development At Grain Filling Stage In Maize (Zea Mays L.)

Maize is one of the three major food crops in the world, and it is also important for feed and industrial raw materials. Nutrients deposited in maize endosperm account for over 80% of the weight of the seed. Maize yield and quality are determined by the development, proliferation and filling of maize endosperm. However, endosperm development is involved in complicated physiological and biochemical processes, including cell division and differentiation, organ formation, as well as material transfer and accumulation. Transcription factors and plant hormones have been reported to regulate endosperm development. MicroRNA (miRNA) plays an important role in the regulation of gene expression in eukaryotes. miRNA regulates growth and development at all stages by cutting or suppressing target gene expression. Therefore, we analyzed the regulation of maize endosperm development on transcriptional leveland post-transcriptional level to enrich our understanding of the regulation network of maize endosperm development.In this study, RNA-seq and miRNA high-throughput sequencing were employed to explore genes and miRNAs expression level and further to resolve the regulatory networks of maize endosperm development (5DAP,7DAP,9DAP,10DAP,11DAP,13DAP,15DAP, 20DAP). The sequencing result were verified by RT-PCR, Yeast one-hybrid system and transient expression. The final results are as follows:(1) In total,7,095 differentially expressed genes (p<0.01) were detected during the process of endosperm development. Most differentially expressed genes were observed between 7DAP and 9DAP,9DAP and 10DAP,which is consistent with beginning synthetic starch at 9DAP. To evaluate the potential functions of the genes that showed significant expressional changes, Gene Ontology categories and KEGG pathway were assigned to the 7,095 differentially expressed genes.The result indicated that differentially expressed genes were involved in Starch and sucrose metabolism, cell wall,Plant hormone signal transduction, oxidation reduction, transcription regulation.(2) 24 motifs are significantly enriched in the promoters of starch synthesis Key enzyme genes.To elucidate the functions of TFs binding with 24 motifs, gene co-expression networks of these TFs was constructed using RNA-seq datasets. MYB127, one of the MYB transcription factor family members, was identified and it affected 8 genes expression involved in starch synthesis. Expression level of Isal was up-regulated by MYB127, which was verified by yeast one-hybrid system and gene gun-mediated transient expression.(3)103 differentially expressed known maize miRNAs belonging to 24 families and 22 novel miRNAs belonging to 7 families were identified by miRNA high-throughput sequencing.70 target genes of known miRNAs were identified by Correlation analysis and miRNA target prediction. Gene Ontology categories and KEGG pathway were assigned to the 70 target genes.The result indicated that target genes were involved in plant hormone signal transduction, transcription regulation, peroxisome.(4) Analysis of miRNA differentially expression shows that 28 miRNAs, which belong to 11 miRNA families, are differentially expressed during maize endosperm development(P<0.01), including miR528、miR393、miR167、miR172、miR1432、miR156、 miR171、miR319、miR159、miR398、miR408. miRNAl67 regulates sugar and starch metabolic pathway by suppressing the target gene ARF, and miRNA 159 and miRNA319 regulated GA signal pathway by suppressing the target gene MYB49.(5) 22 novel miRNAs, which belong to 13 miRNA families, were identified by high-throughput sequencing. The results of stem-loop PCR show that several miRNAs belonging to 7 miRNA families were successfully cloned in maize endosperm.(6) whole genome bisulfite sequencing of the maize genome in unfertilized endosperm and leaf revealed that of all C’s in CG, CHG and CHH context, 73%-81%,56%-68%、2%-3% in endosperm and 82.50%、74.42%、3.05% in leaf, respectively, are methylated. C’s in CG and CHG context methylated of gene region, upstream and downstream of gene are more higher than transcriptional start site and transcription termination.The expression level of 84 genes was regulated by DNA methylation in genes promoters.