| Maize has been an important food crops and has an important position in industrial and agricultural production. The maize endosperm accounts for 80% to 85% of the grain weight and the development, proliferation and full situation of endosperm cells determine the grain weight and quality. Therefore, an in-depth study of the development and formation process of maize endosperm has a vital significance for improving the yield and quality of maize. In this study, different maize inbred lines including high amylose maize(T1), sweet maize(T2), waxy maize(T3), and B73(T4) were taken as experimental materials for constructing the endosperm transcriptome library using RNA-Seq technology based on Ilumilla/Solexa sequencing. Then we screened the endosperm differentially expressed genes between different inbred lines and made analyses of COG functional classification, GO significant enrichment and Pathway significant enrichment for these differentially expressed genes. The main results were as follows: 1. The high amylose maize, sweet maize, waxy maize, and B73 were used for transcriptome sequencing based on Illumina/Solexa sequencing platform. Then we obtained 21127794, 18139530, 18292192 and 21127794 clean reads of four maize inbred lines, respectively. According to the analysis model of B73 genome, we acquired a total of 43540 genes after assembling and jointing these clean reads. The quality evaluation of insert fragment test, comparing statistical analysis, cDNA fragments random experiment test, and gene coverage statistics indicated that the Solexa sequencing quality meet the subsequent data analysis. 2. The analysis of alternative splicing events for different maize inbred lines showed that four maize inbred lines proceeded alternative splicing mainly by exon skipping(ES) and intron retention(IR). The gene number and alternative splicing events in endosperm of high amylose maize, sweet maize and waxy maize were apparently higher than that in endosperm of B73, which showed that the transcriptome components and their relative abundance in endosperm of high amylose maize, sweet maize and waxy maize had large differences and these differentially expressed genes and alternative splicing patterns might affect the gene expression in the process of endosperm development. Meanwhile, we screened out nine splicing regulatory factors(SR) and these SR genes were highly expressed in endosperm of high amylose maize and waxy maize, which indicated that the splicing of related genes in the formation of amylose and amylopectin were subject to their regulation. The splicing pattern of AGPase enzymes in the process of starch synthesis in different maize inbred lines was alternative first exon(AFE). ZmBEIIb(ae) only proceeded alternative splicing by Alternative 3’ splice site in waxy maize endosperm. 3. The formation of maize endosperm is closely related to many gene function groups and regulation networks that contain grain cell signaling in the filling stage, cell defense, antioxidant balance, carbon metabolism, transcriptional regulation, protein degradation, etc. Gene expression profile analysis showed that there were 525 significantly regulated genes including 324 up-regulated genes and 201 down-regulated genes in high amylose maize, sweet maize and waxy maize. We divided these 525 differentially expressed genes into 15 functional categories combining with the relevant functional annotation of them. Moreover, 6 of the 15 functional categories were main functional category gene groups(signal transmission, cell defense, antioxidant balance, carbon metabolism, transcriptional regulation and protein degradation), which suggested that the formation of different maize endosperm was closely associated with main functional category gene groups and regulation networks. 4. Compared with B73 endosperm, we screened out 19, 17 and 24 differentially expressed genes in high amylose maize, sweet maize and waxy maize through constructing starch metabolic pathway of differentially expressed genes. These genes were mainly related to ADP-Glucose focal acidification enzyme, starch synthase, starch Branching Enzymeand starch debranching enzyme in the process of starch synthesis.ZmGBSSIIb in the high amylose endosperm expression is significantly higher than normal maize endosperm, shows that the high amylose maize endosperm amylose synthesis ability is higher than common corn.In addition, soluble starch synthase ZmSSI, ZmSSIIa, ZmSSIIb, and Zm SSIV high expression in waxy maize endosperm, show different subtypes of soluble starch synthase in amylopectin synthesis play a catalytic role is different, and are responsible for the synthesis of branched chain starch.In the sweet corn endosperm ADPG focal phosphorylase ZmAGPL3 and ZmAGPL1(sh2) expression of quantity is significantly lower than the ordinary maize endosperm, show that in the process of starch synthesis ADPG focal phosphorylase activity inhibition or lose, preventing the accumulation of sweet corn starch in the endosperm, promote the formation of soluble sugar. 5. We obtained a total of 100 transcription factors belonged to 19 families of transcription factors through the screening of differentially expressed genes. Next, we build the transcription factors related to starch synthesis gene expression regulation network, combined with the differentially expressed genes expression patterns in different types of maize endosperm, screening get five important transcription factors.ZmNAC116, was cloned from maize inbred line B73.The protein by SDS-PAGE analysis size 68.9 kDa expressed in the supernatant quantity is higher than precipitation. Further expression analysis showed that ZmNAC116 was tissue-specific expression in endosperm.Transient expression showed that the GFP signal was detected only in the nucleus of the onion cell for ZmNAC116-GFP fusion protein using bombarding transformation.ZmNAC116 overexpression transgenic rice showed growth phenotype and controlled plant was no significant difference.In conclusion, this study constructed the endosperm transcriptome library of different maize inbred lines using RNA-Seq technology. Then we screened out differentially expressed genes and made GO analysis and Pathway significant enrichment analysis for these genes. Thus we obtained the significant enrichment entries that were related to the endosperm formation. Meanwhile, we acquired five genes that were specifically expressed in maize endosperm combined with the differentially expressed genes expression patterns in different types of maize endosperm. Our study provides the theoretical basis for the molecular mechanism of maize endosperm formation and is of great significance for creating excellent maize new germplasm. |
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