| Maize(Zea mays,L)as one of the most important cereal crops around the world has an important economic significance.The poor lysine content of maize greatly reduces its nutritional value to monogastric animals.Therefore,it is of great significance to study the synthesis of lysine in maize seed.MicroRNA(miRNA)as a class of important non-coding small RNA,not only can control the expression of genes,but also can mediate the production of the phased small interfering RNAs(phasiRNAs).In addition,the non-coding RNAs that can inhibit the activity of miRNA by binding to miRNA via complementary sequences in plant,are named endogenous target mimics(eTMs).The regulatory network composed of miRNA,phasiRNA and eTM plays important roles in the growth and development of plants.In this thesis,we studied the co-expression network of lysine biosynthesis pathway genes(LBPSs)during maize seed development and identified and characterized the non-coding RNAs(phasiRNAs and eTMs)that interacted with miRNAs in maize seed.The co-expression network of LBPGs was analyzed basing on the RNA一seq data that from 21 different developmental stages of maize seed.First,we identified the LBPGs that were AKI/2/3,ASDs DHDPS1/2,DapB1/2,LL-DAP1/2/3-AT,DapF1/2 and LysA1/2 in maize by the annotation of lysine biosynthesis pathway in the KEGG and the gene annotation of maize in Phytozome.There were two genes encoding DHDPS in maize with significant differences in the expression level and pattern during maize seed development.Further analysis showed that the contribution of the two DHDPSs to lysine synthesis was different,the DHDPS2 plays a major role.Through the functional annotation of the LBPGs co-expression genes,we found that the co-expression genes are closely related to protein synthesis.In addition,we also found some potential regulators that can regulate lysine synthesis,such as the Hub genes which encoded the bZIP,HD-ZIP,MADS and MYB transcription factor,had certain roles in the regulation of genes expression within the module.In addition,by analyzing the 368 inbred lines RNA-seq and single nucleotide polymorphisms data of maize seed,we found that there were 5 LBPGs which had 16 expression quantitative trait loci(eQTL),5 of them werelocal-eQTL and 11 were trans-eQTL.These results indicated that the expression of LBPGs are also regulated by genetic variation.The roles of phasiRNA and eTM during maize seed development were analysed by using the smRNA-seq and degradome sequencing data of 10 days after pollination(DAP)of maize seeds(SeedlO),25 DAP embryo(Emb25)and endosperm(En25).We identified 102 phased siRNA producing loci(PHAS)in maize seed,and 80(78.43%)were expressed preferentially in endosperm,11(10.78%)were in embryo and one was in seed.Some PHAS loci have the same tissue specific expression with the genes in which the loci were located.Further analysis showed that some phasiRNAs could target the 02,PBF and ZmDof3 which could regulate the synthesis of zein and starch and the development of aleurone in endosperm,and the phasiRNA cleavage signal can be found in the degradome data of En25.These indicated the important roles of phasiRNA in maize seed.Furthermore,we identified 151 eTMs for 62 miRNA by analyzing the complementary pairing relationship between the intergenic region of the maize genome and the sequence of miRNAs which were expressed in maize seed.By using the 53 RNA-seq data of seed,embryo and endosperm at different developmental stages,we found that a total of 15 eTMs were expressed in maize seed,and four eTMs were confirmed by semi-qRT-PCR.In addition,we also found that some of the miRNAs that had the expression eTM could regulate the maize seed development.These results suggested that the network composed of eTMs and miRNA might play certain roles in the development of maize seed. |
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