The Expression Analysis Of Glutelin And Mirnas At The Grain Filling Stage In Rice(Oryza Sativa L.)

Rice (Oryza sativa L.) is both an economically important grain crops and a model organism for scientific research. Rice grain filling is a critical and dynamic process, during this periods, large amounts of storage compounds are synthesized and transported into the rice endosperm, which are major determinants of the economic value of rice grain and provide nutrients and calories for humans. The major storage proteins of rice endosperm are the glutelins, which may constitute up to80%of the total protein in the starchy endosperm. Glutelins are encoded by a multigene family, which show highly temporal and spatial specificity expression patterns in rice grain.microRNAs (miRNAs) are negative regulators the mediate eukaryotic gene expression at posttranscriptional levels. In plant, most predicted miRNAs targets are known or putative transcriptional factors with functions in development, pointing to a role for miRNAs at the core of gene regulation networks. miRNA function to regulate diverse developmental process, including meristem and lateral prgan development, root initiation, flowering, timing and phase transitions. More than500miRNAs have been identified in rice and deposited in miRBase (v18.0) to date. miRNAs have been shown to play crucial roles during grain development.In the present study, Rice (Oryza saliva L. cv Nipponbare) was used as material, the differential expression of glutelin genes and miRNAs during grain filling stage were analyzed, and the main results are as follows:1. To determine the site of glutelin gene expression, we constructed transgenic plants carrying the fusion constructs of the glutelin gene promoter region and the GUS reporter gene. Histochemical examination result showed that although all of the eight glutelin genes were expressed specifically in endosperm, their expression patterns within the endosperm varied greatly. The GluA subfamily genes were expressed specific to the outer endosperm in the mature grain, while GluB subfamily genes were expressed in the whole endosperm except for GluB-5. The expression of GluB-5and GluD-1were observed in the inner starchy endosperm rather than in the aleurone and subaleurone layers. GluB-3is a pseudogene, and the GUS activity driven by the GluB-3promoter was limited to the aleurone and subaleurone layers. The expression site of each glutelin genes varied as the seed development, most of the glutelin genes were first expressed in the dorsal vascular bundle and dorsal aleurone, while the expression of GluB-5and GluD-1were first observed in the inner starchy endosperm close to the scutellum and then spread to the whole endosperm towards maturation. The difference in the expression site of GluB-5and GluD-1from other glutelin genes may due to the deviation in the GCN4motif sequence from the cognate GCN4. Quantitative analysis of glutelin promoter showed the potential strength of glutelin promoters exhibit dynamic changes during the grain filling stage. The mRNA level of each glutelin gene during grain filling stage was examined using quantitative RT-PCR, and the result showed that the most of glutelin gene were expressed at maximal level at12days after fertilization (DAF), while the expression levels of GluB-5and GluD-1were highest at17DAF. Western blot analysis revealed a discrepancy between mRNA levels and protein levels of glutelin genes, suggesting a post-transcriptional regulation of glutelin gene expression.2. In this study, high-throughput RNA-sequencing technology was used to reveal miRNAs expression patterns at different developmental stages during rice grain filling. A total of434known miRNAs (380,402,390and392at5,7,12and17days after fertilization, respectively.) were obtained from rice grain. The expression profiles of these identified miRNAs were analyzed and the results showed that161known miRNAs were differentially expressed during grain development, a high proportion of which were up-regulated from5to7days after fertilization. In addition, sixty novel miRNAs were identified, and five of these were further validated experimentally. Additional analysis showed that the predicted targets of the differentially expressed miRNAs may participate in signal transduction, carbohydrate and nitrogen metabolism, the response to stimuli and epigenetic regulation. In this study, differences were revealed in the composition and expression profiles of miRNAs among individual developmental stages during the rice grain filling process, and miRNA editing events were also observed, analyzed and validated during this process. The results provide novel insight into the dynamic profiles of miRNAs in developing rice grain and contribute to the understanding of the regulatory roles of miRNAs in grain filling.