| MicroRNA (miRNA) is a group of short (21-24nt), non-coding RNAs which is widely existed in various tissues, and can modulate biological processes such as growth, development and response to biotic or abiotic stress in plants. The periodjjfrom spikelet filling to maturation is a complex physiological process in rice seed, which is also extremely important for the yield and quality of the production. However, the probable connection of the procedure and the miRNA has not been researched so far. In this study, using two indica varieties of white-core flo2and its corresponding wild type Baifeng B, we isolated several new miRNAs in seeds of10-20days after flowering (DAF) and analyzed the expression profiles of both novel and known miRNAs in this period. The possible role of miRNA in seed development was also investigated through the function prediction and analyzing of the miRNA target genes.We constructed two small RNA libraries of developing spikelet (flo2&Baifeng B), and3240709and3266135miRNAs were collected respectively through high throughput sequencing. By comparing to the9311genome,1363246(74.4%) and1990385(72.6%) matching miRNAs were chosed for further analysis.20conserved families and33nonconserved families could be detected in our libraries, and280more candidate miRNA were also identified.Through microarray strategy, we compared miRNA profiles among3filling stages (the milky, soft dough and hard dough stage). The chip analysis confirmed the expression of142known miRNAs,8novel miRNAs and47annotated miRNA variants. There were45miRNAs with a high signal value. By comparing to signals in10DAF, the results showed that4conserved miRNAs and5nonconserved miRNAs were up-regulated,5conserved miRNAs and4nonconserved miRNAs were down-regulated with available variants (p-value<0.05).8novel miRNAs were also confirmed by chip detection. By comparing to wild type, a number of miRNAs showed reduced or increased signal values in flo2especially in10DAF, which suggest the difference between flo2and Baifeng B mainly exist in the early filling phase.MiRNAs with high and variant expression levels during the spikelet development were selected for the prediction of target genes, the results of which implied the possible involvements of miRNA in various biological processes. Transcription factors of NAC and GAMYB families, regulated by miR164and miR159respectively by prediction, has been reported to participate in the endosperm development and substance accumulation; GBSS1b and BE1, regulated by miR1862d and miR1874-5p respectively by prediction, directly involves in the biosynthesis of starch in seeds. The expression level of miR1862in flo2was higher at milky-ripe stage than Baifeng B and the level of GBSS1was much lower in flo2at milky-ripe stage, which suggested the possible role of miRNA in the regulation of starch biosynthesis in rice seeds. Q-PCR verified the negative correlation between the expression profiles of miRNAs and their target genes. Some miRNAs like miR164and miR167had a positive correlation with their target genes, which suggested a feed-back circuit of miRNA and their targets.By sequencing, the phenotype of flo2in our research was mainly caused by8bp deletion in exon10of FLO2, a gene well reported previously. Target miRNA prediction showed the3’UTR region of FLO2could be affected by miR444and other miRNAs, which expressed in a lower level in flo2than in Baifeng B. The function of FLO2has been less known and its possible regulation mechanism by miRNA needs to be further investigated.Through the identification and expression profiles analysis, we found a group of miRNAs involved in the substance accumulation in rice caryopsis development stages and we found miRNA can have effect on starch sythensis pathway for the first time. Our study grained a deeper understanding of plant miRNAs and offered more imformation on the research of miRNAs in rice filling phase. |
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