Expression Profile Of MicroRNAs During Pollination In Maize

For pollination to succeed, style and pollens must develop normally to ensure the correct interactions between them. This process involves coordination of many factors andgenes. Each step of pollination must be normal, otherwise the pollination may fail. As important factors for regulation ofgene expression, miRNAs play roles in the regulation of signal transduction, morphogenesis, floral development and so on in plants. However, little is known about the expression and function of miRNAs in pollination. In this study, we analyzed the miRNA expression profiles during pollination in maize via microarray and high-throughput sequencing. The main results are as follows.(1) Using immature silks (Im), mature silks (MS), unfertilized ovary (UFO), silks pollinated after 5min (PA5) and 15min (PA15) of zheng-58 inbred line as materials, a set of significantly differential-expressed miRNAs (FDR≤5%, fold change≥2.0) were idetntified. 126 differentially expressed miRNAs were identified by comparing data from silks pollinated after 5min to 15min. A few differentially expressed miRNAs existed by comparing datas from other materials. Using unfertilized ovary as pistil background, we identified 25 significantly differential-expressed miRNAs that were pollination-related by comparing miRNAs expression patterns between different materials, many of which were predicted miRNAs.(2) We selected a few miRNAs for further analysis. We constructed expression vectors overexpressing 12 miRNAs and transformed them into Arabidopsis. We found that 35S::ath-MIR167D and 35S::zma-MIR167D transgenic plants displayed obvious phenotypes, such as dwarfism, abnormal floral development, indehiscent anthers and sterility.(3) We identified three new miRNAs in maize by bioinformatics methods. We detected their mature miRNAs in maize by stem-loop Taqman qRT-PCR. Then, sequences of the 141bp precursor were obtained using RLM-RACE methods. Therefore, the three predicted miRNAs were miRNAs indeed and they made a novel miRNA family.(4) Using mature silks, pollinated silks, mature pollens and in vitrogerminated pollens of B73 inbred line as high-throughput sequencing materials, 109 novel miRNAs were obtained by bioinformatics methods. Targetgenes of these miRNAs encode various proteins including sterol synthase, ribosomal proteins, spermidine synthase, Leucine-rich repeat transmembrane protein kinase, microtubule associated protein, methylesterase and protein kinase. It suggested that the novel miRNAs might have a wide spectrum of function in plant development.(5) A number of significantly differential-expressed miRNAs (chi2 arithmetic, P value≤0.01) were obatained by high-throughput sequencing. 88 miRNAs were considered to be upregulated during pollen tubegrowth including 64 known and 24 novel miRNAs. More importantly, the expression of 48 miRNAs were induced by pollination, including 37 known and 11 novel miRNAs. The targetgenes of these pollination-induced miRNAs encodegRF (growth-regulating factor),gRAS, methyl esterase and laccase. These miRNAs might come from either style or pollens, and therefore they may be involved in the recognition and interactions between stigma and pollens.In conclusion, we identified 25 significantly differential-expressed miRNAs by microarray analysis and 48 pollination-induced miRNAs by high-throughput sequencing, some of them might be candidates of pollination-related miRNAs. Due to the high conservation of miRNAs among different species, these data provide references to the study of miRNAs in plant species.