Identification And Functional Analysis Of Small RNAs And Their Targets In Maize

Small RNAs (sRNAs) are small non-coding RNAs that can regulate gene expression at both transcrip-tional and post-transcriptional levels. Plant sRNAs play important roles in various aspects such as plant development, patterning formation, stress response and signal transduction. There are two major classes of sRNAs, known as microRNA (miRNA) and small interfering RNA (siRNA). There are few reports about sRNAs in maize(Zea mays), therefore, further research on sRNAs can make insight to their devel-opmental roles in maize. In this study, the spatial and temporal expression pattern of sRNA was analyzed, and known and novel miRNAs as well as their targets were identified.The miRNA expression profile of seeds at10days after pollination (DAP),25DAP embryo and25DAP endosperm was well studied based on the high throughput small RNA sequencing of these tissues, and the expression of29miRNA families containing108known miRNAs were confirmed, and54novel miRNAs within43miRNA families were also identified. Based on a previously reported sRNA-seq study, the miRNA expression atlas of various developmental stages of maize tissues were constructed, including embryo, endosperm, young leaf, seedling, root, tassel, ear and pollen. The result indicates that miRNA expression is spatial and temporal specific, and this trend is also found among seeds of different developmental stages. A new type of miRNA named mirtron miRNA was identified in maize, which was not reported previously.The degradome of six tissues in maize, including10DAP seed,25DAP embryo and endosperm, tassel, silk and seedling, were sequenced. Based on these degradome data,40miRNA targets for37known miRNAs were identified with high confidence. Among these newly identified targets, six were confirmed by5’RACE. These targets can be categorized into various types, such as transcription factors and ion binding proteins, indicating the extensive regulation roles of these targets. Further study showed that the binding sites of miRNAs and their targets can also be located in intron, which is new to the current an-notation, in addition to3’UTR and CDS region of target, the locations where they were widely reported previously. In addition to miRNA targets identificaition, degradome sequencing can also be used to iden-tify the cleavage sites of miRNA precursors cleaved by DCL1, and four miRNA precursors were found to be processed in the special "loop-first" manner by DCL1to produce mature miRNAs.The siRNAs in25DAP endosperm showed higher expression in gene region, compared with intergenic and repeat regions, while siRNAs in embryo were not found differed among these regions at the same stage, and siRNAs in10DAP seed showed the most abundant reads among other tissues. To further study the repeat-associated siRNAs (ra-siRNAs) and their roles in seed development, the distribution of ra-siRNAs within repeat regions among ten tissues mentioned above were compared. The result showed that ra-siRNAs were most abundant at the5’end of repeat regions, and most of them originated from LTR-TEs. In addition,24nt siRNAs were enriched in25DAP embryo but were depleted in25DAP endosperm, suggesting that they had different roles during embryo and endosperm development. As inferred by the analysis of ra-siRNAs and transponson superfamilies, ra-siRNAs had different but conserved roles for genome-wide gene silencing during embryo and endosperm development.Furthermore, an miRNAi vector for miR02was constructed and genetically engineered into maize. The expression of miR02in T2generation seeds was0.79times less than that of the WT seeds, while some of the predicted miR02targets were up-regulated. In maize protoplast over-expressing miR02, the expression level of miR02is nearly12times compared to that of WT protoplast, but down-regulation of the predicted targets was not observed. Therefore, further studies are needed to elucidate the function of miR02in maize.