Identification And Characterization Of MicroRNAs From Chinese PCNA Persimmon Based On High Throughput Sequencing

The developmental of tannin accumulation and deastringency naturally of Chinese sweet persimmon is an extremely complex biological process, which involved in a large number of regulation genes. At present, there are some reports on the levels of the genes, transcriptional and proteins. MicroRNAs (miRNAs) play key roles in regulating gene expression at the post-transcriptional level, in plant. The miRNA information on persimmon was scarce. And neither studies demonstrated that miRNA is retated to biosynthesis of tannin. So, in this study, two small RNA libraries established using’Eshi1’fruits (Diospyros kaki Thunb.) collected at15weeks after flowering (WAF)(Stage1), and20WAF (Stage2) were sequenced through the Solexa platform in or to identify miRNAs involved in deastringency of persimmon. At the same time, further verification of miRNAs and theirs targets by the method of qRT-PCR. The main results are as follows:1. A total of6,258,487and7,634,169reads were generated fromthe fruits at Stage1and Stage2, respectively. Based on the sequence similarity and hairpin structure prediction, we identified236known miRNAs belonging to65conserved miRNA families, and33novel miRNAs whose precursors were potentially generated from persimmon transcriptome data. In addition,61miRNAs,17up-regulated and44down-regulated miRNAs, exhibited significant change in the expression levels between Stage1and Stage2. Expression profiles of twelve conserved and ten novel miRNAs were verified using qRT-PCR analysis. The results were basically identical with the sequencing.2. Application software psRNAtarget and WMD3online, there were87and111target gene prediction for the differential expressed miRNAs. Many of conserved miRNA have multiple targets, the number in the range from one to ten. Most the target genes for the miRNAs are wide in plants, for example, miR156target SPL (squamosa promoter-binding-like protein family), miR160target ARF (auxin response factor), miR39target GRF (growth regulating factor), miR398target copper/zinc superoxide dismutase.And SPL9is target gene for dka-miR156j-5p and significiant down regulation in the stage2, but other miR156family members were up regulated; The dka-miR858b, dka-miR395p-3p, dka-miR2911a target gene is MYB, bHLH, ADH, respectively. These four miRNAs were considerated as objective miRNAs. However, results still need further functional verification.3. According to the GO annotation of target genes, target genes encode a broad range of proteins related to transcription regulation, pollen development, gamete production and the organ development. Even more, dka-miR156j-5p, dka-miR858b, miR395p-3p, dka-2911a maybe involved in proanthocyanidins (PAs) accumulation and deastringency of oriental persimmon.Above all, the identification of miRNAs in oriental persimmon is not only abundant in the persimmon miRNA’s information, but also paves way for elucidating the post-transcriptional regulation of the (de)astringency of the persimmon fruits.