Identification And Characterization Of Small Non-coding RNAs In Ovule Of Ginkgo Biloba L.

Ginkgo biloba L., which is known as the "living fossil", is considered as one of the oldest seed plants following the fern species. G.biloba has a long history, its reproductive processes exhibit unique traits, especially the growth and development of the female reproductive organ ovule. So far, many researches have focused on the microscopic morphology of ovule, organization formation, physiological and biochemical changes in G. biloba, but the molecular mechanisms being involved in ovules development rarely were reported. MicroRNAs (miRNAs) are a class of short, single-stranded, endogenous, non-coding RNAs, approximately 22 nucleotides (nt) long that are evolutionarily conserved in eukaryotes. They act as important regulators by guiding target mRNA cleavage or translational inhibition in various biological and metabolic processes in plants, including tissue identity, developmental timing and responses to biotic and abiotic stress, and so on. Therefore, identification and analysis of miRNA on the G. biloba ovule has important significance for further research.In this study, we combined next-generation highthroughput sequencing technology and bioinformatics analysis to identify miRNAs and their target genes in the ovule of G. biloba. The main results achieved as follows:(1) By sequencing, we got 12,121,385 high-quality sequences from ginkgo ovule small RNA libraries. Then after getting rid of the 3’adapter-null,5’adapter-contaminants, insert-null, smaller-than-18nt, poly A sequence, etc, we obtained 11,919,364 (1,991,395 unique) clean reads in the ovule small RNA library.(2) We analyzed, classified and annotated the clean reads in the ovule small RNA libraries. In the libraries, the main length of sRNA was 19-24 nt, which was consistent with the typical length of miRNA. By comparing with the genome, we found that, the ribosomal RNA (rRNA) sequences have 519,513 (31,039 unique), accounting for 4.36% of the total sRNA (1.56% unique); small nuclear RNA (small nuclear RNA, snRNA) 5,769 ESTs (1,014 unique), accounting for 0.05% sRNA of (0.05% unique); small nucleolar RNA (small Nucleolus RNA, snoRNA) 1,858 ESTs (376 unique), the total number of of 0.02%(0.02% unique); transfer RNA (transfer ribonucleicacid, tRNA) 203,471 sequences (6636 unique), accounting for 1.71% of the total; uncommented (unannatation) sequences of sRNA 8,627,585 (1,932,584 unique), accounting for the total number of 72.38%(97.05% unique).(3) We identified all the sRNA sequence of ovule to miRBase database (miRBase20.0) known mature miRNA sequences and precursor sequence. Finally we got 19,746 plant miRNAs unique sequence homology in the small RNA library of ovule, then by strict screening and complex analysis, the final identification to obtain a conservative sequence 144, belonging to 35 miRNA families.(4) In the small RNA library of Ginkgo ovule, there were 99 species of novel RNAs were predicted, and the total expression was 132,843. The first nucleotide base bias analysis of novel miRNAs revealed that first nucleotide base of 19-24 nt were mostly toward Uracil(U), which is the classic features.(5) We used the psRNATarget application software to predict target genes and GO enrichment analysis for miRNAs in ovule. The results showed that,2,062 target genes were predicted for 445 known miRNAs in ovule, and target gene loci to 2,487; 898 target genes were predicted for 123 novel miRNAs in ovule, and target gene loci to 1,331. After analyzing GO enrichment of these target genes, we found that they play important roles in all aspects of plant growth and development in ovule.(6) We selected 14 and 11 miRNAs respectively from known and ovule miRNAs. These miRNAs, which has precursor sequence, were related to photosynthesis, disease resistance, signal transduction and other functions. We used subcloning technology for precursor verification. Finally, we got 10 miRNA precursors which matched Illumina sequencing exactlly and 7 had less than three mismatches. The rest 8 miRNAs can’t amplify from the same period of ovule.(7) We analysised the differences expression level between ovule and female leaves, found that in comprison to female leaves, there were 300 known miRNAs were up-regulated,171 miRNAs were down-regulated, and 73 miRNAs showed no significant difference in ovule. Moreover, there were 46 known miRNAs were up-regulated,47 miRNAs were down-regulated, and 21 miRNAs showed no significant difference in ovule.(8) We predicted target genes from known and novel miRNAs, which related to growth and development, metabolism synthesis, photosynthesis and disease resistance. Then the target miRNA gene function annotation analysised the regulatory functions of those miRNAs. We selected 19 known and novel pre-miRNAs sequences, with two different periods of ovules and female leaves as the test material, in order to analysis the different expression between the same period in different tissues and the same tissue at different times. In summary, by indentifying and characterizing small RNAs, we found that there were various small RNAs in ovule, and these small RNAs were involved in the growth and development of gingko, morphogenesis, signal transduction and other life process. This also indicated the possible existence of complex post-transcriptional regulation, and these results would help further understanding of miRNA regulation in Ginkgo ovule molecular studies.