Identification And Interaction Analysis Of Grapevine Micrornas And Their Targets

Plant microRNAs (miRNAs) are endogenous non-coding single-stranded small RNAs withl9-24nt in length and they are widely distributed in plant genomes. Plant miRNAs can regulate gene expression mainly by degrading targeted mRNAs or repressing gene translation at the post transcriptional level, playing an important regulatory role in controlling plant growth, development, differentiation, apoptosis, metabolism and stress response. Plant miRNAs are one of important research contents of plant functional genomics. Grapevine (Vitis vinifera L.) is among important fruit trees in the world and is also one of the most economically important fruit trees in China. Although some grapevine miRNAs (Vv-miRNAs) have been identified in previous studies, they all belong to wine grapevine cultivars from Vitis vinifera L, and about30%homologous miRNAs between various cultivars from this cultivar population had the sequence discrepancies. Therefore, it is of significance to carry out identification of miRNAs and their target genes in various grapevine cultivar populations and species for gaining insight into grapevine miRNA characteristics, functions, and evolution of various grapevine species. This work can also lay significant foundation for comprehensively carrying out researches on grapevine functional genomics.The table grapevine’Summer Black’, a hybrid of V. vinifera and V. labrusca, and wild Vitis amurensis Rupr. were used as experimental materials in this study where identification and characterization of Vv-miRNAs and their target genes, analysis of the action modes of Vv-miRNAs on their target genes and the spatiotemporal characteristics of these modes were systematically and comprehensively carried out by bioinformatics tools, miR-RACE, Solexa sequencing, Degradome sequencing, qRT-PCR, PPM-RACE and RLM-RACE. The major works and findings were as follows:1. Precise sequences, especially terminal nucleotides of156bioinformatically predicted Vv-miRNAs were determined by miR-RACE in grapevine cv.’Summer Black’, and134target genes were predicted too. The expression of10pairs of randomly selected miRNAs and their target genes were detected in grapevine’Summer Black’leaves, stems, tendrils, inflorescences, flowers, and berries by quantitative RT-PCR (qRT-PCR). Results showed that each of these10pairs of Vv-miRNAs and their target genes had varying expression levels in different tissues, and the expressions levels of both of each pair of Vv-miRNA and corresponding target gene exhibited clear contrasting trends. The result also indicated that the nucleotide discrepancies in the aligned homologous Vv-miRNAs from various grapevine cultivars did not normally change their target genes in the cultivars. However, some nucleotide variations in orthologous miRNAs between Vv-miRNAs and Arabidopsis miRNAs (At-miRNAs) could easily change miRNAs targeting modes.2. A small RNA (sRNA) library of’Summer Black’ flower and berry tissues at different growing stages was constructed and sequenced using Solexa sequencing for more new miRNA identification. Based on sequence similarity and hairpin structure prediction,130conserved Vv-miRNAs and80species specific ones including72novel and8known non-conserved Vv-miRNAs were found. Forty-two (52.5%) of these80unconserved Vv-miRNAs were expressed in various grapevine tissues. Among these42Vv-miRNAs,20were expressed only in grapevine berries. In addition,130target genes for56non-conserved miRNAs could be predicted and the basis of functional annotation indicated that they might play important roles in regulating grapevine berry growth and development as well as response to environmental stresses.3. To further gain insight into action modes of Vv-miRNA on their target mRNAs, a new strategy integrating the modified5’-RLM RACE (RNA ligase-mediated5’rapid amplification of cDNA ends),3’-PPM RACE (Poly (A) polymerase-mediated3’rapid amplification of cDNA ends) and qRT-PCRs of cleavage products was used to validate36potential target genes of21Vv-miRNAs, and to analyze their action modes and target cleavage sites. Totally,31potential target genes of18Vv-miRNAs were verified and the existence of target cleavage modes by Vv-miRNAs and sites of Vv-miRNAs cleaving their targets were determined. It was found that94.4%of the Vv-miRNAs cleaved their target mRNAs mainly at the mRNA nucleotides complementary to the tenth neucleotide of5’-end of miRNAs. Moreover, the expression levels of both the miRNAs and their target mRNAs in the8tissues of’Summer Black’exhibited contrasting variation trends, while expressions of their cleaved targets followed those of their corresponding miRNAs.4. To characterization of miRNA in Amur grapevines, an important wild grapevine originated from China, and to further study evolution of grapevine interspecies based on miRNA sequences, the miRNAs in Amur grapevines (Va-miRNAs) was firstly mined in this study. A total of126conserved miRNAs belonging to27miRNA families and34known but non-conserved miRNAs were found. Significantly,72potential Amur grapevine-specific miRNAs were discovered. The precise sequences of40potential Va-miRNAs were further validated through miR-RACE, and accumulation of18new Va-miRNAs randomly selected in seven tissues of grapevines were detected by real time RT-PCR (qRT-PCR). Va-miRNAs were conserved among28plant species, with some base variations in several of the orthologous miRNAs. Further comparison of orthologous miRNA sequences of Vitis amurensis Rupr. and Vitis vinifera L. revealed the existence of miRNA SNPs (miR-SNPs) and miRNA base variations in length (miR-LDs) and some of these variations could change miRNA targeting modes. Finally,346target genes for the new miRNAs were predicted and they were annotated to be of genes involved in Amur grapevine stress tolerance, anthocyanin systhesis, and sugar metabolism.5. To comprehensively discover the spatiotemporal characteristics of the action modes of Vv-miRNAs on their target mRNAs at genome-wide levels, Solexa, Degradome sequencing and bioinformatics were employed to characterize Vv-miRNAs and their target genes and the spatiotemporal variations of Vv-miRNA regulatory modes on their target genes in grapevine leaves, flowers and berries at different growing stages. A total of242known and132novel Vv-miRNAs were identified, and193target mRNAs including103for known and90for novel miRNAs were validated. The most interesting finding was that over50%of novel miRNAs were expressed exclusively in flowers or berries where they had tissue-specific cleavage roles on their target genes, especially, the breadth of cleavage sites of novel miRNAs on their targets in flower tissues. Moreover, six novel miRNAs in berries were found to response to exogenous gibberellin (GA) and/or ethylene, confirming they were involved in signal paths of GA and ethylene during berry development. Other significant finding was that about93.6%of the known miRNAs exhibited some dynamic regulation on their targets through different action modes during grapevine development. Especially, there happened the phenomena that various Vv-mRNAs of same and/or diverse families co-regulated their target genes and some Vv-miRNA families had one key member that act as the main regulator of their target genes during grapevine development.Our work can immensely enrich the knowledge of grapevine genomics and lay important foundation for further researches on grapevine functional genomics and on mechanism of grapevine flower and berry development.