Investigation Of SNPS Involved In Rice Mirna-mediated Gene Silencing

MiRNAs are key regulators and play inevitable role in plant growth,development and stress tolerance.Single nucleotide polymorphisms(SNPs)that are involved in miRNA-mediated gene silencing might cause serious changes to plant agronomic traits.But current studies of miRNA-related SNP mainly focus on the distribution of SNPs among miRNA regions as well as the influence on secondary structures of pre-miRNAs induced by SNPs,while the studies of how SNPs could influence the interaction between miRNA and target genes are still lacking.To gain the target genes of rice miRNAs,we adopted both bioinformatic methods and degradome experiments conducted in previous studies,and as for filtering the biological relevant target genes,afterward,we tried to employ the public expression data of both miRNAs and target genes to perform the expression correlation analysis.But the result showed that there was no strict negative expression correlation between miRNAs and degradome validated target genes,and this suggests of the complex functioning mechanism between miRNAs and their regulating genes and that the negative correlation cannot be applied in biological relevancy filtering.To have a glimpse into the evolutionary pressure on pre-miRNAs,we calculated and compared the distribution of SNP densities on pre-miRNAs,exons and intergenic regions,and the result showed that pre-miRNA has relatively smaller SNP density than exons and intergenic regions and further,suggests that pre-miRNAs are under stricter evolutionary selection.And to further compare the evolutionary difference between conserved and non-conserved miRNAs,we calculated and compared the SNP density of pre- miRNAs of both kinds and the positional SNP frequencies of the mature miRNAs of both kinds,and the results showed that SNP density in conserved pre-miRNAs was much smaller than that of non-conserved miRNAs and the order of SNP frequencies of positions in mature miRNAs were different between them,and these results indicates the selection pressure on conserved miRNAs are much stricter than on non-conserved ones and the function mechanisms are different among conserved and non-conserved miRNAs.After that,we wanted to examine the relationship of evolution between conserved miRNAs and their target binding sites,and we calculated the SNP frequency on each position of mature miRNA and cognate binding site,and we found the significant correlation of SNP frequencies between mature miRNAs and their binding sites of cognate target genes,and this suggests of the co-evolution between them.To analyze the influence of SNPs to the secondary structure and stability of pre-miRNAs,we performed prediction of secondary structure of all rice miRNA precursors,and calculated the free energy in the meantime.And then,we predicted and did calculation for the SNP-mutated pre-miRNAs and compared the results with non-mutated ones.The results showed that over 90%of all pre-miRNAs had their free energy changes exceeding the threshold of minimum energy that would influence the production of mature miRNAs,and also more than 70%of all conserved miRNAs experienced the energy changes exceeding the threshold among all cultivated rice varieties.This reveals the great difference of miRNA regulation among rice cultivars.To study the influence of SNPs to the interaction between miRNAs and target genes,we extended the haplotype analysis into combined complementarity pattern analysis,and applied the method to all conserved miRNAs and their target genes.And we discovered the complementarity recovery phenomenon in osa-miR818 family,and this suggests of the reciprocal influence between miRNAs and their target genes during their evolutions.Finally,to analyze the relationship between influence caused to their interaction and rice phenotypes,we mainly focused on miRNA binding sites carrying SNPs targeted by conserved miRNAs,and we found 7 target genes carrying 9 SNPs,among which 2 of them were predicted potential to bring about great changes to the regulation of cognate miRNAs.In the meantime,we compared the related phenotypes of different rice cultivars concerning these miRNAs,but we didn't find any apparent differences among them,and this might be explained by the multigene control of plant phenotypes.To summarize,SNP is a good indicator of evolutionary pressure of miRNAs,and can induce influences to miRNA-mediated gene silencing at various stages,including pre-miRNA secondary structure,the sequences of mature miRNAs and binding sites as well as the interaction of miRNA and target genes.These findings would help better understand how SNP could affect the miRNA-mediated gene silencing and how these influences could further induce the miRNA-related plant phenotypes.