| Rice is one of the most important food crops. Whole genome duplication occurred before gramineous specification. Genome duplication plays an important role in evolution, resulting in the formation of gene families. It also plays an important role in differentiation and evolution of miRNAs (microRNAs) families. miRNAs are 21～24 nt short regulatory RNA molecules and mediate post-transcriptional gene silencing (PTGS) by miRNA-induced silencing complex (miRISC). However, little is known about how miRNAs regulate target genes expression during genome duplication in rice. To understand the function of miRNAs among rice of different ploidy levels, we have investigated novel miRNAs identified from haploid, diploid and triploid rice derived from a twin seedlings population with several approaches such as Solexa high-throughput sequencing, RT-PCR,5'-RACE (rapid-amplification of cDNA ends), real time Q-PCR, and transgenic techniques. We have characterized the expression of novel miRNAs between the different ploidy levels and analyzed the cleavage sites of target genes. This study partly illustrates how miRNAs play essential regulatory roles in the evolution of ploidy rice lines.1. Three small RNA libraries from haploid, diploid and triploid rice were constructed and sequenced with Solexa platform. After removing the low quality sequences and adapter sequences,2800402,2196941,2298182 unique reads were obtained with>=18 nt in length, respectively in haploid, diploid and triploid rice.1503535(53.69%), 1138597(51.83%),1210526(52.67%) unique reads had been perfectly mapped onto the genome. The lengths of most small RNAs fell into the range of 21～24nt. At booting stage, miR819j, miR1441, miR1857-5p, miR444d.3, miR1884b, miR819c had higher expression in haploid or triploid rice than in diploid rice. The abundance of miR156, miR166, miR167, miR168 and miR172 was very high in all ploidy levels suggested an essential role for the development.2. Thirty-four novel miRNAs were predicted in the three libraries with bioinformatics analysis. These miRNAs were verified by RT-PCR in haploid, diploid and triploid rice. The novel miRNAs were derived from introns, repeats, retrotransposons, etc, suggesting the polymorphism of distributions of MIRNA gene families and reflecting the various evolutionary origin of miRNAs. We had predicted target genes of the novel miRNAs. The target genes may be involved in development, stress-resistant, disease-resistant signal transduction of rice.3. The expression of miRNA was not strictly relevent with the effect of ploidy in ploidy rice.To understand expression of the novel miRNAs among the rice of different ploidy, we have investigated the expression of miRNAs in different stages and organs by qPCR. miRNAs expression data had been normalized using U6 snRNA, a reference small RNA. At seedling and tillering stages, expression of miR#3, miR#17, miR#23, miR#25 was lower in haploid than in diploid, but was higher at booting and mature stages. So we speculated that these miRNAs may play an important role in the transition from vegetative growth to reproductive growth in haploid. The expression of the five miRNAs was lower in triploid than in diploid at seedling and tillering stages, and was not obvious at booting and mature stages. In brief, the expression levels of miRNAs were dynamic at different stages of rice with different ploidy. Therefore, the results suggested that expression of miRNAs was irrelevant with gene dosage effect exerted by polyploidy, and miRNAs are a complicated regulator in different stages and organs.4. The ployploidization of rice may affected the cleavage sites of target genes mediated by miRNAs.5'-RACE technology is an efficient way to verify targets of miRNAs. Six target genes were verified with 5'-RACE. The cleavage sites mediated by miRNAs in mRNA were polymorphic. Most of sites distributed in upstream or downstream of targets. The cleavage sites by miR827a in target gene LOC_Os04g48390 were different between triploid and diploid. The other cleavage sites by other miRNAs were also different among ploidy rice. Therefore, the results indicated that cleavage sites were affected by ploidy levels.5. The models which miRNAs regulate target genes were varietal.We have investigated co-relation between miRNAs and targets by qPCR. The expression of targets negatively correlated with miRNAs or not. This may be explained by the complex relationship between miRNAs and target genes:one miRNA regulated one target gene, one regulated many, or many regulated one.6. The over express vector was constructed and the Nipponbare was transformed.To investigate function of miRNAs, we had over-expressed miR#9, miR#14 using a pHB vector. This work will help us to understand how miRNAs regulate target genes expression.7. The gene expression may be regulated synergicly by miRNA and methylation.We had also analyzed the DNA cytosine methylation at the 5'-CpCpGpG sites of haploid, diploid, Shuhui 527, Shuhui 363 and their hybrids by using the MSAP (methylation sensitive amplification polyphisim) method, and screened for differentially methylated genes which might be targed by miRNAs. This study will reveal the evidence of relationship between miRNAs and methylated genes. The differentially methylated genes LOC_Os05g22920, LOC_Os01g46750, LOC_Os12g39120, LOC_Os05g26926, LOC_Os05g26902, LOC_Os05g26914 which were identified from haploid, Shuhui 527, Shuhui 363 and their hybrids were potentially regulated by miR399e, miR2925, miR156a-k, miR172b, respectively. Meanwhile, core histone H2A/H2B/H3/H4 domain containing protein gene LOC_Os10g28230 was regulated by the miRNA miR3979 and differentially methylated. Therefore, the data suggested miRNAs may be connected with DNA methylation. |
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