Identification And Functional Analysis Of Microrna From Glycine Maxinvolving Its Interaction With Phytophthora Sojae

Phytophthora. root and stem rot (PRR) of soybean caused by Phytophthora sojae is a destructive disease affecting soybean production regions throughout the world. To breed new varieties with efficient, broad-spectrum and stable resistance to the soybean PRR, understanding of resistance mechanism is of great importance. The utilization of resistant cultivars is the most economical and environmentally safe method for controlling this disease.microRNAs (miRNAs) are20-24nucleotides non-coding RNAs, serving as a class of post-transcriptional regulators in eukaryotic organism. Dicer ribonucleases process miRNAs from their precursor hairpins. Then, the mature miRNA is incorporated into the RNA-induced silencing complex (RISC). Within this complex, miRNA targets the mRNA for cleavage or repression its productive translation based on the match between miRNA muture sequence and mRNA sequence. miRNAs regulate gene network in plants, including development, organ formation, cell proliferation and senescence, biotic and abiotic stresses.Here, we present evidences that plant gene silencing system plays an important role in the resistance during oomycetes-plant interactions. New miRNAs were predicted in Glycine max using bioinformatics approaches. miRNAs of soybean under the stress of P. sojae by artifical inoculation were identified from tolerant, resistant and susceptive cultivars by microarray, and miRNAs related to P. sojae and genotype were assayed, and target genes of miRNA were predicted and functional analyzed. Accordingly, the role of the miRNAs in resistance pathway of PRR was discussed, which lay the foundation for breeding and improving the resistant varieties in soybean by biotechnology method. The main results were as follows:1. Gene silencing system function in plant resistance to ooraycetes infectionRNA silencing is a common strategy shared by eukaryotic organisms to regulate gene expression, and also can operate as a defense mechanism against biotic stress. In Arabidopsis thaliana, several genes have been identified for RNA silencing, including SGS3(suppressor of gene silencing3), A GO1(Argonaute1), HEN1(Hua enhancer1), RDR6(RNA-dependent Polymerase6) and ZIPPY (ZIP). Here, Arabidopsis gene silencing mutants (zippy, ago1-27, sgs3-11and rdr6-11) were inoculated by Pp025and Pc35. The results showed that compared with the wildtype, phenotype of mutants was more resistant. Using PVX-mediated transformation system, p19was transformed into tobacco leaves and found that p19may be interfered plant gene silencing system, p19was transgened into soybean cultivar Williams, the positive roots were inoculated by P6497, and found that the resistance were repressed compared with the control. These results showed that there was relationship between RNA silencing and oomycetes resistance.2. miRNA prediction of Glycine maxSoybean is one of the most important crops with high economic value. Up to now, miRNAs in soybean have been identified by computational analysis or high-throughput sequencing. However, this number is very limited. Soybean ESTs were searched with non-redundant plant mature miRNAs by BLASTn approach, and removed coding gene support; finally,48putative soybean novel miRNAs were identified. To experimentally validate the prediction, poly(A)-tailed RT-RNA was used to amplified the three miRNAs, and the software psRNAtarget was also adopted to search the potential targets of the miRNAs. Based on the gene annotation, we found that many targets coded for different proteins, including resistant proteins and transcriptional factors. In addition, we analysed the source of ESTs containing potential miRNAs. The results showed that some ESTs containing potential miRNAs were involved in stresses, indicating that these miRNAs may be regulated by stresses.3. Expression analysis of conserved miRNAsSeven conserved miRNAs were selected to analyse the expression levels. Eight soybean cultivars inoculated with P6497were extracted small RNA and miRNA was detected by Northern blotting. The results showed that, six conserved miRNAs were expressed in eight cultivars in pre-and post-inoculation.4. Identification of P. sojae involved and genotype-specific miRNAsTo identify miRNAs involving soybean which respond to P. sojae and correlate to the different resistance cultivars, microarray was designed to detect miRNA expression pattern. Three soybean cultivars:Williams (susceptible), Conrad (quantitative resistance) and Williams82(qualitative resistance, containing Rpsl-k) were assayed at two stages:3-day post inoculation (dpi) and mock, microarray data were further confirmed by qRT-PCR analysis.5. Prediction of miRNA targets and functional analysis of miRNAmiRNA targets were predicted by tBLASTn. We took advantage of the available gene expression data of Williliams infected with P. sojae, and compared the expression pattern of miRNAs with that of their potential targets. We used qRT-PCR to validate the results. miRNA fragments are amplified from soybean genomic DNA by PCR, and construct them into overexpression vector pEarleyGate103. By Agrobacterium rhizogenes-mediated transform system, we found that overexpression of miRNA and silencing the target gene may influence plant resistance.In conclusion, gene silencing system plays an important role in plants resistance to oomycetes;48novel miRNAs were identified and characterized in G.max; using microarray, we analysed the miRNA expression pattern involving P. sojae and genotype, and target genes of miRNAs were predicted by Blast. These results suggest that the target genes involving soybean resistance may be regulated by miRNA.