Identification And Validation Of Soybean MicroRNAs Involved In Stress Responses

MicroRNAs (miRNAs) are a class of20-25nucleotides, endogenous, small noncoding single-strand RNAs that play critical regulatory roles in plants growth and development, as well as responses to different environmental stresses. But limited researches have been done on the soybean miRNAs.In order to cloning with stress-related miRNAs in soybean, the soybean seedlings were treated with various stresses, including drought, high salinity, low temperature, abscisic acid (ABA) and soybean mosaic virus (SMV). A mixed small RNA library was constructed and sequenced by Solexa technology. These results provide inprotant clues for the research of soybean stress tolerance.The major results are as following:1. A total of3201342raw sequence reads were obtained by solexa high-troughtput sequencing technology including338403candidate miRNA reads were generated from stress-treated soybean small RNA library. Fanally,228known miRNAs and76putative novel miRNAs were found by bioinformatic analysis, which belongs to60and64miRNA families, respectively.2. A total of22stress-responsive miRNAs were identified using miRNA microarray screenings, including4salinity-responsive miRNAs (miR156a, miR164, miR166a and peu-miR2911),10drought-responsive miRNAs (miR164, miR166a, miR168,482b-5p, miR2118, miR2911, miR1507b, miR1508a, miR319g and miR1450), and8low temperature-responsive miRNAs (miR156a, miR164, miR166a, miR168, miR159a-3p, miR1508a, miR1450and miR2911).3. miR2118expression were upregulated by drought stress, the expression was induced firstly from root, then in root and leaf, the expression was induced in0.5h reached high in1h then declined, while continued increasing expression in shoot and leaf. miR2118expression was not induced by high silinity and low temperature. Using5’RACE (Rapidd amplification of cDNA ends)-PCR, we have identified4target genes of of which coding leucine-rich repeat-containing protein, signal peptidase I, suppressor gene silencing3-like and one unkown protein, respectively4. miRNA1511and miRNA1511were cooperated to cleave the same target gene. miR1511*was validated by analyzing high-troughtput sequencing data and Northern blotting. Using5’RACE-PCR, we identified one target gene belonged to60S ribosomal protein L4family gene Gmrpl4a (Glyma10g05580.1), which were cleaved by both miR1511and miR1511*. The target cleavage site of the miR1511was identified in the first exon of Gmrpl4a, and the miR1511target in second exon of Gmrpl4a.5. Allelic variation of miR1511gene were identified on geographical distribution in China. The MIR1511were conserved in cultivated soybean (Glycine max), but there were several Indels and SNPs in MR1511for perennial wild soybean, though conserved miR1511and miR1511*sequences. There were4allelic variations in MIR1511,2of them were absent of miR1511in annual wild soybeans (Glycine soja), which were confirmed by Northern blotting. One fouth of annual wild soybean accessions were absent of miR1511. We have found with absent of miR1511in Glycine soja, Which were mainly came from lower and middle reaches Yellow River. These results indicated the evolution of MIR1511gene undergo natural selection.