Screening Of Stress-responsive MicroRNAs From Soybean (Glycine Max L.Merr)

microRNAs (miRNAs) are a class of18~24nucleotides, endogenous, small noncoding RNAsthat play critical regulatory roles in plant growth, development and stress resistance. In order to studythe stress-related miRNAs, the high throughput sequencing and the bioinformatics analysis werecombined to identify the salt-related miRNAs, and the target genes were predicted. Meanwhile, theqRT-PCR was performed to test the expression of18miRNAs and17target mRNAs aftersalt/PEG/alkaline treatment. The main results summarized as follows:1. Prediction of stress-related miRNAs and their target mRNAs in soybean. Total RNAs wereextracted from the root of soybean at germination stage treated by salt and normal condition. Thesmall RNA libraries were constructed and sequenced by Solexa technology. A total of162novelmiRNAs were identified by bioinformatic analysis. We predicted907target mRNAs for85novelmiRNAs using the psRNA Target and so on. Of these162novel miRNAs,36miRNAs have themiRNA*;55miRNAs drived from multiple loci, and71have single loci;44novel miRNAs werefound in the RS and RCK libraries simultaneously,54miRNAs expressed only in the RS, and64miRNAs were found only in the RCK, the differential expressions of these miRNAs were related tosalt stress.2. Expression analysis of stress-responsive miRNAs in soybean.18miRNAs were detected insoybean root treated respectively with salt, PEG and alkaline by poly(A)-tailing and primer-extensionRT-PCR.2miRNAs were down-regulated while8were up-regulated under the salt stress;9weredown-regulated while6were up-regulated after PEG treatment; and12showed a decreasedexpression while1showed incresed expression after alkaline treatment.3. Expression analysis of stress-responsive target mRNAs in soybean. The expression changes of17target mRNAs for the9miRNAs studied above in soybean root treated by salt, PEG and alkalinewere analyzed by the real time RT-PCR. Glyma04g10976.1was up-regulated whileGlyma04g29220.3was down-regulated under the condition of salt stress; Glyma08g41270.1wasdown-regulated after stressed by the PEG; Glyma11g37140.2was down-regulated after stressed bythe salt or alkaline; Glyma04g05790.2and Glyma11g01840.1showed a decreased expression aftertreated by the alkaline or PEG; Glyma01g43630.2and Glyma06g18070.2was down-regulated aftersalt, PEG or alkaline treatment.Using the high throughput sequencing and the bioinformatics analysis, a total of162novelmiRNAs and901target mRNAs for85novel miRNAs were identified. And the expression of18miRNAs and17target mRNAs for7miRNAs were analysised by qRT-PCR. This study providesuseful information for the research of stress-related miRNA and their target mRNAs in soybean.