Identification Of Stress Induced MiRNAs And Their Functional Analysis In Soybean

Micro RNAs have turned out to be a new player in plants tolerance to environmental stresses such as drought, salinity, extremely low and high temperature, etc, and many efforts have been devoted to understand their role of response in various crops recently. These single-stranded non-protein coding mi RNAs are ubiquitous throughout the plants genomes, and are involved in post-transcriptional gene regulation. Dozens of mi RNAs have been identified with altered expression profiles and their functional roles have been studied in some mi RNAs in the model plant Arabidopsis and rice under various stresses conditions but very few have been studied in soybean mi RNAs. Although mi RNAs are representing one of the relatively abundant classes of transcripts, the expression levels of mi RNAs vary greatly among species, tissues and differential environmental stresses condition.In the present study, we identified stress induced mi RNAs and their functional analysis. Firstly, we confirmed previous data of microarray analysis in soybean mi RNAs such as gma-mi R166 a, gma-mi R168 a, gma-mi R398 a and gma-mi R1508 a were induced under drought, high salinity and cold stress by northern blot analysis. This result implies that these mi RNAs might play stress response mi RNA in soybean seedling under stresses condition. Consequently, we further detailed analysis on understanding of gma-mi R166 a under various stresses using diverse genotypes of soybean seedlings as soybean has huge biodiversity. Principally, mi R166 a is one of the conserved mi RNAs that exists in many dicot and monocot species, and is plant development related mi RNA as well. Thus far there is no functional result concerned with abiotic stresses of gma-mi R166 a and its target gene. In this study, we validated that Glyma09g02750 and Glyma07g01950 were found to be cleavage by gma-mi R166 a by 5’ RACE among 15 putative target genes under various stresses, and subsequent analysis of this gene Glyma09g02750(Gm PHB), Class III Homeodomain-leucine Zip(HD-Zip III) Transcriptional factor, is 78% similarity to Arabidopsis PHABULOSA(At2g34710). Quantitative real-time PCR revealed that the expression of Gm PHB showed a significance negative correlation with corresponding gma-mi R166 a at different time courses in various stresses which suggested helping further study in the role of adaptation of abiotic stress response in soybean. Furthermore, mild overexpression of gma-MIR166 a in Arabidopsis repressed the transcription of At PHB, enhanced salt stress tolerance and suggested that gma-MIR166 a might function in positive modulation of salt tolerance in soybean.Recently, with the available of public genomic databases, there has been a shift from the labor-intensive development of PCR-based markers to sequence-based genotyping and the development of molecular markers within genes, often coupled with the use of RNA information. But thus far mi RNA-based markers have been only developed in rice, tobacco and cotton. Here we report allelic diversity of gma-mi R1511/1511* by mi R-In Del marker which is the first functional molecular mi RNA marker in soybean for a specific single copy locus used to assess genetic variation by genotyping a total of 1,669 accessions of domesticated soybean(G. max) and its wild progenitor G. soja which are native throughout the China and parts of Korea, Japan and Russia. The results indicate that the gma-mi R1511 locus is distributed in cultivated soybean and has three alleles variation in annual wild soybean. Based on this result, we proposed that mi R-In Del marker technology can be used to assess genetic variation. The inclusion of geo-reference data with gma-mi R1511 In Del marker data corroborated that accessions from the Yellow River basin(Huanghuai) exhibited high genetic diversity which provides more molecular evidence for gene diversity in annual wild soybean and domestication of soybean. In addition, negative correlation of gma-mi R1511 and its target gene Gm RPL4 a under stresses revealed that gma-mi R1511 might have function on soybean development. These results provide evidence for the use of small RNA marker, gma-mi R1511 In Del, as a soybean-specific molecular maker for the study of genetic diversity, genotyping of germplasm and evolution studies because gma-mi R1511 and its target gene were not observe in other legume family so far. This is also the first report of molecular marker developed from soybean mi RNA located within the functional region of pre-mi RNA1511.