Functional Study Of Stress-Responsive MiR169c And Its Target Gene GmNFYA3and MiR394a From Soybean (Glycine Max L.Merr)

Soybean (Glycine max L. Merr.) is China’s leading economic oilseed crop. Due to the low yieldand oil content of soybean in China, and soybean is vulnerable affected by a wide range of abioticstresses, such as drought and salinity, China’s annual needs more than sixty million tons of soybeanimport to meet demand. Therefore, breeding for high yielding, high quality, stress tolerance soybeanvariety is needed to solve major problems in China’s soybean industry. Recent evidence indicates thatplant microRNAs have an important function in adaptive responses to abiotic stresses. Thus, toelucidate the molecular adaptation mechanisms of stress and stress regulatory networks, breeding forhigh yielding, high quality, stress tolerance soybean variety, functional studies of stress-responsivemiRNA in soybean has high scientific significance and application value. In this paper, we described indetail the biological functions of soybean gma-miR169c and its target gene GmNFYA3, andgma-miR394a in drought stresses. This research will explain the mechanism and regulation network ofsoybean drought tolerance, provides the theoretical basis for soybean stress tolerance research andcultivation of new varieties. The main results were listed as follows:1. In this study, we identified and characterized a gene, GmNFYA3, which encodes the NF-YAsubunit of the NF-Y complex in soybeans. Real time RT-PCR analysis indicated that GmNFYA3wasinduced by abscisic acid (ABA) and abiotic stresses, such as PEG, and cold. Subcellular localizationanalysis suggested that GmNFYA3may activate its specific targets in the nucleus. Histochemicalβ-glucuronidase (GUS) staining revealed that the expression of the GUS gene driven by the GmNFYA3promoter occurred in various transgenic Arabidopsis tissues. Coexpression in Nicotiana benthamianaand5’ RACE assays indicated that miR169directs GmNFYA3mRNA cleavage in vivo. Overexpressionof GmNFYA3resulted in Arabidopsis with reduced leaf water loss and enhanced drought tolerance. Inaddition, the transgenic Arabidopsis exhibited increased sensitivity to high salinity and exogenous ABA.Moreover, the transcript levels of ABA biosynthesis (ABA1, ABA2), ABA signaling (ABI1, ABI2) andstress-responsive genes, including RD29A and CBF3, were generally higher in GmNFYA3plants than inwild-type controls under normal conditions. These results suggest that the GmNFYA3gene functions inpositive modulation of drought stress tolerance and has potential applications in molecular breeding toenhance drought tolerance in crops.2. In this study, a soybean gma-MIR394a gene was functionally characterized, especially withregard to its role in drought stress resistance. Expression analysis revealed that gma-MIR394a wasexpressed differentially in various soybean tissues and was induced by drought, high salinity, lowtemperature stress, and abscisic acid treatment in leaves. One target gene of gma-miR394a,Glyma08g11030, was predicted and verified using a modified5’ RLM-RACE (RNA ligase-mediatedrapid amplification of5’ cDNA ends) assay. Overexpression of gma-MIR394a resulted in plants withlowered leaf water loss and enhanced drought tolerance. Furthermore, overexpression of gma-MIR394a in Arabidopsis reduced the transcript of an F-box gene (At1g27340) containing a miR394complementary target site. These results suggest that the gma-MIR394a gene functions in positivemodulation of drought stress tolerance and has potential applications in molecular breeding to enhancedrought tolerance in crops.