Isolation And Characterization Of Early Symbiosis Signal Pathway Genes In Soybean

Soybean is one of the main economic and most important oil crops in the world. The leguminous symbiotic system is the most important way of nitrogen fixation that plays a vital role in agricultural production. The focus of current studies has been done on the symbiotic relationship between legumes and rhizobia for nitrogen fixation in the model plant. The identification and characterization of early nodulation genes in soybean have not yet been studied.In this study, we cloned and identified the early soybean nodulation genes i.e Sym RK, CCaMK, NSP1, NSP2, NIN to explore the Soybean-rhizobia nodulation signaling pathway. The overexpression and downregulation of GmSymRKα/β、GmCCaMKα/β、GmNSP1α/β、GmNSP2α、GmNINα genes were done in the soybean hairy roots to identify their functions in soybean nodulation process. GFP was used as control. The results showed that overexpression of these genes significantly increased the nodulation in Soybean hairy roots than the control. The down-regulation of GmSymRK and GmCCaMK genes through RNA interference significantly decrease the nodulation as compared to control in Soybean hairy roots. It was concluded from these results that GmSymRKα/β 、GmCCaMKα/β、GmNSP1α/β、GmNSP2α、GmNINα genes have an imperative role in soybean nodulation.We also done GmCCaMKα/β、GmNSP1α/β gene complementation in Medicago truncatula mutant hairyroots and overexpression in wild type A17 for further confirmation of these genes role in nodulation. The results showed that complementation of GmCCaMKβ and GmNSP1α/β had recovered their nodulation ability in Medicago truncatula mutant lines mtdmi3 and mtnsp1-1, repactively. Meanwhile, the overexpression of these genes in wild type A17 had significantly increased its nodule number. It was summarized from these results that GmCCaMKβ and GmNSP1α/β efficiently complement the biological function of DMI3 and NSP1 genes in M. truncatula, respactivvely.QRT-PCR were performed to detect the interaction among the gene expression level. Results showed that overexpression of GmSymRK gene up-regulated the GmCCaMK,GmNSP1 and GmNSP2 genes expression while inhibition of GmSymRK gene down-regulated the expression of GmCCaMK,GmNSP1 and GmNSP2 genes. Overexpression of GmCCaMK up-regulated the expression of GmNSP1 and GmNSP2 while inhibition of GmCCaMK expression down-regulated the GmNSP1 and GmNSP2. The GmNSP2 and GmNIN genes were upregulated by overexpressing the GmNSP1. Overexpression of GmNSP2α down-regulated the GmCCaMK and GmNINα genes while GmNINα down-regulated the GmNSP2. It has been suggested from the results that GmSymRK were located in the upstream of GmCCaMK, Gm NSP1 and GmNSP2 while GmNIN were located in the downstream of GmCCaMK. GmNSP2α have a negative feedback for GmCCaMK regulation and GmNINα have negative feedback for GmNSP2 regulation. So the early nodulation signaling pathway is very suitable in soybean for legumes-rhizobia symbiosis process.