Mechanism Of NAC Transcription Factor GmSIN2 And Its Interacting Protein GmNIW In Regulating Biotic And Abiotic Stresses Response In Soybean

Abiotic stress and biotic stress seriously restrict the growth and yield of plants,especially crops.Identification of the key genes and elucidating their mechanisms in regulating stress responses is important to the development of modern agriculture.Plant-specific NAC transcription factors are recognized as important components for the regulation of multiple stress responses.So far,NAC transcription factors that can simultaneously promote abiotic and Biotic stress resistance have not been found in soybean.Former study showed that a multiple stress induced soybean NAC transcription factor,GmSIN2,which improved the yield and salinity tolerance of overexpression transgenic soybean in field.Further,Y2H and BiFC showed that GmSIN2 interacts with GmNIW,a WD40 protein with unknown function in soybean.Both 35Spro:GmSIN2 and 35Spro:NIW transgenic Arabidopsis showed improved salt tolerance and gray mold resistance.Using transcriptomic analysis in 35Spro:GmSIN2 soybean,we found that GmSIN2 positively regulates genes related to JA synthesis and signaling pathway.To explore the mechanism of GmSIN2-GmNIW interaction in regulating abiotic and biotic stress responses in soybean,the following research was carried out in this thesis:1 GmSIN2 promotes JA accumulation and directly regulates the expression of key genes related to JA synthesis and signal transduction pathwayThe JA content measurement of 35Spro:GmSIN2 transgenic soybean showed that GmSIN2 promotes the accumulation of endogenous JA.Furthermore,EMSA was used to identify GmSIN2 specific binding motifs on the GmPHRs(key genes related to JA synthesis and signal transduction pathway)promoters,and to verify their binding with GmSNI2 in vitro binding.ChIP-qPCR was used to demonstrate that GmSIN2 directly binds to the GmPHRs promoter fragments containing specific motifs in soybean,and salt treatment has varying degrees of impact on their binding.Using the transient expression system of LUC reporter gene in Arabidopsis protoplasts,it was demonstrated that GmSIN2 positively regulates the expression of downstream genes driven by GmPHRs promoters,respectively,and the positive regulation requires the GmSIN2 specific binding motifs on the promoters.2 GmNIW interacts with GmSIN2 in vivo and promotes the protein accumulation of GmSIN2.The interaction between GmSIN2 and GmNIW was demonstrated using Co-IP with transgenic soybean hairy roots and GmSIN2 antibody.Using the tobacco transient expression system,it was found that GmNIW promotes the accumulation of GmSIN2 protein.3 GmSIN2 and GmNIW improve plant salt tolerance and gray mold resistance respectivelyOverexpression of GmSIN2 in soybean hairy roots replicated the growth promotion and salt tolerance phenotype of GmSIN2 stable transgenic plants,indicating that the root may be the site of action of GmSIN2 in promoting growth and salt tolerance.On the other hand,the transient expression of GmNIW increased the gray mold resistance in tobacco leaves.These provide new evidences that GmSIN2 and GmNIW regulate plant tolerance to biotic and abiotic stresses.4 GmNIW promotes JA accumulationIn order to better study the functions of GmNIW in regulating salt tolerance and disease resistance in soybean,T2 generation 35Spro:GmNIW-GFP transgenic soybean lines were obtained using agrobacterium-mediated transformation method.It was found that the endogenous JA content was significantly higher in 35Spro:GmNIW-GFP lines than in wild-type,which is consistent with the phenotype of GmSIN2 overexpressing transgenic soybeans.This further suggests that GmSIN2 and GmNIW synergistically promote JA accumulation through protein interaction.In conclusion,this study preliminarily revealed a pathway that GmNIW promotes the accumulation of GmSIN2 through protein interaction,GmSIN2 directly regulates the expression of GmPHRs and promotes JA synthesis to positively regulate the salt tolerance and gray mold resistance in soybean.These results lay a theoretical foundation and provide gene resources for the molecular design breeding of crop with abiotic and biotic stress dual resistance.