Functional Research Of Soybean(Glycine Max [L.] Merr.) MADS-box Gene GmNMH7 In Root Development And Nodulation

Soybean,an important food crop and oil crop,is capable of nitrogen-fixing by root nodules.GmNMH7,a transcription factor of MADS-box family,may be associated with nodules development,but its specific function remained unknown.It is expected that the systematic study of the gene will provide a molecular basis for determining the role of the gene in root development and nodulation regulation.From the aspects of subcellular localization,gene expression determination,functional identification and preliminary mechanism analysis,we got the main research results,shown as follows:1.GmNMH7 gene CDS sequence was cloned in this study,which is 684 bp long.Results of subcellular localization showed that GmNMH7 protein located in the cell membrane and nucleus.2.Relative expression level of GmNMH7 gene measured by Real-time PCR in roots and nodules was much higher than that in other tissues.Expression pattern of GmNMH7 gene in 10 days after rhizobia inoculation was shown to be similar to GmENOD40-1,Gm ENOD40-2,and GmNFR5α,several genes regulating early development of nodules.The expression peak of GmNMH7 gene even presented earlier than the other genes indicating that the gene was related to the Nod Factor signaling pathway and that the gene was likely to have function at the early development of nodules.3.Over-expressing GmNMH7 gene in hairy roots reduced the nodule number and the root length significantly.In the positive transgenic hairy roots,over-expressing GmNMH7 gene down-regulated the expression levels of GmENOD40-1,GmENOD40-2 and GmNFR5αsignificantly.4.Results of bioinformatics analysis for GmNMH7 promoter revealed that there were many cis-acting elements associated with nodulation and phytohormone response in it.We constructed engineering strains K599::pC13P1-GmNMH7pro::GUS,which were used for inducing transgenic hairy roots harboring p C13P1-GmNMH7pro::GUS plasmids.GUS staining results showed that prior to rhizobia inoculation,GmNMH7pro::GUS was characterized by vascular bundle specific expression,which may be related to some material transportation.Gm NMH7pro::GUS expressed abundantly in nodules after 10 days of inoculation,meaning that GmNMH7pro lost the specificity of the expression of vascular bundle induced by rhizobia.The expression was hardly observed neither in nodules nor vascular bundle after 28 days of rhizobia inoculation,suggesting a weak expression of GmNMH7pro::GUS in mature nodules.5.This study demonstrated regulation interactions between GmNMH7 gene and ABA and GA₃.The expression of GmNMH7 gene was capable to response to ABA and GA₃ treatment in the roots of Zigongdongdou seedlings.In the positive transgenic hairy roots,over-expressing GmNMH7 gene reduced the content of ABA,but increased the content of GA₃.Transgenic Arabidopsis thaliana responded to GA₃ more apparently than to ABA.Therefore,we draw a conclusion that GmNMH7 gene participates in the Nod Factor signaling pathway and regulates nodulation negatively probably through regulating the content of GA₃.