Functional Analysis Of GmCRN1 In Root Architecture Modification And Nodulation Of Soybean

Soybean is rich in abundant protein and oil,and thus plays an important role in human daily life.In addition,soybean is also the typical biological nitrogen fixation（BNF）crop,providing sufficient and clean nitrogen source for plant growth and development,and it is of great significance for the development of green and sustainable agriculture.Nitrogen（N₂）fixing nodules are specialized organs formed on the host roots after rhizobia infection.Previous studies have shown that soybean varieties with shallow root system displayed the better three-dimensional architecture,it can not only better absorb nutrients from the soil,but also more efficient for nodulation with rhizobia.Furthermore,inoculation with rhizobia was able to change root architecture as well,suggest that root architecture modification is closely related to nodulation and N₂ fixation.However,the genetic and molecular mechanisms of root architecture（RA）modification and nodulation are still unclear.Previously,QTL mapping was performed using 175 F_9:11 recombinant inbred lines（RIL）constructed by parents JD12 and NF58 with significant differences in root architecture and nodulation traits,and many QTLs for controlling RA and nodulation were found in this population.Among them,q BNF-C2 showed the highest LOD value,which is an important genetic loci for controlling RA modification and nodulation.However,the major candidate gene cloning and its function in this loci remain unknown.Therefore,the project is proposed on the basis of previous works and combine with the bioinformatics technology to clone the major candidate gene for q BNF-C2,and named GmCRN1（Control Root Architecture and Nodulation 1）according to its possible biological function,and to further explore the biological function of GmCRN1 in root architecture modification and nodulation in soybean.The main results are as follows:（1）Using the soybean transgenic composite plants to analyze the phenotype of overexpressing GmCRN1 and RNAi lines,and found that overexpression of GmCRN1significantly promoted soybean nodulation,increased nodule number,improved the nitrogen fixation capacity,promoted root growth,and thus soybean biomass,while RNAi of GmCRN1showed the opposite phenotype.It is revealed that GmCRN1 is indeed the major gene for q BNF-C2.（2）Through GmCRN1 sequence analysis from parental soybean,it was found that a SNP variation caused the amino acid change from arginine to threonine.Subcellular localization showed that GmCRN1 belongs to JD12-type was localized in nucleus,and the mutanted protein Gmcrn1 was partially retained in cytoplasm.It implied that the SNP variation on the exon of GmCRN1 may be the key reason to cause the phenotypic differences in RA and the nodulation in the RILs.（3）Bioinformatics analysis showed that GmCRN1 is highly conserved and it is a unique transcription factor in legumes,and there is no homologous gene in other non-legume plants.Soybean contains two GmCRN1 homologous and the amino acid sequence similarity was showed over 90%.（4）GmCRN1 was expressed in flowers,leaves,pods,roots and nodules.GmCRN1promoter drive GUS in transgenic composite plant was further used to analyze its histochemical localization in roots and nodules,and found that the expression of GmCRN1started from the vascular tissues of roots,and then GmCRN1 mainly expressed in the junction of roots and nodules at the early stage of nodule formation.With the development of nodules,GmCRN1 is mainly localized in nodule epidermis,cortex and bacteroids.These results indicated that GmCRN1 expression plays a role root growth and nodule development in soybean.（5）Hydroponic experiments of near-isogenic lines showed that functional GmCRN1significantly increased nodule number,nodule dry weight,number of infected cells in the bacteroids,and thus improved the N₂ fixation capability of nodules.In addition,compared with Gmcrn1 non-functional type,GmCRN1 significantly promoted root growth,increased root length,root surface area and average diameter,suggest that GmCRN1 synergistically regulated the growth and development of roots and nodules in soybean.（6）GmCRN1 complementation in Gmcrn1 line showed that complementing GmCRN1restored soybean nodule number,nodule dry weight,N₂fixation capability of nodules,as well as significantly promoted root growth,and thus plant biomass.（7）Grafting experiments showed that the promotion of GmCRN1 on soybean nodulation was not dependent on the GmCRN1 in shoots.When the rootstock is functional GmCRN1,it significantly promoted soybean nodulation and root growth,increased the number of nodules,nodule dry weight,and improved the N₂fixation capacity of nodules,and thus plant biomass.In summary,this study is proposed on the basis of previous works in our laboratory on the QTL for RA and nodulation traits to further study the functional analysis of the candidate gene GmCRN1 on regulates soybean nodulation and RA.The results showed that GmCRN1promoted soybean nodulation,root growth and increased plant biomass.This study is preliminarily exhibited that GmCRN1 plays a synergistic regulation in root architecture modification and nodulation in soybean.These results could provide candidate genes or fragments for genetic improvement of soybean with high BNF efficiency,and also provide a theoretical basis for the study of the cooperative regulation mechanism of root architecture modification,nodulation and N₂ fixation in soybean.