Effect Of Nanometer Molybdenum Oxide On Co-expression Network Of Genes Related To Symbiotic Nodules And Flavonoid Synthesis In Soybean

Molybdenum（Mo）is an important component of nitrogenase in leguminous crops,and the lack of molybdenum leads to adecrease in soybean yield and quality.The results of previous studies showed that nano-molybdenum oxide was better than conventional molybdenum fertilizer in promoting nodulation and yield increase of legume crops,but its regulatory mechanism was unclear.In this paper,the quartz sand cultivation test was used to confirm that nano-molybdenum oxide improved the nitrogen fixation capacity of soybean nodulation.The effects of molybdenum oxide on the composition of key genes and signaling substances flavonoids in soybean nodulation nitrogen fixation were comprehensively analyzed bydynamic transcriptome and targeted metabolome technology.The role of nano-molybdenum oxide in promoting the formation of soybean nodules and biological nitrogen fixation was explored,and the effects anddifferential mechanisms of nano-molybdenum oxide and ionic molybdenum on symbiotic nitrogen fixation in soybean were revealed.The main results obtained were as follows:1.The Effect of MoO₃NPs on the Growth Rate and Morphology of Rhizobia.Compared with the control,MoO₃ NPs increased the OD value of rhizobia（32-48hours）within the concentration range of 0.01-10 mg/L,and the morphology of rhizobia was more full under low concentration（0.1 mg/L）MoO₃NPs treatment.Underhigh concentrations（500 mg/L and 1000 mg/L）of MoO₃ NPs treatment,the OD values（12-48hours）of rhizobia were significantly lower than control,and the morphology of rhizobia was slender,with some rhizobia cellsdisintegrating.This indicated that low concentration of MoO₃NPs has a promoting effect on the growth of rhizobia,whilehigh concentration of MoO₃NPs showed a significant inhibitory trend on the growth of rhizobia.2.MoO₃NPs enhanced soybean nodulation and nitrogen fixation ability.At Mo levels of 0.01 mg/kg and 0.10 mg/kg,the application of ionic molybdenum fertilizer and nano molybdenum oxide could both increase the number of soybean root nodules,fresh weight of root nodules,and nitrogenase activity.however,the increasing was greater when the Mo level is 0.10 mg/kg,and the effect of applying MoO₃NPs was better than that of ionic molybdenum fertilizer.The expression levels of nitrogenase component proteins nif K,nif H,and leghemoglobin LB in MoO₃NPs and ionic molybdenum fertilizer after inoculation with rhizobia for 21,28,and 35days werehigher than control.Moreover,the expression levels of key proteins in nitrogen fixation processes under nano-molybdenum oxide treatment werehigher than those under ionic molybdenum fertilizer.When the Mo level was 0.10mg/kg,the nitrogen accumulation of plants cultured with MoO₃NPs and ionic molybdenum fertilizer increased by 56.9%and 31.5%,respectively.These results manifest that molybdenum application significantly increased the nodulation and nitrogen fixation ability of soybeans,and under the same molybdenum level,the effect of nano molybdenum oxide on promoting nodulation and nitrogen fixation of soybeans was better than that of ionic molybdenum fertilizer.3.MoO₃NPs regulated the expression of nitrogen fixation related genes in soybean roots.Transcriptome analysis showed that exogenous application of molybdenum mainly regulates the expression of nodule symbiosis related genes and flavonoid synthesis related genes.After 3d of inoculation,both MoO₃NPs and ionic molybdenum fertilizer upregulated the expression of tumor related genes such as Gm N56,Gm NIC2,LBC,Noulin-23,and Noudlin-27.Among them,ionic molybdenum fertilizer treatment significantly upregulated the expression of tumor related genes such as ENOD93 and LBA,while MoO₃ NPs upregulated the expression of tumor related genes such as ENOD55-2 and LBC3.Mo mainly upregulated the expression of genes such as CHS5,LOC114412891,and Gm IRCHS on the 7thday of inoculation.On the 11thday of inoculation,it upregulated the expression of genes such as CHS8 and CHR6.MoO₃NPs mainly upregulated the expression of genes such as Gm IRCHS and LOC100801369 on the 7^th day of inoculation.On the 11thday of inoculation,it upregulated the expression of genes such as CHS6 and F3’H.4.MoO₃NPs affected the composition and content of root flavonoids in soybean.The composition and content changes of flavonoids were analyzed by targeted metabolomics technology.Compared with the control group,229 kinds of flavonoids showed significant changes in content after nano molybdenum oxide treatment,and 194 kinds of flavonoids showed significant changes after ionic molybdenum treatment.Among them,MoO₃NPs treatment significantly regulated the content of apigenin,epiaf catechin,genistein,rutin and other compounds at two time points,and ionic molybdenum treatment significantly regulated the content of quercetin,sumacin,daidzein,kaempferol and other compounds at two time points.5.Dynamic transcriptome and targeted metabolome joint analysis identified the co-expression network and core genes of molybdenum regulating flavonoid synthesis.Based on thedifferentially expressed genes and thedynamic changes of flavonoid components,the co-expression network of MoO₃NPs and ionic molybdenum fertilizer response was constructed,and five key core genes regulating flavonoids were screened.The key gene responsible for the co response of MoO₃NPs and ionic molybdenum fertilizer is Gm CHS9,which encodes chalcone synthase.The key genes specifically responsive to ionic molybdenum fertilizer were Gm CHS5 and Glyma.08G110420.Wm82.a4.v1,which encode genes chalcone synthase and flavonoid synthase,respectively.The key genes Glyma.16G033700.Wm82.a4.v1 and LOC10077825 that specifically respond to NMo were responsible for encoding UDP glycosyltransferase andd FR flavonone 4-reductase,respectively.After inoculation with rhizobia,the expression levels of Gm CHS9 and Gm CHS5 treated with NMo were significantlyhigher than those of ionic molybdenum and control at all time points.It is speculated that MoO₃NPs can regulate the synthesis of flavonoids by affecting chalcone synthase activity.In summary,MoO₃NPs and ionic molybdenum can affect the nitrogen fixation ability of soybean-rhizobium symbiosis by regulating the expression level of root genes and the secretion and composition of flavonoids,which elucidates the key factors that promote the symbiotic nitrogen fixation of soybean-rhizobium.The results of this study can provide reference for the agricultural application of MoO₃NPs oxide,and provide a new way to improve the symbiotic nitrogen fixation efficiency of legume crops.