Study On The Role Of Lj5NG4 And LjbHLH93 In The Establishment Of Nodule Symbiosis In Lotus Japonicus

The root nodule symbiosis system of legumes is the most important source of biological nitrogen fixation in nature.Nodulation factor secreted by rhizobia when they perceive flavonoids produced by legumes is regarded as the initiation of symbiotic signal transduction.The nodulation factor receptors NFR1 and NFR5 are important in this process.The study of signal transduction mechanism in the process of leguminous root nodule formation,especially during the establishment of early symbiosis,is of great significance for revealing the cooperative cross-regulation between symbiotic signaling and immune response in legumes.At present,the downstream signaling mechanism of NFR1 has not been fully clarified,identifying protein interacted with LjNFR1 and revealing their symbiotic signal regulation network are the focus of research.In this study,using the root nodulation factor receptor kinase LjNFR1 as a bait and a yeast membrane two hybrid library constructed with transcripts harvested at different stages of nodule symbiosis,we identified an auxin related transporter protein Lj5NG4.Then taking Lj5NG4 as the object,the function and mechanism of symbiotic nitrogen fixation in Lotus japonicus were analyzed,the main results were as follows:1.Phylogenetic tree analysis of 5NG4 proteins from different species showed that Lj5NG4 belongs to the nodulin MtN21/EamA-like transporter protein family,which was regulated by auxin induction and homologous protein was identified as auxin transporter.In addition,phylogenetic tree analysis of Lj5NG4 and the known auxin transporters showed that they were in different evolutionary branches.2.In order to observe the expression pattern of Lj5NG4 in symbiosis,the microscopic observation of tYFP fluorescence protein signal confirmed that Lj5NG4 was strongly expressed in root hairs at the early stage of infection,but not not in inner mature nodules,suggesting that Lj5NG4 was more functional in the early stage of symbiosis infection.3.In order to further verify the existence of interaction between Lj5NG4 and LjNFRl in Lotus japonicus,yeast two hybrid and Split-Luc in tobacco were used to confirm that.4.CRISPR/Cas9 was used to knockout Lj5NG4,and stable transformation of Lotus japonicus was conducted.After two generations of screening,homozygous stable mutants in T2 generation were obtained.The statistical results of symbiotic phenotype of mutant strains showed that Lj5NG4 inhibited the formation of infection threads in the early stage of symbiotic nitrogen fixation,and negatively regulated the infection in the early stage,but had no significant effect on nodule development And the result was further confirmed by complementary experiments.5.Given that Lj5NG4 may be an auxin transporter,the effect of auxin can be further confirmed by exogenously applying auxin of different concentrations.The data showed that low concentrations(10-7,10-8,10-9 M)of auxin will weaken Lj5NG4 mutant phenotype,play positive regulation function in nodule symbiosis development.Soybean saponins are of great significance in regulating soybean traits and qualities.?-Amyrin synthase(AMS)is a key speed-limiting enzyme in the saponin synthesis pathway,which is of great significance for research.Using the promoter of GmAMS as bait,teacher Jin's research group obtained Glyma04G and Glyma06G?which belong to the bHLH protein family.Due to the gene multiplication of soybean genome,which brings a lot of inconvenience to the research,the homologous gene Lj1g3v2098310.1 was used for research?and named as LjbHLH93.With this as the research object,the research results were as follows:1.Microscopic observation with the tYFP fluorescent protein signal confirmed that LjbHLH93 was not expressed in the young nodules,but was strongly expressed in the infected cells inside the mature nodules,indicating that LjbHLH93 may affect the nodules by regulating the infected cells of mature nodules.2.The interaction between LjbHLH93 protein and LjAMS could not be verified by EMSA,and LjAMS was found to be self-activated in yeast,and the interaction between LjbHLH93 and LjAMS could not be verified by yeast one hybrid either.3.LjbHLH93 was knocked out by CRISPR/Cas9 technology,and stable transformed mutants could not be obtained,but the nodule numbers of mutants obtained by hair root transformation was significantly reduced.However,no significant changes in the symbiotic phenotype of mutants which were observed by overexpression technique.