Function Analysis For LjSERKS Gene And Activity Identification Of Ubiquitin-conjugating Enzyme LjE2

In legumes,symbiosis and immunization have gradually become the focus of discussion,and related researches have also been widely carried out.Although there are many reasonable speculations about symbiosis and immunity,no breakthrough was made in the experiment.In our study of the immune key kinase LjSERK,the symbiotic key kinase LjSym RK was able to phosphorylate with LjSERK2 and LjSERK3 in vitro.This suggests that LjSERK may be involved in regulating the symbiotic nitrogen fixation pathway,and that LjSym RK may also be involved in the regulation of immune responses.Using these studies as a background,this study will explore the effects of LjSERK2 and LjSERK3 on symbiotic nitrogen fixation by observing the symbiotic phenotypes of LjSERK2 and LjSERK3 mutants.In the ubiquitination reaction,it is generally believed that the type of modification of the substrate is mainly determined by E3.However,recent studies have shown that the specificity between E2 and E3 also plays an important role in the type of substrate modification.Therefore,the traditional in vitro ubiquitination system can no longer meet the needs of research.It is very necessary to establish a specific in vitro ubiquitination assay system for the species itself.This article will analyze and clone the potential E2 ubiquitin-binding enzyme in Lotus corniculatus,and detect its protease activity in vitro by ubiquitination assay.The aim is to provide experimental materials for the establishment of Lotus japonicus-specific in vitro ubiquitination system.The results of the study are as follows:1.Overexpression of LjSERK2 and LjSERK3 in Atbak1-4 mutant found: the plant height of LjSERK2 transgenic plants was significantly increased and the flowering period was advanced.The petiole of the LjSERK3 transgenic plant was significantly longer and the single leaf area became larger.The phosphorylation of MAPK3/6 in transgenic plants was significantly increased after treatment with flg22.The results showed that LjSERK2 and LjSERK3 can partially restore the Atbak1-4 mutant phenotype,and there may be functional redundancy between LjSERK2 and LjSERK3.2.LjSERK2,LjSERK3 knocked out in Lotus japonicus using CRISPR-Cas9 system found: In the LjSERK2,LjSERK3 single knockout and LjSERK2/LjSERK3 double knockout mutants,the total number of nodulated tumors was significantly reduced at 2 weeks after rhizobia inoculation.However,there was no significant change in the nodule density.At the same time,mutants showed developmental phenotypes such as plant dwarfism and short root system.The results showed that LjSERK2 and LjSERK3 had no significant effect on the nodulation density,but played an important role in plant growth and development.3.Overexpression of LjSym RK in wild-type Arabidopsis found: Compared with the empty vector control,transgenic plants overexpressing LjSym RK were insensitive to flg22 treatment,both the phosphorylation level of MAPK3/6 and the transcription level of immune-related Marker genes were significantly lower than those of wild-type.These results indicate that overexpression of LjSym RK in Arabidopsis can partially inhibit the flg22-mediated immune response.4.Using At E2 sequence,14 LjE2 genes were predicted by homologous sequence alignment and phylogenetic analysis was performed.5.The 11 fusion expression vectors p ET-LjE2:His were successfully constructed and 10 LjE2 proteins were induced and purified by prokaryotic expression system.6.Successfully identified 9 LjE2 with ubiquitin-conjugating enzyme activity by in vitro ubiquitination experiments successfully.