Mechanism Analysis Of Symbiosis Receptor-like Kinase Interacted With SERKs Proteins And Application Of CRISPR/Cas9 In Symbiotic Nitrogen Fixation Research

The mechanism of symbiosis signal transduction has been an advance hotspot in this field.Currently,a series of regulators involved in symbiotic nitrogen fixation network has been identified.SymRK is a critical gene in symbiotic signaling pathway,its mutant leads to the interruption of early signal transduction.Previous research in our lab verified that LjBAK1/SERK3 interacted with the protein kinase domain of SymRK by using the yeast two-hybrid system.Base on the above background,in this report,we focus on LjBAKl/SERK3 andLjSERK2 and their functions in the symbiotic nitrogen fixation pathway of Lotus japonicus,the results were shown below:1.Higher structure imitation and domain analysis of SERK proteins of Lotus japonicus reveals that both two of them are highly homologous with AtBAKl/SERK3 and AtSERK2,respectively(up to 90%),so we name these 2 proteins LjBAKl/SERK3 and LjSERK2.2.Kinase activities of LjSERK2 and LjBAK1/SERK3 are identified by using autoradiography.Phosphorylation assay indicates that LjBAKl/SERK3 is phosphorylated by SymRK,but no phosphorylation between LjSERK2 and SymRK.3.LjSERK2 didn't interact with SymRK,whereas the kinas-negative mutant of LjBAK1/SERK3 interacted with the protein kinase domain of SymRK and its kinase-negative mutant.Combined with the result about phosphorylation in vitro,it suggested that LjBAKl/SERK3 may play a role in signal transduction of SymRK in symbiotic pathway.4.After incubation with M.loti five days,we found that the number of infection threads and nodule primordia in LjBAK1/SERK3 LORE1 inserted mutants was significantly increased with wild type.CRISPR/Cas9 system is newly developed sequence-specific nucleases in genome editing.This system recognition the target sites of specific gene by RNA-DNA complementary base pairing.For this reason,the CRISPR/Cas9 system is a simple,inexpensive and versatile tool for genome engineering in plants.In the second chapter of this article,we combined CRISPR/Cas9 system with hairy root transformation.CRISPR/Cas9 is applied to edit multiple sites in Lotus japonicus.The main results show below:1.Referring to PTG-Cas9 system,we transformed the expression of sgRNA casette into polycistronic tRNA-gRNA model and change the AtU6-26 promoter with LjU6 promoter,which was suitable for Lotus japonicus.2.Combined with hairy root transformation,CRISPR/Cas9 is applied to edit multiple sites in nod factor receptor 1(NFR1)of Lotus japonicus.3.Analysis the phenotype of transgenic hairy roots,we found that the editing efficiency of target 1 in LjNFRl is 58.3%,but no mutation is detected on target 2.4.Further sequencing analysis demenstrates that targeted mutation by CRISPR/Cas9 system is insertion or deletion.The mutation of target 1 was 58.3%,while no mutation was found in target 2.