| The symbiotic nitrogen fixation established in legumes and rhizobia is widely studied and applied,which has significant application value in agriculture,environment and economy.At present,numerous researchers have begun to update and improve the molecular mechanisms of symbiotic nitrogen fixation in rhizobia-legumes from different aspects such as rhizobia,host plants,and the external environment.As a key gene in the symbiotic signaling pathway,nod factor receptors(NFR)play a crucial role.Nod factor receptors are a class of LysM receptors that recognize nod factors(NF)and activate symbiotic signaling pathways,eventually causing rhizobium to invade host plants and form nodules.For the later nodule senescence,in addition to the influence of external factors,a series of physiological and biochemical changes were also triggered,including the expression of Cysteine proteases.Based on the background of co-transformation of LjNFR1 and LjNFR5 into Medicago truncatula that inoculated with M.loti MAFF303099 which can form nodules,in the first chapter of this article,the Glycine max,Medicago truncatula and Lotus japonicus nod factor receptor mutants were used as material to explore the structures and functions of NFR in Glycine max and Lotus japonicus,aiming to expand the host range of rhizobia and to perfect the symbiotic signaling pathway.In the second chapter,MG20 were used as materials to explore the role of cysteine protease genes in the nodule senescence of Lotus japonicus,providing theoretical basis and experimental materials for the later experiment.The main results are as follows:1.Co-transformation of LjNFR1-LjNFR5 complements Gmnfr5 mutant,which could form nodules when inoculated with Bradyrhizobium japonicum rhizobium,but could not form nodules when inoculated with Mesorhizobium loti rhizobium.However,GmNFR1α-GmNFR5α co-transformation complement Mtnfp mutants could not develop nodules when inoculated with Bradyrhizobium japonicum rhizobium.2.Using GmNFRs and LjNFRs as research subjects,we changed the intracellular domain and extracellular domain structures of NFRs.It was shown that GmNFR1α does not fully complement Ljnfr1 mutants,whereas GmNFR5α can fully complement Ljnfr5 mutants to form normal nodules.It also shows that the extracellular domain of LjNFR is an absolutely conserved structure and plays a vital role in the symbiosis process,affecting whether symbiosis can proceed normally.3.Using biological software analysis,8 LjCYPs that may be associated with nodule senescence were obtained.Based on this,Real-time PCR was used to detect the expression of 8 LjCYPs in the later stage of Lotus japonicas nodules.The results showed that LjCYP1,LjCYP3,LjCYP5,Lj1g3v4047290.1 were up-regulated expression in the later stage of Lotus japonicas nodules(7 to 10 weeks).In particular,the up-regulation expression of LjCYP5 was evident.4.Using the CRISPR-Cas9 system and Lotus japonicus hairy root transformation method to kcock out individual LjCYP3 and dual LjCYP1-LjCYP5 respectively,we found that the genomes of Lotus japonicus nodules were edited,mainly generating base deletion and insertion,which finally affected the nitrogen fixation efficiency and senescence of Lotus japonicus nodules.Firstly,using TUNEL assay to detect 7 weeks nodules,single knockout of LjCYP3 and double knockout of LjCYP1-LjCYP5 both could effectively delay root nodule apoptosis and senescence.Secondly,using acetylene reductase method to detect nitrogenase activity of 9 weeks nodules,single knock-out LjCYP3 and double knock-out LjCYP1-LjCYP5 both could significantly increase nodules nitrogenase activity.Thirdly,from the point of view of nodule senescence,the number of red nodules was significantly more than control when LjCYP3 was knocked out.When double knock-out LjCYP1-LjCYP5,the number of red nodules was significantly more than that of the control,and the senile nodules was significantly less than the control. |
