Function And Molecular Mechanisms Of Symbiotic Nitrogen Fixation In Lotus Japonicus And Its Application In Rice

Legume-rhizobia symbiotic nitrogen fixation is the most efficient biological nitrogen fixation process,which is important in agricultural production,such as soybeans and Medicago truncatula.Because of these advantages such as smaller genome,shorter growth period and stronger matching with rhizobia,Lotus japonicus is widely used as a model of legume in the study of symbiosis.In the early symbiotic period,rhizobia express Nod genes and synthesize nod factors（NF）by perceiving flavonoids which are secreted from plant roots.In the root hairs,nod factor receptors（NFR）could recognize the nod factor to initiate the symbiotic signaling,which induces calcium spiking and expression of symbiosis-relate genes via a common symbiosis pathway（CSP）.To establish RNS,rhizobia need to reside into nodules in the root cortical cells via infection threads（IT）.The infection threads and the nodule primordia（NP）almost simultaneously initiate and develop in the process.The released Rhizobia are enveloped by plant cell membrane to form symbionts,which grow and develop in plant cells and eventually form bacteroids.Right now,the nodule primordia also develop into ripened nodules with nitrogen fixing ability.In root nodules,rhizobia utilize nitrogenase system to convert nitrogen into ammonia for plant growth,while plants provide carbohydrates and other carbon sources for rhizobia via photosynthesis.However,root nodule symbiosis（RNS）is limited in legumes and very few non-legumes,and the main food crops（rice,etc.）do not have the ability to symbiotic with rhizobia,which greatly limits the application of root nodule symbiosis in agricultural production.In order to solve these problems,this study explored the molecular mechanisms of two symbiotic genes in Lotus and explored the feasibility of symbiotic nitrogen fixation in non-legumes.In chapter 1,we briefly introduced the nitrogen cycle on the earth,the significance of biological nitrogen fixation,the origin of root nodule symbiosis,and the molecular mechanism of root nodule symbiosis.In chapter 2,The role of the various domains of the symbiosis receptor kinaseSYMRK in the RNS was studied using domain swap.We found that the intracellular domain of SYMRK played a key role in symbiosis,while the extracellular domain participated in very fine tune regulation in the invasion process of Rhizobia.MLD and LRR domains might work together to be involved in symbiotic signaling and the LRR domain might play a negative role in Lj SYMRK^GDLC-mediated RNS.Bymutagenizing the conserved amino acids on LRR domain,five serine residues were found to be required for the function of Lj SYMRK^GDLC in RNS.These findingprecisely refine the molecular mechanisms of SYMRK function in symbiotic signaling in L.japonicus.In chapter 3,nine symbiosis related genes were regulated by different promoters,and then six multigene expression vectors were constructed using Transgene-Stacking II.We transformed these vectors into rice and verified the expression of the target gene in transgenic plants.We will explore the symbiotic effect between transgenic rice and rhizobia.In chapter 4,On the basis of previous studies,the molecular mechanism of CCa MK-interacting protein CIP73 was discovered.CIP73 as a transcriptional regulatory factor negatively regulated the expression of NIN.CIP73pro:Gus Histochemical staining of transgenic plants revealed that the expression of CIP73 was opposite to that of NIN,which confirmed that CIP73 negatively regulates the transcription of NIN.In Lotus,over-expression of CIP73 could significantly inhibited the expression of NIN and negatively regulated the infection of rhizobia,the formation of nodule primordium,and nodulation.