Phenotypic Identification Of GAP2 Related Symbiotic Mutants In Lotus Japonicus And Functionalanalysis Of NAD1 Gene In Medicago Truncatula

Symbiotic nitrogen fixation of legumes and nodules is the most efficient form of biological nitrogen fixation,the symbiotic relationship between the legumes and rhizobium will be established in the root of the plant to form a special root nodule.The first step of symbiotic nodulation is the release of flavonoid from legume plant,which can induce the rhizobium to producce nod factors.Nod factor receptor 1 and 5 form a heterodimer in the membrane can directly bind with nod factor in Lotus japonicas,and then transfer signals to downstream proteins.Small G protein ROP6 interacts with NFR5 and plays an important role in the nodulation.LjGAP2 is the activator protein of ROP6.The first chapter of this study focuses on the expansion of the LjGAP2 gene and its function in the symbiotic nitrogen fixation pathway,and the results are showed as below.1.In the study of LORE1 insertion mutants of LjGAP2,the M1 and M2 generations did not get the homozygous mutants,but the heterozygotes did not find a significant symbiotic phenotype.2.The stable transformation method of LjGAP2 for overexpression and RNA interference,RT-PCR results show:in wild type MG20 as control,stable transformation of the expression levels of genes in plants over expressing plants was the lowest for 12.12 times of the control,the highest was 72.15 times;3 'RNAi plants in the lowest for 18%of the control;5' RNAi plants in the lowest for 19%of the control.3.Statistical the length of root and stem,the number of nodules and the density of nodules after 14 days of inoculation.We found that over expression LjGAP2 lead to root length decreased significantly,nodulation number was significantly reduced,nodulation density decreased significantly;5 'RNAi plants had no significant differences;3'RNA interference mostly show the number of nodules per plant and nodulation density was significantly increased,the interference effect lower than the strongest effect had no significant differences.The results showed that the LjGAP2 gene played a negative role in the regulation of LjROP6.NAD1 may be a regulator of the immune defense response in the legume plant.The second chapter of this study focuses on the work of the MtNAD1 gene in two parts.1.From the perspective of evolutionary biology,use structural domain of non leguminous plants Alnus glutinosa NAD1-like protein replacing Medicago truncatula NAD1 protein domains.According to the results of the experiment of the hairy root transformation,these results showed that:NAD1-NT,Loop,CT(terminal)domain section was conservative in symbiotic nitrogen fixing plants(legumes and actinomycetes).NAD1 transmembrane domain(TM1 TM2)and the adjacent C end region of 9 amino acids of TM2 in the legume had a specific.Two specific amino acid sites were found in the transmembrane domain of NADI:TM1(CFCF)and TM2(F67VV).2.Mtnadl mutant was used to screen the rhizobium mutant library,and to identify the genes that inhibited the phenotype of Mtnadl mutant.We found a white nodule which has similar size with red nodule,but failed to isolate from the rhizobium.