Research On The Biological Function Of NIN Interacting Protein NIP2 In Lotus Japonicus

Symbiotic nitrogen fixation between leguminous plants and rhizobia is the most efficient process of the nitrogen fixation.LjNIN is a key gene in the process of symbiotic nitrogen fixation,and it effects in the nodulation signaling transduction pathway.Through the homologous comparison and structure prediction,we found that LjNIP2 is likely to be a methyltransferase,and it interacted with the PB1 domain of LjNIN.How does it affect the process of symbiotic nitrogen fixation,it is not clear.This research is focused on the expansion of the LjNIP2gene and its function in the symbiotic nitrogen fixation pathway.The main results were as follows:1.In the study of LORE1 insertion mutants of LjNIP2,the F1 and F2 generations did not get the homozygous mutants,but the heterozygotes did not find a significant symbiotic phenotype.And the ratio of heterozygotes and wild type is close to 2:1.The homozygous mutants are likely to be death.2.15 days after the inoculation,the number of the root nodules of LjNIP2 over-expression plants increase significantly,compared to the control.After 3 weeks of the inoculation,the RNAi efficiency of the LjNIP2 positive root decreases with the positive number of root nodules.All the results above indicate that LjNIP2 gene have positive regulating effects in the symbiotic nitrogen fixation process.3.2 weeks after the inoculation,compared to the control,the positive root nodule number of LjNIP2 RNAi plant declines to some extent,but there is no significant difference.The average non-positive root nodule number of LjNIP2 RNAi plant equals to that of the control,and no significant difference was found.These may be results from different RNAi efficiency of each positive root.4.By testing the LjNIN and LjNPL expression quantities of LjNIP2 RNAi plant and the positive roots of the control,we find that both LjNIN and LjNPL expression quantities descend slightly with the decrease of LjNIP2 expression quantity,and the descending degree is related to the expression quantity of ijNIP2.5.Since the mutant of LjNIP2 has no homozygotes,the LjNIP2 over-expression plants bloom in advance compared to the control,both numbers of blooming flowers and capsules have increased.The LjNIP2 gene may well be related to the fertility of Lotus japonicas,and have positive regulating effects.6.Using the full-length ofNIN,structural domains of PB1 and RWP-RK as baits,no new proteins is fished from Lotus japonicas AD-cDNA A library.