Symbiotic Function Characterization Of A Digalactosyl Diacylglycerol Synthase Gene MtDGD1 In Medicago Truncatula

Digalactosyldiacylglycerol(DGDG),which is widely present in the lipid membrane of chloroplasts,is the main lipid of thylakoid membrane.The presence of DGDG was also detected in the peribacteroid membrane of legume plants,but it requires more experimental evidence to elucidate the functional and mechanism of DGDG in symbiotic nitrogen fixation.DGD2 and DGD1 are the two major synthetase of DGDG.Under normal physiological or phosphorus deficiency condition,DGD1 is responsible for the synthesis of more than 80% of DGDG.DGD2 participates in synthesis of DGDG only when the DGD1 pathway is blocked.This work is intended to study and characterize the effect of MtDGD1 on the symbiotic nitrogen fixation in Medicago truncatula.The results are obtained and summarized as follows.1.The full length of MtDGD1 was obtained basing on the analyses of data available for M.truncatula genome,transcriptome and splicing.2.The expression vector containing a His-tag of MtDGD1 was constructed and the antibody was prepared.Immunofluorescence and immunoelectron microscopic experiments for the localization of MtDGD1 was conducted.The results showed that MtDGD1 was co-localizated with the infected cell in nodules,and located to the symbiosome membranes.3.By using of RNAi interference method,MtDGD1 gene expression was suppressed.The symbiotic phenotype of the transformed plants are observed,the results showed that the RNAi plants were different from the control.The average weight of nodules of RNAi plants was less than that of the control group,nodule nitrogenase activity was also decreased.The total lipids of nodule were extracted and analyzed through LC-MS analysis.It showed that the total amount of DGDG of nodules in RNAi plants was reduced by 50% compared to that in the control group,and also there was a corresponding reduction for different species of DGDG.Real_time PCR showed that the expression of DGDG synthase gene MtDGD1 was inhibited in RNAi plants.The above results indicate that DGD1 gene contributes to the symbiotic nitrogen fixation by affecting the synthesis and content of DGDG in nodules,and the findings in this study provide some clues for the determination of the synthetic site of DGDG in nodules.