Symbiotic Function Characterization Of DGDG Synthase Gene MtDGD1 And Polygalacturonase Gene MtPG In Medicago Truncatula

Digalactosyldiacylglycerol（DGDG）,which is the main nonphosphorus lipid of thylakoid membrane of chloroplasts,was also present in the peribacteroid membrane of legume plants.DGD1 was responsible for majority DGDG synthesis in high plants.In the preliminary work,the full length of Mt DGD1 was obtained and the localization of Mt DGD1 was identified by Immunofluorescence and immunoelectron microscopic experiments,which showed that Mt DGD1 was co-localizated with the infected cell in nodules,and located to the symbiosome membranes.This study focused on Mt DGD1,the major synthase of DGDG in Medicago truncatula,to identify and functionally characterize the effect of Mt DGD1 on the symbiotic nitrogen fixation based on the preliminary study.The results are obtained and summarized as follows,1.Real-time PCR of the spatial and temporal transcript levels of Mt DGD1 in different tissues and M.truncatula transformation based on promoter-GUS vector was carried out and demonstrated Mt DGD1 was proably involved in nitrogen fixation.2.The associated symbiotic phenotype of over-expression and RNA interference（RNAi）transformed plants showed that Mt DGD1 could affect the development of nodules from Medicago truncatula through decreasing total amount of DGDG,with a corresponding reduction for different species of DGDG.3.Mt DGD1 was up-regulated and DGDG was accumulated in roots and nodules in phosphate starvation experiment,which implied DGDG might also contribute to symbiotic nitrogen fixation by conserving phosphate for other essential cellular processes during nodulation.These results indicate that DGD1 gene may contribute to symbiotic nitrogen fixation by affecting the synthesis and content of DGDG in nodules,and this is required for effective nodule organogenesis and nitrogen fixation.In this study,bioinformatics analysis was carried out on the polygalacturonase（PG）gene of Medicago truncatula,the results showed that the length of the gene was 4150 bp,its coding region was 1278 bp,encoding 425 amino acids.The encoded protein contains two conserved domains,PL-6 superfamily and Glyco_hydro₂8,which are related to degradation of cell wall pectin.What’s more,the homology between PG and some other leguminous plants,such as chickpeas and red bean were more closer.Based on the analysis of gene expression profile and RT-PCR detection,the results showed that Mt PG expression was significantly enhanced in the root nodules but very low in non-symbiotic tissues such as roots and leaves.In addition,three Tnt1 transposons inserted mutants（NF0999,NF5561 and NF4746）were screened by PCR and got one homozygous mutant material NF4746.The phenotype identification and other results above all suggested that the Mt PG gene played an important role in the early infection of symbiotic nitrogen fixation in legumes.