Functionally analysis of legume symbiotic specific genes in transgenic Lotus japonicus

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(1)For surveying symbiotic differentiation of key organelle, mitochondria, soybean nodule and root mitochondria were compared after two dimensional electrophoresis and the proteins were characterized in combination with MALDI-TOF MS. We also surveyed the soybean nodule bacteroid by the methods of proteome analysis.(2)We identified a Sed5-like clone LjSYP32-1 which contributes to nodule tissue formation and plant growth in Lotus japonicus. Promoter analysis and in situ hybridization revealed that LjSYP32-1 expressed significantly in inner cortex cell layer surrounding the infected zone of young nodules and at meristem area of developing lateral root. The anti-sense plants showed the nodules were much smaller and some nodules fused each other by sharing the vascular bundles. Such irregular nodules were observed in the rate of two times more comparing to those of wild type plant. The data suggest that LjSYP32-1 is contributing to support plant growth and normal nodule tissue differentiation.(3)Using antisense technique, we diminished the expression levels of the nodule enhanced PEPC gene (Ljpepc1) in a determinate legume plant (Lotus japonicus) for surveying the influence to symbiotic phenotype and biochemical parameters. In nodules of Asppc plants, PEPC activity reduced to ca. 10% of non-transformants and the plants showed a typical nitrogen deficient symptom without nitrogen nutrient supply, and returned to normal growth with supply of 2.5 mM nitrate. Significant reduction of sucrose, succinate, asparagine, asparatate and glutamate contents also decreased in Asppc nodules. The data was discussed on a role of PEPC for the carbon/nitrogen metabolic flux in nodules.(4)We isolated the some novel protein kinase possessing the subdomain VIB sequences characteristic of Ser/Thr kinase. To determine the function of the gene, we have made transgenic Lotus japonicus by RNAi methods.