Functionally analysis of legume symbiotic specific genes in transgenic Lotus japonicus

（1）为了研究大豆根瘤中关键细胞器的共生分化，采用双向电泳技术比较了大豆根瘤、线粒体和根线粒体的差异，并结合MALDI-TOF MS对蛋白质进行了分析。(2)We鉴定了一个与Sed 5相似的克隆LjSYP 32 -1，该克隆有助于百脉根根瘤组织的形成和植株的生长。启动子分析和原位杂交结果表明，LjSYP 32 -1在幼根瘤侵染区周围的皮层细胞层和发育侧根的分生组织中有显著表达。反义植株的根瘤明显变小，部分根瘤通过共用维管束相互融合。与野生型植物相比，观察到的这种不规则结节的比率是两倍多。这些数据表明LjSYP 32 -1有助于支持植物生长和正常的根瘤组织分化。（3）利用反义技术降低根瘤增强型PEPC基因（Ljpepc 1）在有限生豆科植物百脉根中的表达水平，研究其对共生表型和生化指标的影响。在Asppc植物的根瘤中，PEPC活性降低至约100%。10%的非转化体和植物在没有氮营养供应的情况下表现出典型的氮缺乏症状，并且在供应2.5mM硝酸盐的情况下恢复正常生长。Asppc根瘤中蔗糖、琥珀酸、天冬酰胺、谷氨酸和谷氨酸的含量也显著降低。对PEPC在结核碳/氮代谢通量中的作用的数据进行了讨论。(4)We分离出了具有Ser/Thr激酶特征的VIB亚区序列的新型蛋白激酶。为了研究该基因的功能，我们利用RNAi技术获得了转基因百脉根。

(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.