Studies on the physiological mechanisms of isoflavonoids on the nod-gene expression of Bradyrhizobium japonicum

为了研究携带rj基因的大豆品种接种亲和或不亲和菌株时，无效或有效根瘤的共享点，本研究解剖分析了日本慢生根瘤菌菌株Is-1（与CNS不亲和，与Hill不亲和）和Is-34（与CNS亲和，与Hill不亲和）感染的rj_2rj_3和rj_4基因型大豆CNS和Hill的根瘤发育情况。亚甲基蓝染色法检测根毛卷曲，光镜下观察根毛皮层分裂，荧光显微镜下检测根瘤菌Is-1和Is-34的荧光抗体对根瘤菌的诱导作用。结果表明，不亲和性菌株侵染的限制机制可能是在播种接种后5 d内根瘤菌侵入皮层下初生分生组织。基于不同根瘤菌间大豆根瘤中异黄酮成分的差异，以及不同根瘤菌间染料木素、大豆苷元和根瘤提取物产生的代谢产物种类的差异，我们寻找了一个作用于无效或有效根瘤共享点的信号分子。这些大豆根部的大豆苷元和甘油I含量在7 d前保持在正常水平。接种Is-1和Is-34后3天和5 d的CNS和Hill根中诱导根瘤菌节点基因表达的主要异黄酮染料木素和大黄酮含量也没有差异。然而，在山根提取物中发现了一个特定的荧光点，而在CNS中没有。为了分析nod代谢产物的组成和数量，采用^<14> c -乙酸作为示踪剂，用染料木素、大豆苷元、每种大豆根提取物或Hill的特定荧光点孵育Is-1和Is-34。这些结果表明，Is-1和Is-34的节点代谢物分离模式在各处理间差异不大，但在Is-1和Is-34之间，节点代谢物的数量和强度差异较大。几丁质酶处理使结瘤代谢物的数量减少，从而证实结瘤因子包括在结瘤代谢物中。因此，寄主植物根系对nod代谢物的不同反应决定了大豆品种与日本双歧杆菌菌株之间的亲和性。少

In order to investigate the point shared into ineffective or effective root nodules when soybean cultivars harboring Rj-gene were inoculated with a compatible or incompatible strain, we analyzed anatomically the nodule development in the roots of Rj_2Rj_3-and Rj_4-genotype soybean, CNS and Hill respectively, infected by Bradyrhizobium japonicum strains Is-1 (incompatible to CNS and compatible to Hill) and Is-34 (compatible to CNS and incompatible to Hill). The root hairs curlings were assayd by the staining method with methylene blue, the cortex divisions were examined with a light microscope, the invitation of rhizobia were investigated by the fluorescent antibody of rhizobia strains Is-1 or Is-34 with a fluorescence microscope. These results showed that the restriction mechanism of the infection with incompatible strains might operate at the step of rhizobia invasion into the primary meristem directly under cortex within 5 days after sowing and inoculation.On the assumption that the … More various composition of isoflavonoids were different in those soybean roots and the species of nod metabolites produced by genistein, daidzein and the root extracts were different among rhizobia strains, we searched a signal molecular which operate at the point shared intoineffective or effective root nodules. Daizein and glyceollin I contents in those soybean roots were level up to 7 days. Also the roots of CNS and Hill of 3 and 5 days' seedling inoculated with Is-1 or Is-34 were no difference in the contects of genistein and daidzen known as main isoflavonoids which induced the nod-gene expression of rhizobia. However a specific fluorescent spot was identified in the root extract of Hill and not in that of CNS.In order to analyze the composition and amount of nod metabolites, Is-1 and Is-34 were incubated using ^<14>C-acetate as tracer with genistein, daidzein, each soybean root extract or a specific fluorescent spot of Hill. These results shown that the separation pattern of nod metabolites of Is-1 or Is-34 were little difference among each treatment, but in comparison between Is-1 or Is-34, the number and intensity of nod metabolites were large difference. The chitinase treatment of those nod metabolites decreased the number of nod metabolites, thus it was confirmed that nod factors were included those nod metabolites. Hence, it was the different composition of nod metabolites between strains determined the compatibility of soybean varieties and B.japonicum strains, through different responses of host plant roots to nod metabolites. Less