Elucidation of the symbiotic mechanism through the point of osmoregulation of root nodule bacteria.

1.甜菜碱产生菌(GB株)的构建：将球型节杆菌gB合成基因(Coda)导入pTE3表达载体，构建豆科细菌gB菌株。慢生根瘤菌和中根瘤菌GB菌株的胆碱氧化酶活性分别为0.25单位/mg蛋白和0.35单位/mg蛋白。BjGB菌株胞内GB含量(μg/mg细胞干重)为0.56.2。结瘤试验：BjGB菌株比亲本菌株更早开始结瘤。结瘤数、根瘤重、固氮活性(ARA)和单株重均提高到亲本的1.3-5倍左右。在较低温度(20℃/10℃)下结瘤能力较强(根瘤数、根瘤重与亲本的百分比分别为145.0、152.4、222.6和107.9)。3.GB含量测定：BjGB菌株形成的根瘤中GB含量为43.5Gfw.(亲本中未检测到GB)。根据纳滤法的标记酶分析，BjGB产生的大约87%的Gb被释放到植物的细胞质中，其中一些有可能整合到植物的细胞器中，如线粒体和液泡。4.BjUSDA110基因的全局表达分析：对大豆种子提取物(SSE)响应的BjUSDA110基因的全局表达分析结果表明，位于共生岛上的克隆如NOD和III型(TTSS)明显上调，这些克隆约占全基因组上调克隆的45%。表达谱与金雀异黄素诱导相似。此外，还有一些与渗透调节有关的共生克隆(ABC转运蛋白等)。也被上调了监管。然而，固氮基因(nif、fix等)都没有表达出来。

1.Construction of glycinebetaine production strain (GB strain) : GB synthetic gene (codA) from Arthrobacter globiformis was introduced into pTE3 expression vector and GB strains of leguminous bacteria were constructed. Choline oxidase activities of Bradyrhizobium japonicum and Mesorhizobium loti GB strains were 0.25 and 0.35 units/mg protein, respectively. Intracellular GB content (μg/mg dry wt of cell) of BjGB strain was 0.56.2.Nodulation test : Nodulation of BjGB strain started earlier than that of the parent strain. Besides, the number and weight of nodules, nitrogen fixation activity (ARA), and plant weight were increased to around 1.3-5 times that of the parent. The nodulation ability seemed to be superior in lower temperature (20℃/10℃) (number and weight of nodules, ARA, and plant weight against parent (%) : 145.0, 152.4, 222.6, and 107.9, respectively).3.Measurement of GB content : GB contents in nodules formed with BjGB strain were around 43.5μg/gFW (no GB was detected in the parent). According to the marker enzyme assay of NF methods, GB of around 87% produced by BjGB (bacteroids) was released to plant cytosol and some of them had a possibility to be incorporated into plant organelles such as mitochondria and vacuoles.4.Global expression analyses of Bj USDA110 genes : Results obtained from global expression analyses of Bj USDA110 genes which respond to soybean seed extracts (SSE) showed that clones located within symbiosis island such as nod and type III (ttss) were conspicuously up-regulated and these clones occupied around 45% of up-regulated clones in the whole genome. The expression profile was similar to that of the induction by genistein. Besides, some clones out of symbiosis which was related to osmoregulation (ABC transporter, etc.) were also up-regulated. However, genes for nitrogen fixation (nif, fix etc.) were not expressed.