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

1.甜菜碱产生菌（GB菌株）的构建：将球形节杆菌（Arthrobacter globiformis）的GB合成基因（codA）导入pTE 3表达载体，构建了豆科细菌GB菌株。大豆慢生根瘤菌（Bradyrhizobiumjaponicum）和百脉根中慢生根瘤菌（Mesorhizobiumloti）GB菌株的胆碱氧化酶活性分别为0.25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菌株的结瘤开始早于亲株。此外，根瘤数和重量、固氮活性（ARA）和植株重量增加到亲本的1.3-5倍左右。在较低温度（20℃/10℃）下，BjGB菌株的结瘤能力表现为上级（结瘤数、结瘤重、ARA和株重分别为145.0、152.4、222.6和107.9%）。3. GB含量测定：BjGB菌株结瘤后，GB含量约为43.5μg/gFW（亲本中未检测到GB）。根据NF法的标记酶测定结果，BjGB产生的GB约占87（类菌体）被释放到植物细胞质中，并且其中一些有可能被并入植物细胞器如线粒体和液泡中。对大豆种子提取物（SSE）应答的Bj USDA 110基因的全局表达分析结果表明，位于共生岛内的克隆如nod和III型（TTSS）显著上调，这些克隆占全基因组上调克隆的约45%。表达谱与染料木黄酮诱导的表达谱相似。此外，一些脱离共生关系的克隆也与植物生长调节（ABC转运蛋白等）有关。也被上调了。然而，固氮基因（nif，fix等）没有表达。

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.