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

从根瘤细菌的渗透调节角度阐明共生机制。

• 批准号: 15580054
• 负责人: OHWADA Takuji
• 金额: $ 2.11万
• 依托单位: Obihiro University of Agliculture and Veterinary Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15580054/
• 关键词: root nodule bacteria; nodulation; glycinebetaine; osmoregulation; global gene expression of root nodule bacteria; 低温ストレス; 窒素固定

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.

1.甘氨酸甜菜碱生产菌株(GB菌株)的构建：将球形节杆菌的GB合成基因(codA)导入pTE3表达载体，构建豆科细菌的GB菌株，日本缓生根瘤菌和中慢生根瘤菌GB菌株的胆碱氧化酶活性分别为0.25和0.25。细胞内GB含量分别为0.35单位/毫克。 BjGB菌株的（μg/mg细胞干重）为0.56.2。结瘤试验：BjGB菌株的结瘤开始时间早于亲本菌株。此外，结瘤的数量和重量、固氮活性（ARA）和植株重量增加至亲本的1.3-5倍左右，在较低温度（20℃/10℃）下，结瘤能力似乎更优越（根瘤数量和重量， ARA、相对于亲本的植株重量(%)：分别为145.0、152.4、222.6和107.9)。3.GB含量的测定：BjGB菌株形成的根瘤中的GB含量约为43.5μg/gFW(在BjGB菌株中未检测到GB)。根据NF方法的标记酶测定，约87%的GB是由BjGB产生的。 （类菌）被释放到植物细胞质中，其中一些有可能并入植物细胞器，如线粒体和液泡。 4. Bj USDA110 基因的全局表达分析：从响应于 Bj USDA110 基因的全局表达分析获得的结果大豆种子提取物 (SSE) 显示，位于共生岛内的克隆，例如 nod 和 III 型 (ttss) 显着上调，并且这些克隆的表达显着上调。约占全基因组上调克隆的45％，其表达谱与金雀异黄素诱导的表达谱相似，此外，一些与渗透调节相关的非共生克隆（ABC转运蛋白等）也上调。然而，固氮基因（nif、fix等）没有表达。