MONITORING OF RHIZOBLA ON THE ROOT SYSTEM AND ANALYSIS OF THE MECHANISM OF NODULATION IN SOME LEGUMES USING A GUS MARKED STRAIN

利用GUS标记菌株监测根系根瘤并分析部分豆类结瘤机制

• 批准号: 10660017
• 负责人: DAIMON Hiroyuki
• 金额: $ 1.54万
• 依托单位: OSAKA PREFECTURE UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10660017/
• 关键词: Green manure; Gus gene; Legume; Nitrogen fixation; Rhizobia; Aus 遺伝子

The diversity in the infection process of rhizobia to some legumes was investigated by using a gus-marked strain. The results are summarized as follows.1. To obtain gusA-marked strains of Bradyrhizobium sp. USDA3024, B.sp. U9709-A, and Azorhizobium caulinodans U9709-SRS, conjugation experiments were performed with the strain of E.coli S17-λ pir with plasmid containing the β-glucuronidase operon (mTn5SSgusA21 on pCAM121). Root nodules formed by each gus-marked strain developed blue color on incubation in X-Gluc buffer in Clotalaria, Arachis and Sesbania.2. The gus-marked strain did not differ from the parent strain in nodulation capability and nitrogenase activity in each legume.3. Growth pouch method was adequate for infection test of a gus-marked strain in Crotalaria seedling.4. With a microscopic observation, an infection thread-like structure was found in the root hairs of tap root in 2 day-old seedling of Crotalaria juncea.5. Number of root nodule and nitrogenase activity in peanut plants were inhibited by application of nitrate to culture solution.6. Stem nodules of Sesbania rostrata were initiated at the site of the cotyledonary node and followed by forming on the surface of stem.7. Further investigation of the diversity in the rhizobia-infection process and the effect of various environmental conditions on nodulation and nitrogen fixation is now in progress using the gus-marked strain. Another marker gene, GFP, would be also used for those experiments.

采用淋球菌标记菌株，研究了根瘤菌对部分豆科植物感染过程的多样性。研究结果如下：1.为了获得gusA标记的慢生根瘤菌USDA 3024、B.sp. U9709-A和茎瘤固氮根瘤菌U9709-SRS菌株，用含有β-葡萄糖醛酸苷酶操纵子（pCAM 121上的mTn 5 SSgusA 21）的质粒与大肠杆菌S17-λ pir菌株进行接合实验。在X-Gluc缓冲液中培养时，各gus标记菌株形成的根瘤在Clotalaria、Arachis和田菁中均呈现蓝色. gus标记的菌株在各豆科植物中的固氮酶活性和固氮能力与亲本菌株没有差异.生长袋法适用于gus标记菌株在猪屎豆幼苗中的侵染试验.显微镜下观察发现，猪屎豆2日龄幼苗主根根毛中存在侵染线状结构.硝酸盐对花生根瘤数和固氮酶活性有抑制作用.长喙田菁茎瘤起源于子叶节部位，随后在茎表面形成.目前正在利用gus标记的菌株进一步研究根瘤菌感染过程的多样性以及各种环境条件对根瘤菌生长和固氮的影响。另一种标记基因GFP也将用于这些实验。

项目成果

Yohioka M.: "The effect of nitrate nitrogen on nitrogenase activity in early developmental phase of peanut."Root Research. 7 (4) (in Japanese). 125 (1998)

Yohioka M.：“硝态氮对花生早期发育阶段固氮酶活性的影响。”根研究。

大門弘幸: "ラッカセイの根粒着生および窒素固定活性の硝酸態窒素による阻害を検討するための水耕栽培の試み"近畿作育研究. 45(印刷中). (2000)

Hiroyuki Daimon：“尝试通过水耕栽培来检查花生中硝酸盐氮对结瘤和固氮活性的抑制”Kinki Crop Research 45（出版中）。

Daimon, H.: "The effect of nitrate nitrogen on growth, nodulation and nitrogen filing ability in peanut"Proceedings of the 3rd Asian Crop Scince Conference. 594-602 (1999)

Daimon, H.：“硝态氮对花生生长、结瘤和氮填充能力的影响”第三届亚洲作物科学会议论文集。

Daimon H.: "Formation of infection thread-like structure in rhizobial infection in Crotalaria juncea."Japanese Journal of Crop Science. 68 (suppl.2) (in Japanese). 266-267 (1999)

Daimon H.：“猪屎豆根瘤菌感染中感染线状结构的形成。”日本作物科学杂志。

Daimon H.: "The effect of nitrate nitrogen on growth, nodulation and nitrogen fixing ability in peanut."Proceedings of the 3rd Asian Crop Science Conference. 594-602 (1998)

Daimon H.：“硝态氮对花生生长、结瘤和固氮能力的影响。”第三届亚洲作物科学会议论文集。