固氮斯氏假单胞菌四碳二羧酸转运调控基因dctB的功能研究

Research on the function of dctB gene encoding C4-dicarboxylate transport regulation protein from Pseudomonas stutzeri A1501

为研究固氮斯氏假单胞菌(Pseudomonas stutzeri)A1501四碳二羧酸转运相关基因dctB的生物学功能,构建了携带功能完整的dctB基因片段的广宿主质粒pLL2922,通过三亲接合将该质粒转移至dctB突变株及野生型菌株A1501,并对其以琥珀酸、延胡索酸、苹果酸等四碳二羧酸为唯一碳源的培养基中的生长情况与固氮活性进行了测定.结果表明:dctB突变株不能利用琥珀酸和延胡索酸进行生长和固氮,但能以苹果酸为唯一碳源进行生长和固氮.携带功能完整dctB的广宿主质粒pLL2922,可以互补dctB位点的突变,并部分恢复突变株的四碳二羧酸转运和固氮能力.将该质粒转入A1501野生型菌株可以提高A1501的固氮活性,在琥珀酸,延胡索酸,苹果酸为唯一碳源的培养基上固氮活性比野生型菌株提高62.8%,33.9%,27.8%.表明dctB基因对于四碳二羧酸的转运及固氮能力均具有重要作用.

To investigate the function of dctB which related to dicarboxylic acids transport in nitrogen - fixing Pseudomonas stutzeri A1501, a broad host range plasmid （pLL2922） was constructed. By tri - parental mateing the plasmid was transferred into dctB mutant and A1501. Nitrogenase activity and growth curve were assayed in media containing different dicarboxylic acids as sole carbon scources. Results indicated that a dctB insertion mutant could not utilize fumarate and succinate as the sole carbon source, but the strain grew with malate. The strain had a Nif ＋ phenotype when malate was the carbon source. Nitrogenase activity was not detected with succinate or fumarate as the carbon source. Introduction of a broad host range plasmid （pLL2922） carrying the dctB region into the dctB mutant restored growth and nitrogenase activity in media containing succinate or fumarate. In addition the nitrogenase activity of the strains which containing plasmid（pLL2922） were increased by 62.8%, 33.9%, 27.8% using succinate, fumarate and malate as the sole carbon sources.