耐盐高效大豆根瘤菌株的构建

THE CONSTRUCTION OF SALT-TOLERANT HIGH EFFECTIVE STRAINS OF SOYBEAN RHIZOBIA

快生型大豆根瘤菌RT19是一株耐盐而固氮能力差的菌株,在含有0.3 mol/L NaCl的YM培养液中生长良好,代时为3.5h,甚至在0.6 mol/L NaCl条件下仍可生长。慢生型大豆根瘤菌USDA110对盐敏感,在含有0.1 mol/L NaCl的YM培养液中不生长,但结瘤固氮能力强。本实验以RT19的DNA转化USDA110细胞,在以0.3 mol/L NaCl为选择标记的YMA培养基上,挑取322个耐盐和生长快的菌落,将其中101个菌落纯化,并回接到大豆'112-2-4',得到两株耐盐、生长快而且固氮活性强的转化子RTt19和RTt50。供体菌RT19和转化子RTt19、RTt50在0.4 mol/L NaCl培养基中生长时,细胞内积累大量谷氨酸,而且丙氨酸和缬氨酸有不同程度的增长。 RTt19和RTt50经过20代的转管移植,其性状保持不变。

Fast-growing soybean rhizobium RT19 is a salt-tolerant but less efficient nitrogen fixing strain. It has a doubling time of 3.5h in YM liquid medium with 0.3mol/L NaCl and even grows in concentrations of up to 0.6mol/L NaCl, while slow-growing soybean rhizobium USDA110 is severely inhibited in medium containing 0.1 mol/L NaCl, but nodulates and fixes nitrogen effectively. Two transformants, RTt19 and RTt50, which are salt-tolerant, fast-growing and more efficient nitrogen fixing, were obtained by transforming the DNA from strain RT19 into the cells of strain USDA110 and selected on YMA medium with 0.3 mol/L NaCl as selective marker. Intracellular free glutamate was found to increase rapidly in strain RT19 and transformant RTt19 as well as RTt50 grown on medium containing 0.4 mol/L NaCl. Alanine and valine were found to be increased at lower level. The characteristics of transformants RTt 19 and RTt50 are maintained even after 20th, transfer of the cultures.