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本试验是用粘末端连接法把卡那霉素抗性基因(Kan~r)插入到大豆根瘤菌B.j110的苹果酸脱氢酶(mdh)基因中,使mdh基因失活。首先,通过EcoRI切点把Kan~r连接到pTZ18U载体上,经转化把这一重组DNA转入大肠杆菌XLI-Blue中,利用含有氨苄青霉素和卡那霉素的选择培养基选出转化菌株,提取pTZ18U-Kan~r质粒DNA;然后,用Sall分别切已克隆在pTZ19U中的B.j110mdh基因及pTZ18u-Kan~r,经粘末端连接把卡那霉素抗性基因连接到苹果酸脱氢酶基因中间,从而使其失活。再将所得到的这一重组DNA用电脉冲法(Electroporation)转入大豆根瘤菌B.j2143中,用以检验经插入失活后的苹果酸脱氢酶基因对大豆固氮作用的影响。
In this experiment, the kanamycin resistance gene (Kan ~ r) was inserted into the malate dehydrogenase (mdh) gene of soybean rhizobia B.j110 by the mucoadhesive ligation method, inactivating the mdh gene. First, Kan ~ r was ligated into pTZ18U vector by EcoRI cleavage site, and the recombinant DNA was transformed into E. coli XLI-Blue by transformation. The transformed strain was selected by selection medium containing ampicillin and kanamycin, The pTZ18U-Kan ~ r plasmid DNA was extracted. Then, the B.jl10mdh gene and the pTZ18u-Kan ~ r cloned in pTZ19U were respectively ligated with Sall, and the kanamycin resistance gene was linked to malate dehydrogenase Enzyme gene in the middle, making it inactivated. The resulting recombinant DNA was transferred into soybean rhizobia strain B.j2143 by electroporation to examine the effects of the malate dehydrogenase gene after inactivation on nitrogen fixation of soybean.