费氏中华根瘤菌042BS结瘤调节基因的克隆及功能检测

The Cloning and Functional Analysis of Sinorhizobium Fredii 042BS Regulaltory Modulation Genes

费氏中华根瘤菌 (Sinorhizobiumfredii) 0 4 2BS可以在大豆和苜蓿上结瘤。用费氏中华根瘤菌USDA2 5 7的nodD1和nodD2基因分别作为探针 ,与 0 4 2BS总DNA进行Southern杂交 ,发现其DNA经EcoRI酶切后分别在 3 0kb和 6 0kb处各有一条阳性带。回收这两条阳性带附近的DNA片段 ,建立部分基因文库 ,克隆到带有nodD1基因的 3 0kb片段 ,以及带有nodD2基因的 6 0kb片段。对nodD1和nodD2进行序列分析 ,结果表明 0 4 2BS的nodD1与费氏中华根瘤菌根瘤菌USDA2 5 7和USDA1 91的同源性高达 99% ,而nodD2与USDA2 5 7的同源性为1 0 0 %。再将nodD1的片段克隆到pBBRIMCS 5载体上 ,导入豌豆根瘤菌蚕豆生物变种 (Rhi zobiumleguminosarumbv.viciae)LPR5 0 5 4中进行功能检测 ,显示 0 4

The total DNA of Sinorhizobium fredii 042BS was digested by EcoRI for Southern blotting with probes of nodD1 and nodD2 from S.fredii USDA257. The 3kb positive band hybrided with nodD1 probe and 6kb positive band with nodD2 probe were found, respectively. Partial gene library were constructed using pUC18 as vector, and the clones with the nodD1 and nodD2 genes were obtained. The sequence of nodD1 and nodD2 of 042B showed that they are highly homologous with nodD1 and nodD2 of S.fredii. The fragment with nodD1 was cloned into the vector pBBRIMCS-5 and introduced into R.leguminosarum bv.viciae LPR5054 to study the function of the nodD1. The results showed that nodD1 of 042B can be induced by genistein and luteolin secreted by the seedlings of soybean and alfalfa respectively.