紫云英根瘤菌基因文库的构建及含完整结瘤基因的重组质粒pRaZ15的分离

Construction of Gene Library and Isolation of pRaZ15 Containing Complete Nodulation Genes in Rhizobium astragali

以紫云英根瘤菌菌株7653R为材料,制备总DNA,经EcoRI限制酶部分酶解,通过10—50%蔗糖梯度离心,分离到20—30 kb的DNA片段。利用能在革兰氏阴性菌中转移和复制的广谱奇主载体——pLAFR1质粒,构建了紫云英根瘤菌基因文库。通过与苜蓿根瘤菌1021菌株中8.7kb的共同结瘤基因(作探针DNA)杂交,从基因文库中分离到紫云英根瘤菌共同结瘤基因片段。以紫云英根瘤菌不结瘤突变株7653R+1(7653R消除共生质粒)为受体、构建的7653R基因文库(E.coli C600)为供体,通过协助转移质粒pRK2013(LE392)进行三亲交配,在含四环素的根瘤菌合成培养基(SM)上选择接合转移子。将得到的所有接合转移子混合在一起接种植物,通过植物结瘤试验,分离到含紫云英根瘤菌结癌基因的重组质粒pRaZ15。将该质粒用EcoRI完全酶切,得到25kb左右的外源DNA片段,该片段携带完整的结瘤基因簇。

Total DNA partially digested by EcoRI was prepared from Rhizobi-um astragagli strain 7653R, from which 20--30kb DNA fragments werecollected from sucrose-gradient (10--50%) centrifugation. Gene libraryof Rhizobium astragali was constructed with cosmid pLAFR1. Commonnod genes were identified by DNA hybridization with a 8.7kb nodfragment of R. meliloti 1021. Tri-parental mating experiments wereconducted with recipient strain 7653R+1 (Nod mutant of 7653R devoidof sym plasmid), donor stain 7653R gene library and helper strainLE392 (pRK2013). Hybrids were grown on SM containing tetracycline(10μg/ml), mixed and then used as inoculate for plant nodulation teston A. sinicus. Nodules were formed. Symbiotic bacteria were isolated(pRaZ15) containing complete set of nod genes. pRaZ15 was digestedcompletely by EcoRI, 25kb DNA inserts on pLAFR1 were obtained.