摘要
用植物筛选法从紫云英根瘤菌7653R基因文库中分离到能互补7653R+1(原菌株7653R消除共生质粒的突变株)共生缺陷功能的重组质粒pRaZ15。pRaZ15质粒克隆有紫云英根瘤菌共同结瘤基因簇。采用三亲交配—植物试验法,将pRaZ15质粒转移到豌豆根廇菌菌株T83K3和菜豆根瘤菌菌株3622—15中,用以回接紫云英植株,并在紫云英上结瘤,证实了pRaZ15质粒上克隆有紫云英根瘤菌寄主专性结瘤基因。将pRaZ15质粒再次转移到紫云英根瘤菌不结瘤突变株7653R+1中,回接紫云英植株,用乙炔还原法测定出所结根瘤的固氮酶活性。用α—^(32)p-dCTP标记克隆有肺炎克氏杆菌nif HDK基因的pSA30,以此作为探针,与pRaZ15质粒的EcoR1片段杂交,证实了紫云英根瘤菌nif HDK基因也存在于pRaZ15质粒上。
Previous work showed that the recombinatant plasmid pRaZ15 construed in this laboratory contained the common nod genes of Rhizobium astragali. Incorporation of pRaZ15 into R. astragali Nod^- mutant 7653R+1 (a nod^- mutant of 7653R devoid of Sym-plasmid) recovered its nodulation ability. In this report, evidances presented showed that the host-specific nod genes (hsn) and nitrogenase structural gencs(nifHDK) were also carried by pRaZ15. Transconjugants were obtained by thihyhridization incorporated pRaZ15 into Rhizobium leguminosarum strain T83K3 and Rhizobium phaseoli strain 3622-15, of which the original recipents were unable to nodulate Astragalus sinicus. Both transconjugants were found to nodulate A. sinicus. The experiments prove that the pRaZ15 contains the hsn genes specific to A. sinicus nodulation. The pRaZ15 was again transfered to the Sym-plasmid cured mutant R. astragali strain 7653R+1 and used to inoculate A. sinicus. Nodules were formed and its nitrogenase activities were detected by the aectylene-reduction method. ^(32)P--dCTP labeled pSA30 cloned with K. p. nifHDK genes was hybridized with Sall fragments and EcoRI fragments of pRaZ15. Radioactivity exposure suggested that pRaZ15 contained nif genes.Evidences gather so far indicate that pRaZ15 contains all the necessry genes for symbiotic nitrogen fixation as contained in the sym-plasmid of R. astragli.
出处
《华中农业大学学报》
CAS
CSCD
北大核心
1989年第4期285-290,共6页
Journal of Huazhong Agricultural University
基金
国家自然科学基金
关键词
紫云英
根瘤菌
固氮基因
结瘤基因
R. aslragli
has genes
nifHDK
gene pRaZl5
thihybridization
