采用PCR-RFLP技术在不同水平上鉴定大豆根瘤菌(英文)  

Characterization of soybean rhizobia at different levels using PCR based techniques

作　　者：张学贤[1] M.木论贝格 李德安 周俊初[1] 李阜棣[1] 

机构地区：[1]华中农业大学微生物系,武汉430070 [2]DepartmentofMicrobiology,WageningenAgriculturalUniversity

出　　处：《生物多样性》1999年第4期277-284,共8页Biodiversity Science

摘　　要：采用16SrRNA基因PCR扩增与限制性酶切片段多态性分析（RFLP）技术对选自弗氏中华根瘤菌（S．fredii）、大豆慢生根瘤菌（B．japonicum）和埃氏慢生根瘤菌（B．elkanii）的19株代表菌进行了比较分析，根据用3种限制性内切酶的RFLP分析结果，可将供试菌株分为S．fredii，B．japonicum，B.elkaniiⅡ和B．elkaniiⅡa等4种基因型。各类菌株之间没有交叉，因此本研究采用的PCR-RFLP技术不失为一种快速鉴别大豆根瘤菌的新方法。采用本技术已将分离自中国的22株快生菌和19株慢生菌分别鉴定为S．fredii和B．japonicum。对供试参比菌株和野生型菌株进行的16S～23S基因间隔DNA（IGS）的PCR-RFLP分析结果表明：S.frediii和B.japonicum菌株的IGS长度不同，所有供试S．fredii菌株的IGS为2.1kb，而供试B.japonicum菌株则为2．0kb。依据RFLP的差异，可将来自中国两个不同地区的S.fredii株区分为2个基因型，而来自中国东北黑龙江地区的19株B．japonicum菌株则可分为11个基因型。对上述野生型菌株还进行了REP－PCR和ERIC-PCR分析并确定其具有菌株水平的特异性。Nineteen standard USDA strains representing three recognized soybean rhizobia, Sinorhizobium fredii,Bradyrhizobium japonicum and B. elkanii, were examined by restriction fragment length polymorphism (RFLP)analysis of 16S rRNA genes amplified by polymerase chain reaction (PCR). Four composite genotypes were obtained from the combined data of the RFLP analysis with three endonucleases. S. fredii, B. japonicum, B. elkanii Ⅱ and Ⅱa reference strains fell into these four genotypes respectively. No genotype was shared by two species. Therefore,16S rDNA PCR-RFLP is a rapid tool for the identification of soybean rhizobia. According to 16S rDNA PCRRFLP, 22 fast-growing and 19 slow-growing soybean rhizobia strains from China were identified to S. fredii and B . japonicum , respectively.The reference strains and wild type isolates were also examined by 16S～23S intergenic spacer PCR-RFLP analysis. PCR results indicated that the length of the intergenic region between S. fredii and Bradyrhizobium sp.is different. All fast-growing S. fredii strains produced one larger band (2.1 kb), and all slow-growing Bradyrhizobium strains produced one smaller band (2.0 kb). Based on 16S～23S RFLP analysis, 22 S. fredii isolates can be further separated into two "genotypes" that originated from two different regions in China; 19 B. japonicum strains from Heilongjiang Province (northeast of China) can be separated into 11 genotypes. These wild type isolates were also characterized at strain level by REP (repetitive extragenic palindromic) and ERIC (enterobacterial repetitive intergenic consensus) PCR fingerprinting.

关 键 词：大豆 根瘤菌 遗传多样性 PCR-RFLP 

分 类 号：Q939.114[生物学—微生物学]