【作者】 张伟涛；

【导师】 周俊初；

【作者基本信息】 华中农业大学 ， 微生物学， 2006， 硕士

【摘要】 通过16S rRNA PCR-RFLP，16S rRNA序列分析，16S-23S rRNA PCR-RFLP和富含G-C的RAPD技术对分离自中国主要大豆产区的44株慢生根瘤菌进行遗传多样性和系统发育的研究。16S rRNA PCR-RFLP，16S rRNA序列分析表明：供试菌株和代表菌株可以分为4种基因型，基因型Ⅰ包括18株分离自北方的菌株，他们与慢生根瘤菌B．japnicum，B．liaoningense和B．elkanii．代表菌株的基因型均有差别。基因型Ⅱ有23株，其基因型与除了USDA6的所有B．japonicum的代表菌株以及B．liaoningense．的代表菌株都一样。基因型Ⅲ单独包括B．japonicum的代表菌株USDA6。基因型Ⅳ包括3株供试菌株以及B．elkanii，的代表菌株。16S rRNA序列分析表明HAS5与B．elkanii的代表菌株聚在一起，其与代表菌株USDA76的序列差异为0.4％。DD3，BQ3，GXD1，JZ2与其它所有B．japonicum的代表菌株归为一大类，有很高的同源性，其序列差异均小于1％。16S-23S rRNA PCR-RFLP分析表明：群Ⅰ为16S rRNA RFLP分析的基因型Ⅰ的菌株；16S rRNA PCR-RFLP分析中的基因型Ⅱ的菌株在16S-23S rRNA PCR-RFLP分析中分为群Ⅱ和群Ⅲ，群Ⅱ包括B．liaoningense和部分基因型Ⅱ的菌株，群Ⅲ包括B．japnonicum和基因型Ⅱ剩余的部分菌株，大部分来自长江流域；群Ⅳ由3株来自红安的菌株以及B．elkanii．组成。RAPD分析表明供试菌株和代表菌株在72％的相似水平上同样分为Ⅰ、Ⅱ、Ⅲ、Ⅳ四个群，与16S-23S rRNA PCR-RFLP分析一致。其在更高的相似水平上分群更加细化，反映地理因素对分群的重要影响。 通过16S rRNA PCR-RFLP，16S rRNA序列分析，16S-23S rRNA PCR RFLP和富含G-C的RAPD等技术对分离自中国55株豇豆根瘤菌和进行遗传多样性和系统发育的研究。16S rRNA PCR-RFLP，16S rRNA序列分析表明：供试菌株和代表菌株分为6种基因型，属于慢生根瘤菌的包括基因型Ⅰ、基因型Ⅱ、基因型Ⅲ，共48株。其中基因型Ⅰ有16株菌株，与B．japonicum和B．liaoningense基因型完全相同；。基因型Ⅱ包括25株与B．japonicum和B．liaoningense以及B．elkani均有差异；基因型Ⅲ有7株菌株与B．elkani基因型完全相同的。属于快生根瘤菌的包括基因型Ⅳ、基因型Ⅴ、基因型Ⅵ，其中基因型Ⅳ有一株DdE4与豌豆根瘤菌的基因型完全相同；基因型Ⅴ有4株与费氏中华根瘤菌完全相同；基因型Ⅵ有两株与费氏中华根瘤菌略有差异，各基因型的供试菌株与参比菌株的序列差异均小于1％。16S-23S rRNA PCR-RFLP分析表明：供试菌株有48株为慢生根瘤菌，7株为快生根瘤菌。慢生根瘤菌在80％的相似水平上分为Ⅰ，Ⅱ，Ⅲ和Ⅳ群，Ⅰ群有30株；Ⅱ群包括5株供试菌株和大豆慢生根瘤菌B．japonicum；群Ⅲ包括6株供试菌株和辽宁慢生根瘤菌B．liaoningense；群Ⅳ包括7株供试菌株和埃氏慢生根瘤菌B．elkanii．的代表菌更多还原

【Abstract】 Genetic diversity and phylogeny of 44 indigenous strains of Bradyrhizobium from soybean (Glycine max L.) cropping zones of China were investigated through 16S rRNA PCR RFLP, 16S rRNA sequencing, 16S-23S rRNA PCR-RFLP and G-C rich RAPD assays. Results of 16S rRNA PCR-RFLP and sequence analysis shown that strains tested and reference strains were clustered into 4 genotype, Genotype I consisted of 18 isolated strains mainly from the north of China.,which appeared genetic differences from B. japnicum, B. liaoningense and B. elkanii. Genotype II comprised 23 isolated strains, all reference strains but USDA6 of B. japonicum, and the type strains of B. liaoningense. Genotype III solely consisted of type strains of B. japonicum USDA6 Genotype IV comprised three isolated strains and the reference strains of B. elkanii.l6S rRNA sequences shown that HAS5 clustered with the reference strains of B. elkanii, and the sequence divergence between HAS5 and USDA76 was 0.4%. Strains of DD3, BQ3, GXD1, JZ2, and reference strains from other species formed a large homological cluster sharing a high homology in 16S rRNA sequence, and their sequence divergence was less than 1%.Results of 16-23S rRNA PCR-RFLP shown that group I comprised the same strains of Genotype I in 16S rRNA RFLP assay. Strains of Genotype II in 16S rRNA RFLP assay were divided into group II and group III. Group II contained B. liaoningense and partial isolates from Genotype II. Group III consisted of B. japnonicum and the remains of genotype II, and mainly isolated strains from central China Yangtze River drainage basin .Group IV consisted of three strains from Hongan and B. elkanii.Results of RAPD analysis shown that test strains and reference strains were also divided into I , II, III,IVgroups in the similarity of 72%.They are similiarily tol6-23S rRNA PCR-RFLP assays. At higher similarity, strains tested from different area were further clustered which reflected the effect of geographical factor on rhizobia genetic diversity.16S rRNA RFLP, 16S rRNA sequencing, 16S-23S rRNA Intergenetic Spacer (IGS) RFLP and G-C rich RAPD assays were conducted to clarify genetic diversity and phylogeny of 55 indigenous cowpea [Vigna unguiculata (L.) Walp.] rhizobia from different geographic regions of China. Isolated cowpea rhizobia comprised six 16S rRNA genespecies. Genotype I consists of 15 isolated strains from different geographic region. Together with reference strains of B. japonicum and B. liaoningense, they share the same RFLP patterns. Genotype II comprises 26 isolated strains, which was genetically separate from B. japnicum, B. liaoningense, and B. elkanii. GenotypeIII contains 7 isolates and the reference strains of B. elkanii. Genotype IVwas solely consisted of strain DdE4,更多还原