【作者】 陈卫民；

【导师】 韦革宏；

【作者基本信息】 西北农林科技大学 ， 微生物， 2009， 博士

【摘要】 采用表型分析(全细胞脂肪酸)和遗传型分析(16S rRNA PCR-RFLP、nodC PCR-RFLP、nodA PCR-RFLP、nifH PCR-RFLP;16S rRNA、nodC、nodA、nifH、持家基因dnaK、dnaJ、recA和glnA基因全序列分析;G+Cmol%;DNA-DNA同源性等)多相分类技术,对分离自我国西北干旱半干旱地区豆科植物骆驼刺、黄芪以及包括欧洲和北美洲在内的刺槐根瘤菌进行了多样性分析和系统发育研究。16S rRNA PCR-RFLP及其序列分析结果表明,分离自甘肃和新疆的33株骆驼刺根瘤菌存在明显的遗传多样性。主要分为3基因型,多数按地区聚群,归属于中慢生根瘤菌属,基因型Ⅰ和Ⅱ的菌株与已知中慢生根瘤菌的序列相似性最大仅为97.8%,与中华根瘤菌属各种间序列最大仅为相似性为96.0%,与根瘤菌属各种间序列最大仅为相似性为93.1%,属于新种群;基因型Ⅲ主要由来自甘肃安西的根瘤菌组成,属于M. tianshanesnse。持家基因dnaK、dnaJ、recA和glnA全序列分析,结果与16S rDNA序列分析结果一致,并能将系统发育关系较近的菌株区分开,可以用来进行根瘤菌的系统分类研究。通过共生基因nodA、nodC和nifH PCR-RFLP分析,共得到4种nodA基因型、5种nodC基因型、5种nifH基因型。其中,分离自新疆的根瘤菌共生基因型单一,揭示了根瘤菌与寄主的专一性及其地区的专一性;分离自甘肃却表现出丰富的多样性及其地区的特异性。序列分析表明,它们在系统发育树上与中慢生根瘤菌的共生基因关系最近,且都形成了独立的分支,属于新的共生基因,也验证了两个新种的可能性。全细胞脂肪酸分析结果表明,骆驼刺根瘤菌16S rRNA基因型Ⅰ和Ⅱ的代表菌株CCNWXJ12-2和CCNWXJ40-4均含有16 : 0、17 : 0、17 : 0 iso、18 : 0、19 : 0 cyclo 8c以及summed feature 3(包括12 : 0、未知ECL 10.928、16 : 1 iso 1/14 : 0 3-OH),不含20 : 3 6、9和12cis,属于中慢生根瘤菌属。与已知参比菌株相比,基因型Ⅰ和Ⅱ代表菌株CCNWXJ12-2和CCNWXJ40-4含有很高18 : 1 7c。当这两个菌株相比较时,基因型Ⅰ的代表菌株CCNWXJ12-2不含18 : 1 9c,并且11-Methyl 18 : 1 7c和20 : 1 7c远高于基因型Ⅱ代表菌株,基因型Ⅱ的代表菌株CCNWXJ40-4不含17 : 0 cyclo、17 : 1 8c和20 : 2 6,9c,证明两个菌株是完全不同的新种。16S rRNA基因型Ⅰ的菌株平均Tm值为78.64℃,G+Cmol%是62.06%,其代表菌株CCNWXJ12-2与群内各菌株的DNA-DNA平均同源率为92.32%,与中慢生根瘤菌属各参比菌株的同源率在0.56~44.25%之间。基因型Ⅱ的菌株平均Tm值为78.82℃,G+Cmol%是62.06%,与各参比菌株同源率在3.85～43.33%之间。基因型Ⅰ与基因型Ⅱ菌株DNA-DNA杂交同源率37.87%。可以确定基因型Ⅰ与基因型Ⅱ的菌株为中慢生根瘤菌属中不同的新种。对55株分离自陕西、甘肃、宁夏和新疆4个省、31个采样点的12种黄芪植物的根瘤菌进行了16S rDNA遗传型分析,共得到11种16S rDNA基因型,序列分析表明,这些黄芪根瘤菌归属于Agrobacterium、Mesorhizobium、Sinorhizobium、Mesorhizobium和Rhizobium5个属,其中,从基因型Ⅱ到基因型Ⅷ的7个基因型属于中慢生根瘤菌,约占总数的54.5%;基因型Ⅺ的20株菌都属于Agrobacterium tumefaciens,且均分离自新疆。通过序列比较,同时发现黄河对黄芪根瘤菌的分布起着重要的影响。黄芪根瘤菌的nifH、nodA和nodC基因PCR-RFLP分析,共得到14种nifH基因型、18种nodA基因型和18 nodC基因型,揭示了黄芪根瘤菌的共生基因具有丰富的遗传多样性。nodC基因序列分析显示,大多数黄芪根瘤菌的固氮基因位于中慢生根瘤菌属中,并且聚集在一起,系统发育关系十分接近,揭示了不同地域的不同黄芪根瘤菌之间可能发生了基因转移。同时,回接和互接试验,证明了斜茎黄芪具有广泛的结瘤范围,它可以与不同来源的根瘤菌形成高效的固氮体系。对起源于北美洲经过欧洲引进到中国分布于西北地区的刺槐根瘤菌进行了系统发育研究及共生基因比较分析,16S rDNA PCR-RFLP及序列分析揭示了西北地区刺槐根瘤菌具有很高的多样性,而nodC、nodA、nifH PCR-RFLP及序列分析,发现三个地点的共生基因非常相似,专一性较强,可能是西北土著根瘤菌通过横向转移的方式获得了刺槐携带而来的根瘤菌的共生基因,使得刺槐能在不同的生态环境中很快的形成有利于自己生长的共生体。更多还原

【Abstract】 The rhizobial strains isolated from the nodules of Alhagi sparsifolia, Astragalus sp. and Robinia pseudoacacia growing in the arid and mid-arid regions of northwest of China were characterized by the genetic and phenotypic methods, including analysis of 16S rDNA, nodC, nodA, nifH genes PCR-RFLP and their sequences, G+Cmol%, the DNA-DNA hybridization, the whole fatty acids and four housekeeping genes.Thirty-three rhizobia were isolated from the legume Alhagi sparsifolia growing in the Gansu and Xinjiang provinces. They fell into three groups by restriction analysis of their 16S rDNA, and these, together with dnaK, dnaJ, recA and glnA genes, were sequenced from representative isolates to assess their taxonomic position by phylogenetic analysis. The bacteria in each group belonged to different lineages that might represent three different Mesorhizobium species, two of which form a novel clade very distinct from other species in the genus. The strains belonging to genospeciesⅠandⅡshared less than 97.8% with defined Mesorhizobium species, 96.0% with Sinorhizobium species and 93.1% with Rhizobium species.Four nodA types, five nodC types and five nifH types were obtained through the PCR-RFLP analysis of symbiotic genes. Sequencing anylysis showed that the strains corresponding to genospeciesⅠandⅡharboured closely related nodA, nodC and nifH genes forming new lineages. They fell into Mesorhizobium group. The strains from Xinjiang were specific to geographical site and host plants, however strains from Gansu had high diversity of symbiotic gene types.CCNWXJ12-2 and CCNWXJ40-4 belong to Mesorhizobium because they contain 16 : 0, 17 : 0, 17 : 0 iso, 18 : 0, 19 : 0 cyclo 8c and summed feature 3, without 20 : 3 6, 9, 12cis. Both of them contain 18:1ω7c except all defined Mesorhizobium species. The difference between CCNWXJ12-2 and CCNWXJ40-4 is that the forward has 17 : 0 cyclo, 17 : 1 8c and 20 : 2 6, 9c, or the later has 18 : 1 9c. Therefore, they are two different novel species.For the strains of the genospeciesⅠ, their Tm is 78.64℃, G+Cmol% is 62.06%, DNA-DNA hybridization (<45%). The strains of the genospeciesⅡ, their Tm is 78.82℃, G+Cmol% is 62.06%, DNA-DNA hybridization (<43.3%). The DNA homology between CCNWXJ12-2 and CCNWXJ40-4 is 37.87%, which suggests they are two different new species.A total of 55 isolates from root nodules of 12 different Astragalus species growing in four provinces in northwestern China, covering 31 sampling sites, were characterized by PCR-RFLP analysis of 16S rDNA and symbiotic genes. Eleven genotypes were identified by sequence analysis of 16S rDNA, they belong to Agrobacterium, Mesorhizobium, Sinorhizobium, Mesorhizobium and Rhizobium related strains, eight of them were Mesorhizobium-related species, accounting for 54.5% of all isolates, and genospeciesⅪconsisting of 20 isolates is a biggest group, they belong to Agrobacterium tumefaciens and all of them are from Xinjiang.Eighteen nodA types, eighteen nodC types and fourteen nifH types were attained through the PCR-RFLP analysis of symbiotic genes. The nodC gene phylogeny placed them into Sinorhizobium, Mesorhizobium and Rhizobium branches, most Mesorhizobium-related strains shared the similar symbiotic genes independence of their original host plants and sites, it indicated that genes transfer occurred within genus or genera, which played an important role in diversification of rhizobia and in adapting to an introducing environment. The presence and absence of Astragalus rhizobial species in upstream or downstream suggested that their distribution should be influenced by irrigation of Yellow.Thirty three isolates from Robinia pseudoacacia were characterized using RFLP and sequencing of 16S rRNA, nodA, nodC and nifH genes. Their 16S rRNA gene patterns and sequences placed them in three clades: 85% of isolates were related to the Mesorhizobium mediterraneum/temperatum group, while the remainders were similar either to M. amorphae or to Sinorhizobium meliloti. However, despite their diverse taxonomic positions, the nodA, nodC and nifH genes phylogenies indicated that these R. pseudoacacia symbionts share similar symbiosis genes, implying gene transfers and a degree of host specificity. Comparison of R. pseudoacacia symbiotic diversity in native and other invaded areas suggests that most Chinese symbionts may not have arrived with the seed but were local bacteria that acquired specific symbiotic genes from American native rhizobia.更多还原