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1、河北大学 硕士学位论文 含羞草根瘤菌的数值分类及种群鉴定 姓名：戴燕燕 申请学位级别：硕士 专业：微生物学 指导教师：刘晓云 20100601 摘 要 I 摘 要 本论文采用表型性状研究、SDS-PAGE、16S rDNA 全序列分析、DNA-DNA 杂交 测定及 G+C mol%等方法对分离自云南省西双版纳地区的 66 株含羞草根瘤菌进行了种 群地位的研究及环境污染物耐受性方面的测试，同时从含羞草根瘤菌的分类、研究方法 及种群的鉴定等方面对含羞草根瘤菌的研究进行了综述。 对 16S rDNA PCR- RFLP 所获得的 5 大类群的代表菌株进行 16S rDNA 全序列克隆 测序。 20
2、株代表菌株在系统发育树中归于 3 个系统发育分支， 其中代表菌株 HBU66044、 HBU66029 等 10 个菌株位于 Burkholderia 属；代表菌株 HBU66322、HBU66194 等 9 株 菌位于 Cupriavidus 属；菌株 HBU66289 位于 Rhizobium 属。 对66株供试菌株进行了89项生理生化和抗性研究，结果表明分离自含羞草植物的根 瘤菌具有较丰富的表型多样性，且大多为快生根瘤菌，归于Rhizobium、Burkholderia和 cupriavidus三个系统发育分支，同时发现了一批可以耐高盐和高抗生素的抗逆性菌株。 通过SDS-PAGE全细胞
3、蛋白电泳、BOX-PCR分析进行研究，发现这两种方法获得图谱类 型较多，且菌株图谱类型大多依宿主及采集地点不同而表现出差异。结合表型生理生化 结果，将多种方法所得性状、特征相同菌株归为一种类型，最终将所有66株含羞草根瘤 菌归分为55个表型和遗传型菌株类型。 经 DNA-DNA 杂交及 G+C mol%测定结果可知，菌株 HBU66286 与模式菌株 Burkholderia phymatum LMG21445 杂交同源性达到 83.4%，G+C mol%也在 Burkholderia 属的 G+C mol%范围内（6467%），符合种内水平，说明是该菌株为瘤状伯克霍尔德 菌（Burkhold
4、eria phymatum） 。菌株 HBU66322，HBU66194，HBU66166，HBU66232 与其 16S rDNA 全序列同源性最高的模式株 Cupriavidus taiwanensis LMG 19424 DNA-DNA 杂交值在 060%之间，且 G+C mol%也接近模式株 G+C mol%（67.3%） 。 菌株 HBU66049，HBU66029，HBU66044，HBU66305 与其同源性较高的模式株 Burkholderia phymatum LMG 21445 的 DNA-DNA 同源性在 060%之间，G+C mol%也 在 Burkholderia 属
5、的 G+C mol%范围内。 综合以上研究表明，分离自云南版纳地区的 66 株含羞草根瘤菌归于根瘤菌的 3 个 属，分别为 Rhizobium、Burkholderia、Cupriavidus，其中菌株 HBU66322，HBU66194， 摘 要 II HBU66166，HBU66232、HBU66029，HBU66044 可能是不同于已知种的 DNA 杂交新 种。各类群代表菌株对重金属及苯酚有较强的耐受性，而且部分菌株还可耐高盐、耐酸 碱并能耐一定浓度的抗生素，为根瘤菌应用于土壤修复应用与研究提供了理论依据。 关键词 含羞草 根瘤菌 种群鉴定 数值分类 DNA-DNA 杂交 Abstrac
6、t III Abstract This thesis reviewed recently developed rhizobial systematics, summarized the main methods applied in rhizobia identification and studied on the heavy metal and phenol tolerance. The phylogenetic positions of representative strains symbiotic with mimosa were revealed by 16S rRNA gene
7、sequences. It indicated that all strains separated into Rhizobium、 Burkholderia and cupriavidus.The representative strains HBU66117 、 HBU66074 、 HBU66003、HBU66305、HBU66304、HBU66247、HBU66049、HBU66044、HBU66029、 HBU66286 were located in the subbranch of Burkholderia.The strains HBU66294、 HBU66322、HBU66
8、194、HBU66166、HBU66232、HBU66133、HBU66131、HBU65028、 HBU66045 distributed in the cupriavidus branch, and HBU66289 in the Rhizobium genus. Sixty-six rhizobial strains were studied by numerical analysis of 89 phenotypic characteristics.The results showed that 45 of all tested strains were clustered into
9、5 groups at a similarity level of 80%. The strains in this study with a high level resistance to saline、pH、 antibiotics were found. Some strains can resist to 5% NaCl, and grow at YMA medium with pH12.0, some strains even resisted to 300 g/mL of antibiotic. This result showed that there is a great p
10、henotypic diversity among the isolates. In order to indentify the strain furtherly, whole cell protein SDS-PAGE and BOX-PCR were applied to central rhizobial strains. The patterns of SDS-PAGE and BOX-PCR showed that it can get more message from the strains. Based on the results of above methods, all
11、 strains were classified 55 different styles of all strains . Central strain HBU66286 which were related to Burkholderia were chosen for determination of DNA base composition and DNA-DNA hybridization in comparison with the type strains of related described rhizobial species. The DNA relatedness bet
12、ween HBU66286 and B.phymatum was 83%, which demonstrated that HBU66286 belonged to B.phymatum. And the DNA homology of the representative strains HBU66322、HBU66194、 HBU66166、HBU66232 with reference strain C.taiwanensis LMG19424T ranged from 0 60%.the DNA homology of the representative strains HBU660
13、49、HBU66029、HBU66044、 HBU66305 with reference strain B.phymatum LMG21445T ranged from 0-60%.the G+C Abstract IV mol% of them were all in the G+C mol% range of the genu Burkholderia or cupriavidus.It showed that they may be new distinct new species. To determine that the strains HBU66322，HBU66194，HBU
14、66166，HBU66232， HBU66049，HBU66029，HBU66044，HBU66305 that isolated from Mimosa spp. related to Burkholderia、cupriavidus species, the ability to renodulate the 10 species of leguminous plants were checked, Over a four-week period, all of test strains only can formed nodules with the Leucaena glaucapla
15、nt on the roots of plant. Nine rizobium strains from mimosa were used to analyse resistance and the maximum resistance level （MRL） to heavy metals and phenol.The result showed that most of strains had strong resistance for heavy metals and phenol.Some strains even were tolerant to Zn2+of 5 mmol/L、Hg
16、2+ of 0.5 mmol/L、Cu2+ of 0.6 mmol/L、phenol of 1700 mg/L. Meantime the strains of Burkholderia have stronger resistance than the strains from cupriavidus. From above analysis, we discovered that strains isolated from nodules of Mimosa have great diversity. The strains isolated from mimosa were mainly distributed in Rhizobium、 Burkholderia and cupriavidus branches respectively.Meantime 8 strains may be new speises. These finding also shows that the strains of Burkholderia have stronger resi
