【作者】 冯春生；

【导师】 韦革宏；

【作者基本信息】 西北农林科技大学 ， 微生物学， 2008， 硕士

【摘要】 本文采用16S rDNA PCR RFLP、数值分类、nifH PCR RFLP、nodA PCR RFLP、系统发育分析(16S rDNA、nifH、nodA基因序列分析)等技术对67株分离自我国西北地区天蓝苜蓿根瘤菌进行了系统发育研究。16S rDNA PCR RFLP及序列分析表明,所有待测菌株共分为6种酶切类型,系统发育研究表明各酶切类型代表菌株形成了根瘤菌属(Rhizobium),土壤杆菌属(Agrobacterium),中华根瘤菌属(Sinorhizobium)3个系统发育分支;大部分菌株属于草木犀中华根瘤菌(S. meliloti)。数值分类结果在88%的相似水平上分为3个群。群1中华根瘤菌属;群2为土壤杆菌属的;群3为根瘤菌属。结果表明表型分群方法和遗传分群方法有较好的一致性。生理生化结果表明,供试菌株在抗生素试验当中,77.8%的菌株在青霉素300μg/mL中均可生长。99%的菌株均可以在任何浓度的林可霉素中生存。72.2%的菌株可以耐受4%NaCl。90%的菌株可以生长在初始pH 11、12的YMA平板上。所有菌株可经受60℃1 0min的热冲击。nifH PCR RFLP和序列分析结果表明nifH DNA序列反映的系统发育关系与16S rDNA序列的系统发育关系有比较大的差异。然而,将nifH DNA序列翻译为蛋白质序列以后,除了CCNWGS0061、CCNWNX0047外,nifH蛋白质序列相似性为100%。该结果论证了大部分菌株的nifH所编码的固氮酶是完全相同的。nodA PCR RFLP和序列分析结果表明除了CCNWGS0056和CCNWNX0041例外,基于nodA反映的系统发育关系与16S rDNA序列的系统发育关系基本是一致的。综合以上结果可得出以下结论:与天蓝苜蓿共生的根瘤菌相当专一;供试天蓝苜蓿具有较强的抗逆性;表型分群方法和遗传分群方法有较好的一致性;nifH和nodA基因的高度保守性很好地解释了天蓝苜蓿与草木犀中华根瘤菌(S. meliloti)共生专一性的现象。更多还原

【Abstract】 Sixty-seven bacterial isolates obtained from root nodules of the legume of Medicago lupulina mainly grown in the Northwestern of China were characterized by 16S rDNA PCR RFLP、numberical taxonomy、nifH PCR RFLP、nodA PCR RFLP、phylogenic studies( the sequences analysis of 16S rDNA、nifH、nodA genes).16S rDNA PCR RFLP and sequences analysis showed that all the tested strains were divided into six diferrent genotypes and belonged to Sinorhizobium, Rhizobium, Agrobacterium respectively. Most of them could be identify as Sinorhizobium meliloti.All the tested strains were divided into 3 phena at the level of 88% by numberical taxonomy. The group 1 were Sinorhizobium; the group 2 were Agrobacterium;the group 3 were Rhizobium. The result of phenotypic sdudies had a consistency with genetic sdudies.Phenotypic studied showed that 77.8% or 99% of isolates were resistant to 300μg/mL panicillin or lincomycin respectively. 72.2% isolates could grow on YMA plated supplied with 4%NaCl. 90% of isolates could grow on YMA plates at PH11、12. All the tested strains could sustain the heat shock of 10 minutes at 60℃.nifH PCR RFLP and sequences analysis showed that nifH sequnece analysis existed discrepancy with 16S rDNA phylogeny. While, after the nifH DNA were translated into protein sequence, all the nifH protein sequence have similarity of 100%, except for CCNWGS0061、CCNWNX0047. The result demonstrated that the nitrogenase codeed by nifH were totally identical.nodA PCR RFLP and sequences analysis showed that the phylogeny results between nodA and 16S rDNA were quite agreed with each other, except for CCNWGS0056、CCNWNX0041.From the results mentioned above, conclusions be drawed as follow: the microsymbionts of Medicago lupulina were quite specific; the tested strains showed strong stress resistance; the phenotypic taxonomy and genetic taxonomy were quite agreed with each other. The symbiotic specificity between Medicago lupulina and S. meliloti could be explained by the conservative character of nifH and nodA.更多还原