【作者】 陈晓琴；

【导师】 陈强；

【作者基本信息】 四川农业大学 ， 微生物学， 2007， 硕士

【摘要】 以从流沙河流域不同海拔高度土壤中分离获得的53株土壤自生固氮菌为研究材料，采用数值分类、BOX-PCR、RAPD和16S rDNAPCR-RFLP分析方法，对其表型和遗传多样性进行了研究。生理生化分析表明，供试菌株在营养特性、抗生素抗性、化学药物耐受性、耐盐及耐酸碱能力等方面存在明显的差异。其中，有8株菌耐4％的NaCl，有4株菌能忍耐pH4.0的环境，有13株菌能在4℃条件下正常生长；菌株1262能以钼酸铵作唯一氮源，且对测试的6种抗生素的抗性达300μg／mL。数值分类将所有供试菌株在82.6％的相似水平上分成17个独立的表观群，其中有7个菌株单独成群，第5和第12表观群最大，分别由9个和14个菌株组成，分布在海拔722m～1440m和747m～1800m的生境中，分离土类有紫色土、黄壤、水稻土、黄棕壤等多种类型，表明它们是该区域的优势自生固氮菌。各表观群在土壤类型和海拔高度无明显相关性，表现出丰富的表型多样性特征。BOX-PCR分析获得了较丰富的扩增带谱，能较好地揭示出不同菌株间存在的遗传差异。在68％的相似水平上供试菌株被分为10个遗传类群，除菌株1051独立成群外，其余菌株聚成9个遗传群，各群的菌株在2～14株之间。聚群菌株与分离地海拔高度和土壤类型相关性较小，揭示了供试固氮菌在菌株水平上具有不同水平的遗传多样性。RAPD分析选用6个随机单引物，各引物分别产生了2～17条带，扩增产物表现出丰富的谱带类型；RAPD聚类图在70％的相似水平处将全部供试菌株分成9个RAPD遗传群，其中有32株菌聚入群2、4和群5，群8由2株菌组成，其余遗传群分别由3～6株菌组成。分析结果表明，RAPD能有效揭示供试菌株的遗传多样性。所有供试菌株的16S rRNA PCR-RFLP分析中，4种限制性内切酶(MspⅠ、HaeⅢ、HinfⅠ和AluⅠ)的酶切图谱共获得27种遗传型，其中有16个为唯一遗传型，遗传型6和12分别有10个和5个菌株，其它遗传型则只包含2～3个菌株，表明供试菌株在系统发育上仍存在丰富的多样性特征。在78.6％相似性水平处，所有菌株被分为8个16S rRNA遗传群。总的来说，本研究所采用的表型分群和遗传型分群的结果在总体趋势上是一致的，BOX-PCR的群2、4和10分别与数值分类中的第5、13和12表观群一致；RAPD的群3、4和8分别与数值分类的群6、12和8相对应，16S rRNA PCR-RFLP中的群5、6和8则数值分类的群6、12和10完全吻合，说明本实验所获得的研究结果可信。由于本实验未选取标准菌株，要进一步确定供试菌株的确切分类地位和系统发育关系，还需要通过代表菌株的16S rDNA全序列测定及DNA-DNA杂交来确定。更多还原

【Abstract】 Fifty-three free-living Dizotrophs strains isolated from soils at different altitude inLiusha river valley were analyzed by phenotypic features, BOX-PCR, RAPD and 16SrRNA PCR-RFLP to reveal their phenotypic and genotypic diversity.The results of phenotypic features showed that the tested strains had differentphysiological and biochemical characteristics, including carbon and nitrogen sourcesusage, antibiotic resistance, chemical drug tolerance, salt and corrosion resistance, etc.and there were 8 strains could grow on the medium containing 4% NaCl, 4 strains couldgrow in the environment of pH4.0, and 13 strains could grow under 4℃conditions.Furthermore, strain 1262 was the only strain using ammonium molybdate as solenitrogen source, and could tolerate 6 kinds of antibiotics tested at concentration of300ug/mL.Dendrogram of numerical taxonomy showed that all the tested strains were dividedinto 17 independent groups at the boundary of 82.6% similarity, and 7 individual groupswere included; group 5 and group 12 were the two largest among these groups, and wereformed by 9 and 14 strains, respectively; these strains distributed in the habitats at thealtitude from 722m～1440m and 747m～1800m, and in purple soil, yellow soil, andpaddy soil, and so on, which suggested that strains in these two groups were thedominant ones in these areas. There was no obvious correlation between the phenotypicgroups and soil types and altitude strains distributed, which demonstrated the phenotypicdiversity among the strains was existed.Abundant bands were amplified in analysis of BOX-PCR, which could be used toreveal the existence of genetic diversity. All the strains clustered into 10 different geneticgroups at the similarity of 68%, except strain 1051 grouped individually, the other strainsaccumulated 9 genetic groups with 2～14 strains for each.Six RAPD primers were chosen for RAPD analyzing, each primer was produced 2～17 bands separately. The results of RAPD analysis indicated that all the strains weredivided into 9 groups at the similarity of 70%, and 32 strains grouped into group 2, 4 and5; there were only 2 strains in group 8, and the others were contained 3-6 strains, respectively. The results also showed that RAPD revealed the genetic diversity amongstrains effectively.The 16S rDNA PCR-RFLP analysis resulted in 27 different genotypes by 4 kinds ofendonuclease (MspⅠ, HaeⅢ, HinfⅠand AluⅠ), there were 16S rDNA genotypes formed,and genotype 6 and 12 had 10 and 5 strains respectively, and the other genotypes contained2-3 strains, which indicated that these strains had great diversity in phylogeny. The 16SrDNA PCR-RFLP dendrogram showed that there were 8 genetic groups formed at 78.6%similarity level, and group 4 and 6 were the largest two groups, and were constructed by 11and 14 strains, respectively.Overall, the analysis of phenotypic and genotypic diversity was good agreementwith each other, which approved the research results were credible and stable. Forexample, the group 2, 4 and 10 of BOX-PCR were good agreement with group 5, 13 and12 of numerical analysis, and the group 3, 4 and 8 of RAPD were equal to group 6, 12and 8 of numerical analysis, and the group 5, 6 and 8 were identical with group 6, 12 and8 of numerical analysis, which indicated the 4 methods used could reveal the biodiversityamong the tested free-living nitrogen fixation strains.Since no reference strain was used in the study, the 16S rDNA full sequence andDNA-DNA hybridization should be performed to determine the exact taxonomic positionand phylogeny of free-living Dizotrophs.更多还原