含羞草、山蚂蟥等几种豆科植物根瘤菌的数值分类及16S rDNA PCR-RFLP分析

Numerical Taxonomy and 16S rDNA PCR-RFLP of Rhizobia of Some Legumes Isolated from Mimosa spp. and Desmodium spp.

为揭示云南省豆科植物根瘤菌的资源多样性,采用数值分类、16S rDNA PCR-RFLP和16S rDNA全序列分析技术对云南省8个地区含羞草、山蚂蝗等几种不同豆科植物分离的48株根瘤菌菌株进行了遗传和表型多样性研究。16S rDNA PCR产物经HinfⅠ、MspⅠ、RsaⅠ和HaeⅢ4种内切酶消化后产生22种遗传图谱类型;数据经MVSP3.0软件聚类后,所有供试菌株在70%的相似性水平上分为4个分支,经16S rDNA全序列分析,确定分支Ⅰ为伯克霍尔德属(Burkholderia)类群,含1株菌株;分支Ⅱ为贪铜菌属(Cupriavidus)类群,含5株菌株;分支Ⅳ为根瘤菌属(Rhizobium)类群,包括29株菌株;而分支Ⅲ没有和参比菌株聚在一起,经测序为狭长平胞属(Stenotrophomonas)类群。通过对菌株的抗生素抗性、耐盐性、生长温度范围、BTB产酸产碱反应以及对3-酮基乳糖利用情况共计28个表型性状测定,结果显示所有菌株耐盐性较强,多数菌株可耐受60℃的高温。实验表明,分离自云南省的这些菌株具有丰富的遗传和表型多样性。

In order to characterize the diversity and phylogeny of rhizobium in Yunnan of China, 48 strains isolated from root nodules of Mimosa spp. and Desmodium spp. in 8 regions of Yunnan Province were studied with numerical taxonomy, 16S rDNA PCR-RFLP and 16S rDNA gene sequencing analysis. 23 genotype profiles were obtained from the fingerprinting of the 16S rDNA PCR-RFLP by four restriction endonucleases Hirnc I , Msp I, Rsa I and Hae 1~. These strains were clustered into 4 branches at the similarity level of 70% by MVSP3.0 software. 16S rDNA sequencing revealed that branch I belonging to the genera of Burkholderia, include 1 strain; branch II belonging to the genera of Cupriavidus, include 5 strains; branch IV belonging to the genera of Rhizobium, include 29 strains; branch 111 belonging to the genera of Stenotrophomonas with no reference strains. The analysis of 28 phenotypie characteristic Of these strains showed that most of these strains had a high level resistance to saline, some strains could grow＇under severe environment such as high content ration of antibiotics （300 ug/mL） and high temperature （60~C）. All the results demonstrated a high genetic and phenotypic diversity of rhizobium nodulating legumes of Yunnan Province.