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作 者 ：彭贤超,张小平*,徐开未,黄昌学,K．Lindstrom

期 刊 ：生态学报 2008年 28卷 11期 页码：5469~5481

关键词：数值分类；BOXAIR-PCR；16S rDNA；PCR-RFLP；GSⅡ基因；序列分析；

Keywords:numerical taxonomy, BOXAIR-PCR, 16S rDNA, PCR-RFLP, GSⅡgene, sequences analysis,

摘 要 ：研究利用数值分类、BOXAIR-PCR指纹图谱、16S rDNA PCR-RFLP、16S rDNA和GSⅡ序列分析等方法，研究了分离自金沙江干热河谷区的86株胡枝子根瘤菌的多样性和系统发育，结果表明金沙江干热河谷区胡枝子根瘤菌蕴涵丰富的生物多样性。通过数值分类，供试未知菌株表现出极大的表型性状多样性，能耐高温(60 ℃)和低pH (4.0)，在低温(10 ℃)或者高pH (9.0)条件下生长很差，耐盐性也很差。供试未知菌株的16S rDNA用HaeⅢ、MspⅠ、HinfⅠ和TaqⅠ酶切后具有16种遗传图谱类型，其中10株供试未知菌的16S rDNA遗传图谱类型不同于所选用的已知参比菌株。BOXAIR-PCR的分群结果分散，很多在16S rDNA PCR-RFLP中具有相同遗传类型的菌株也表现较大差异，表明了供试菌株在基因组水平上差异很大。序列分析结果表明，6株代表菌株分布于Rhizobium、Sinorhizobium、Mesorhizobium、Bradyrhizobium 4个属，16S rDNA序列与GSⅡ序列分别构建的系统发育树在属水平上基本一致，但16S rDNA序列的同源性比GSⅡ高，6株代表菌株间16S rDNA序列的同源性在87.5%～99.5%之间，GSⅡ序列的同源性在79.4%～89.8%之间；而代表菌株与亲缘关系最近的参比菌株间的16S rDNA序列的同源性为99.9%～100%，GSⅡ的同源性为88.9%～99.6%。

Abstract:The diversity and phylogeny of 86 rhizobial strains which isolated from the root nodules of Lespedeza Michx in arid-hot valley of JinSha river in Sichuan, China, were studied by numerical taxonomy,BOXAIR-PCR fingerprinting,16S rDNA PCR-RFLP, 16S rDNA and GSⅡ sequences. The result of numerical taxonomy showed that there was a great phenotypic diversity among the isolates. The strains tested could grow normally at 60℃ or pH＜4.0 culture conditions. However, they could not grow at 10℃ or pH＞9.0. They were not endured salt tolerance. The same strains used in numerical taxonomy were analyzed by 16S rDNA PCR-RFLP with four kinds of restriction endonucleases (HaeⅢ、MspⅠ、HinfⅠand TaqⅠ). The strains had 16 genotypes and 10 unique genotypes different from the reference strains. Based on the dendrograms generated by BOX-PCR, the strains were divided into 37 groups at the similarity of 82%, some strains belonged to one phenon in 16S rDNA PCR-RFLP are in different phenons in BOXAIR-PCR, which showed that there was a great diversity in genes among the stains. The results of analysis on 16S rDNA and GSⅡsequences indicatded that six representatives of strains were classified into four groups on genus Bradyrhizobium、Rhizobium、Sinorhizobium and Mesorhizobium, and 16S rDNA sequencing homology was higher to GSⅡ sequencing homology. GSⅡ sequencing homology and 16S rDNA sequencing homology among six representatives of strains were respectively between 79.4%-89.8% and 87.5%-99.5%. Further, GSⅡ sequencing homology and 16S rDNA sequencing homology between the representatives of strains and phylogenetically closest reference stains were respectively from 88.9% to 99.6% and from 99.9% to 100%.

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