金沙江干热河谷区胡枝子(Lespedeza Michx)根瘤菌多样性及其系统发育

Diversity and phylogeny of rhizobial strains isolated from root nodules of Lespedeza Michx in arid-hot valley of Jinsha River

研究利用数值分类、BOXAIR-PCR指纹图谱、16SrDNA 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、Bradyrhizobium4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%。

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 GS Ⅱ 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 ,Msp Ⅰ , Hinf Ⅰ and Taq Ⅰ）. 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 GS Ⅱ sequences 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 GS Ⅱ sequencing homology. GS Ⅱ 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, GS Ⅱ 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%.