刺槐根瘤菌遗传多样性与系统发育的研究
摘要
从甘肃、陕西两省的3个采样点分离获得84株刺槐共生根瘤菌,采用数值分类、RFLP和序列测定多相分类技术,对16S rDNA、recA、nifH和nodC基因进行遗传多样性和表型多样性分析。首次采用多点采样的方法系统地对中国刺槐根瘤菌进行遗传多样性究研。
16S rDNA PCR-RFLP和全序列系统发育分析结果表明,中国刺槐根瘤菌共有9种酶切图谱类型组合,即9种基因型;16S rDNA全序列分析表明,9株代表菌株位于系统发育进化树的8个分支上,分别是M. loti/ciceri,M. tianshanense,M. huakuii,M. amorphae,R. giardinii,S. xinjiangense,S. morelense和B. liaoningense。其中16S rDNA RFLP基因型Ⅰ(M. amorphae)为刺槐优势根瘤菌基因型,占待测菌株的62.2%,广泛分布于3个采样点。
nodC和nifH的研究结果表明,刺槐根瘤菌具有相对保守的共生基因。序列分析结果显示,nifH基因位于系统进化树M. albiziae,S. americanum和B. elkanii的分支上。nodC基因虽然在RFLP分析结果中为四种基因类型,但是序列系统发育分析表明,4种nodC基因型在系统发育树上聚在同一分支(彼此间相似性达98.6%—99.4%),与M. amorphae ACCC19665的nodC基因相距最近,即同源性最高。而且与以前研究的中国刺槐根瘤菌,北美刺槐根瘤菌和欧洲刺槐根瘤菌的nodC基因位于同一系统发育树分支上,同源性为98.9%—100%之间。
recA基因序列分析结果中,位于B. liaoningense和R. giardinii分支的刺槐根瘤菌,其16S rDNA序列的聚类结果也分别位于B. liaoningense和R. giardinii,分析结果在种的水平上达到一致。但是,位于中慢生根瘤菌属的根瘤菌recA和16S rDNA聚类分析结果仅在属的水平上达到一致,在种的水平上稍有差异,其中recA系统发育树中位于M. ciceri,M. caraganae和M. amorphae分支上的代表菌株,在16S rDNA结果中依次位于M. loti,M. tianshanense和M. amorphae分支上。
数值分类结果表明,在66%的水平上待测菌株聚成3个表观群,在83%水平上聚成5个亚群。其中亚群1、2和3主要由属于M. amorphae的待测菌株组成,构成表观群Ⅰ;亚群4 (M. huakuii)和亚群5 (M. tianshanes)一起构成表观群Ⅱ;表观群Ⅲ的待测菌株主要属于Sinorhizobium。表型多样性研究表明,西北干旱半干旱地区的刺槐根瘤菌可以利用绝大多数的碳源和氮源,而且它们具有很高的耐盐性、耐碱性、抗性。
84 isolates from Robinia pseudoacacia in 3 sampling sites in Shaanxi and Gansu were characterized using restriction fragment length polymorphism and sequencing of 16S rDNA, recA, nodC and nifH genes, as well as numerical taxonomy of physiological and chemical characteristics.
16S rDNA patterns and sequences placed the tested strains in 9 RFLP groups, which located in 8 branches of the phylogenetic tree of 16S rDNA sequences. The 8 branches were M. loti/ciceri, M. tianshanense, M. huakuii, M. amorphae, R. giardinii, S. xinjiangense, S. morelense and B. liaoningense, among which the largest group was M. amorphae with 62.2% of tested stains from all the three sampling sites. The results revealed the rich genetic diversity of rhizobia nodulating Chinese Robinia.
The results of nodC and nifH revealed that the tested strains nodulating Robinia shared the common symbiotic genes. The nifH gene sequences showed that tested strains were clustered into M. albiziae, S. americanum and B. elkanii in the nifH phylogenetic tree. The nodC PCR-RFLP result showed 4 genotypes, but phylogenetic tree by sequencing indicated they were on a single branch independently; the similarity between tested strains ranged from 98.6% to 99.4%. The similarity between group of the tested strains and the nearest reference strain, M. amorphae, ranged from 98.9% to 100%.
The result of recA agreed with the study of 16S rDNA sequence analysis on the tested strains in branches of B. liaoningense and R. giardinii. Tested strains in groupes of M. ciceri,M. caraganae and M. amorphae in recA phylogenetic tree were different from the results of 16S rDNA phylogenetical tree, in which they belonged to M. loti,M. tianshanense and M. amorphae respectively.
The physiological and chemical results showed that the tested strains were divided into 3 Groups on the level of 66% similarity and 5 subgroups on 83% similarity level. Group I included subgroups of 1, 2 and 3, where the most tested strains belonged to M. amorphae; Group II consisted of subgroups of 4 and 5, which belonged to M. huakuii and M. tianshanes respectively; most of tested strains in GroupⅢare Sinorhizobium.
16S rDNA PCR-RFLP和全序列系统发育分析结果表明,中国刺槐根瘤菌共有9种酶切图谱类型组合,即9种基因型;16S rDNA全序列分析表明,9株代表菌株位于系统发育进化树的8个分支上,分别是M. loti/ciceri,M. tianshanense,M. huakuii,M. amorphae,R. giardinii,S. xinjiangense,S. morelense和B. liaoningense。其中16S rDNA RFLP基因型Ⅰ(M. amorphae)为刺槐优势根瘤菌基因型,占待测菌株的62.2%,广泛分布于3个采样点。
nodC和nifH的研究结果表明,刺槐根瘤菌具有相对保守的共生基因。序列分析结果显示,nifH基因位于系统进化树M. albiziae,S. americanum和B. elkanii的分支上。nodC基因虽然在RFLP分析结果中为四种基因类型,但是序列系统发育分析表明,4种nodC基因型在系统发育树上聚在同一分支(彼此间相似性达98.6%—99.4%),与M. amorphae ACCC19665的nodC基因相距最近,即同源性最高。而且与以前研究的中国刺槐根瘤菌,北美刺槐根瘤菌和欧洲刺槐根瘤菌的nodC基因位于同一系统发育树分支上,同源性为98.9%—100%之间。
recA基因序列分析结果中,位于B. liaoningense和R. giardinii分支的刺槐根瘤菌,其16S rDNA序列的聚类结果也分别位于B. liaoningense和R. giardinii,分析结果在种的水平上达到一致。但是,位于中慢生根瘤菌属的根瘤菌recA和16S rDNA聚类分析结果仅在属的水平上达到一致,在种的水平上稍有差异,其中recA系统发育树中位于M. ciceri,M. caraganae和M. amorphae分支上的代表菌株,在16S rDNA结果中依次位于M. loti,M. tianshanense和M. amorphae分支上。
数值分类结果表明,在66%的水平上待测菌株聚成3个表观群,在83%水平上聚成5个亚群。其中亚群1、2和3主要由属于M. amorphae的待测菌株组成,构成表观群Ⅰ;亚群4 (M. huakuii)和亚群5 (M. tianshanes)一起构成表观群Ⅱ;表观群Ⅲ的待测菌株主要属于Sinorhizobium。表型多样性研究表明,西北干旱半干旱地区的刺槐根瘤菌可以利用绝大多数的碳源和氮源,而且它们具有很高的耐盐性、耐碱性、抗性。
84 isolates from Robinia pseudoacacia in 3 sampling sites in Shaanxi and Gansu were characterized using restriction fragment length polymorphism and sequencing of 16S rDNA, recA, nodC and nifH genes, as well as numerical taxonomy of physiological and chemical characteristics.
16S rDNA patterns and sequences placed the tested strains in 9 RFLP groups, which located in 8 branches of the phylogenetic tree of 16S rDNA sequences. The 8 branches were M. loti/ciceri, M. tianshanense, M. huakuii, M. amorphae, R. giardinii, S. xinjiangense, S. morelense and B. liaoningense, among which the largest group was M. amorphae with 62.2% of tested stains from all the three sampling sites. The results revealed the rich genetic diversity of rhizobia nodulating Chinese Robinia.
The results of nodC and nifH revealed that the tested strains nodulating Robinia shared the common symbiotic genes. The nifH gene sequences showed that tested strains were clustered into M. albiziae, S. americanum and B. elkanii in the nifH phylogenetic tree. The nodC PCR-RFLP result showed 4 genotypes, but phylogenetic tree by sequencing indicated they were on a single branch independently; the similarity between tested strains ranged from 98.6% to 99.4%. The similarity between group of the tested strains and the nearest reference strain, M. amorphae, ranged from 98.9% to 100%.
The result of recA agreed with the study of 16S rDNA sequence analysis on the tested strains in branches of B. liaoningense and R. giardinii. Tested strains in groupes of M. ciceri,M. caraganae and M. amorphae in recA phylogenetic tree were different from the results of 16S rDNA phylogenetical tree, in which they belonged to M. loti,M. tianshanense and M. amorphae respectively.
The physiological and chemical results showed that the tested strains were divided into 3 Groups on the level of 66% similarity and 5 subgroups on 83% similarity level. Group I included subgroups of 1, 2 and 3, where the most tested strains belonged to M. amorphae; Group II consisted of subgroups of 4 and 5, which belonged to M. huakuii and M. tianshanes respectively; most of tested strains in GroupⅢare Sinorhizobium.
引文
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