摘要
从河北省内保定、高邑和沙河3个试验地19个刺槐种源分离获得了57株刺槐根瘤菌,采用16S rRNA PCR-RFLP和16S rRNA基因全序列分析技术对其进行了遗传多样性和系统发育研究,并对其中30株刺槐根瘤菌进行了抗逆性指标试验,筛选出了一批抗逆性强的菌株。
57株供试菌株和6株参比菌株通过16S rRNA PCR-RFLP多态性分析共产生了39种16S rRNA基因遗传图谱,有些菌株的遗传图谱类型完全一致,而且大部分菌株都来自同一试验地,表明刺槐根瘤菌受环境的影响表现出地区分布的差异性。聚类分析结果表明,刺槐根瘤菌遗传多样性丰富,大部分供试菌株按地区聚开,没有以刺槐种源聚类的倾向,说明刺槐根瘤菌遗传多样性受试验地环境的影响较大,宿主种源对其影响较小。相关分析结果显示,不同试验地刺槐根瘤菌的遗传距离之间以及跟种源地理距离之间没有显著相关关系,进一步表明刺槐根瘤菌遗传多样性受刺槐种源的影响较小。
16S rRNA基因全序列分析结果显示,GY-2属于中华根瘤菌属(Sinorhizobium)系统发育分支,与S.morelense LMG20571的亲缘关系最近,序列相似性达到99.6%。确定了部分供试菌株的系统发育地位。进一步证明刺槐与根瘤菌的共生固氮体系因其所处的区域地理环境不同而具多样性。
对30株刺槐根瘤菌进行了抗逆性试验,结果表明,刺槐根瘤菌具有广泛的生长适应性。7株刺槐根瘤菌能在盐浓度50 g/L的培养基上生长,耐盐性很强;25株刺槐根瘤菌能在pH值12.0的碱性培养基上生长,耐碱性很强;20株刺槐根瘤菌能在37℃下生长,具有一定的耐热性;43.3%的刺槐根瘤菌对50μg/ml的氨苄青霉素有抗性。筛选出了12株耐酸碱能力较强的刺槐根瘤菌,他们均能耐受盐浓度为40 g/L的盐环境和pH值为12.0的碱性环境,这些抗逆性强的根瘤菌种质资源可为改善盐碱地环境中刺槐接种根瘤菌使用。
57 strains rhizobia isolated from Robinia pseudoacacia L in 19 different provenance grown in Baoding、Gaoyi and Shahe,they were analyzed using 16S rRNA PCR-RFLP polymorphism and the sequence analysis of 16S rRNA gene for genetic diversity and phylogenic study.30 Robinia pseudoacacia rhizobia were tested by stress resistance indexes,and selected some strains had strong resistance.
57 tested strains and 6 reference strains were generated 39 genetic map patterns using 16S rRNA PCR-RFLP polymorphism.Some strains had the same genetic map,and most strains isolated from the same region.The results indicated that Robinia pseudoacacia rhizobia affected by the environment to show differences in regional distribution.Cluster analysis showed that there existed a high degree of genetic diversity for the Robinia pseudoacacia rhizobia.Most strains separated by region,and had not the tendency to cluster for Robinia pseudoacacia provenance.Genetic diversity influenced by the geographical environment.Correlation analysis showed that there was no significant correlation between genetic distance and geographic distanc among provenances.
Sequencing analysis of 16S rRNA revealed the representative strain GY-2 shared 99.6% sequence Similarity with S.morelense LMG20571,and belonged to Sinorhizobium in the phylogenetic classification status.Further proved the symbiotic nitrogen fixation of Robinia pseudoacacia and rhizobia with the difference of regional geographical environment.
30 Robinia pseudoacacia rhizobia were tested by stress resistance indexes,the results showed that Robinia pseudoacacia rhizobia had wider rang of adaption for growth environment.7 strains can grow in YMA medium with 50 g/L NaCl,and had strong salt resistance.25 strains can grow in YMA medium with pH 12.0,and had strong alkali resistance.20 strains can grow in YMA medium with 37℃,and had a certain degree of heat resistance.43.3% strains had resistance to Ampicillin with 50μg/ml.12 strains have strong resistance were selected,they can grow in YMA medium with 40 g/L NaCl and pH 12.0.These strains can be inoculated with Robinia pseudoacacia which growed in saline land to promote growth.
引文
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