一株晋大53号大豆中慢生根瘤菌的分离鉴定及抗逆分析

Isolation,Identification and Stress Resistance Analysis of A Mesorhizobium Isolated from Soybean Variety Jinda 53

为筛选适于在山西省主栽品种晋大53号推广应用的根瘤菌资源,以晋大53号大豆根瘤为研究对象,采用组织块分离法、平板划线法、震荡培养法以及分子生物学方法,对根瘤菌进行分离、纯化和鉴定,并研究其产酸、碱能力、耐盐特性以及结瘤能力。结果表明:从晋大53号大豆根瘤中成功分离并纯化出1株根瘤菌,编号为53-4,具有刚果红染料抗性,产酸快,可耐5%浓度NaCl。测序获得该菌株的16S rDNA序列,经NCBI在线数据库比对后,发现53-4菌株同Rhizobia sp.(HQ589024.1)、Beijerinckia fluminensis(NR 116306.1)和Mesorhizobium sp.(JN622153.1)等具有高达99%的序列同源性。NJ系统发育树显示该菌株同Mesorhizobium sp.(JN622153.1)聚为同一支,表明53-4菌株属于中慢生根瘤菌(Mesorhizobium sp.)。对该菌株结瘤基因nodA进行PCR扩增,经电泳检测能够扩增出明显条带,说明该菌株基因组中具有根瘤菌关键结瘤基因nodA。回接53-4的晋大53号的株高、地上部鲜重、根鲜重、植株干重和根瘤鲜重均高于接种优良大豆共生根瘤菌株USDA110,其中地上部鲜重和根瘤鲜重显著高于接种USDA110处理,分别提高20.0%和85.4%。中慢生根瘤菌53-4的耐盐特性、产酸、碱能力以及共生效果均优于USDA110,表明其在晋大53号大豆种植过程中具有较好的应用潜力。

To screen the rhizobia resources suitable for the promotion and application on the main cultivar Jinda 53 in Shanxi Province,we took the root nodules of Jinda 53 soybean as the research object and seperate and purified the rhizobia,and then studied the capacity of its acid or alkali,salt resistance property and nodular capacity with the methods of tissue separation,flat line,shake culture and molecular biology.The results showed that 1 strain of rhizobia,coded as 53-4,was successfully isolated and purified from Jinda 53 soybean root nodules.It was resistant to congo red dye and 5%NaCl salt concentration with fast acid production.The 16S rDNA sequence of the strain was sequenced and Blast by NCBI online database,and it was found that the strain 53-4 had a high sequence homology with Rhizobia sp.(HQ589024.1),Beijerinckia fluminensis.(NR 116306.1)and Mesorhizobium sp.(JN622153.1),with a homology of 99%.After constructing NJ phylogenetic tree with MEGA5.0,it was found that the strain was clustered into the same strain with Mesorhizobium sp.(JN622153.1),indicating that the strain 53-4 belonged to Mesorhizobium sp..PCR amplification of nodulation gene nodA of the strain was performed,and bright bands were detected by electrophoresis,indicating that nodA was a key nodulation gene of rhizobia found in the genome of the strain.The results of re-inoculation experiments showed that the plant height,shoot fresh weight,root fresh weight,plant dry weight,nodule fresh weight of Jinda 53 soybean were higher than that of USDA110 inoculated.The shoot fresh weight and nodule fresh weight were significantly higher than that of USDA110 inoculation,increasing by 20.0%and 85.4%,respectively.Compared with USDA110,Mesorhizobium 53-4 also had better salt tolerance,acid or alkali production capacity and symbiotic effect,which indicated that it had good application potential in the planting of Jinda 53.