川西高寒山区野生药用黄芪根瘤菌的遗传多样性

Genetic Diversity of Rhizobial Bacteria Nodulating Medicinal Plants of Astragalus Grown Wildly in West of Sichuan,China

【目的】揭示川西高寒山区(四川甘孜藏族自治州和阿坝藏族羌族自治州)野生药用黄芪根瘤菌的遗传多样性,为优质菌株筛选提供依据。【方法】从该地区10种野生药用黄芪植物根瘤中分离纯化根瘤菌,用BOXAIR、16S r DNA-RFLP及PCA(Principal Component Analysis)分析药用黄芪根瘤菌的遗传多样性;通过16S rRNA基因序列同源性确定菌株的系统发育地位;测定菌株的耐盐性、初始pH生长范围及生长温度范围分析药用黄芪根瘤菌的抗逆性。【结果】从7个县11个采样点采集到的10种药用黄芪植物的根瘤中共分离纯化出35株根瘤菌。BOXAIR-PCR分析表明,在84. 6%的相似性水平处,供试菌株聚成了6个遗传群; 16S r DNA PCR-RFLP分析结果显示,除SCAU549、SCAU568和SCAU596单独成群外,其余菌株聚成了4个遗传群(相似系数83%);BOXAIR-PCR药用黄芪根瘤菌16S r DNA Simpson遗传多样性指数D=0. 916。35个菌株分别属于Rhizobium (20/35株)、Ochrobactrum(3/35株)、Rhizobium-Agrobacterium(12/35株) 3个系统发育分支。生理性状测定试验表明,27/35的菌株能在4～45℃的温度范围下生长,8/35的菌株能在10～37℃的范围生长,所有菌株经60℃处理10 min,28℃仍能正常生长; 28/35的菌株能在pH 4～10的培养基上生长,5/35的菌株能在pH 4～8的培养基上生长,而SCAU533、SCAU536仅能在pH6～8的范围内正常生长。除12/35的菌株不能在1%Na Cl的YMA培养基上生长外,18/35菌株能在2%～7%Na Cl的YMA培养基上生长,而SCAU542、SCAU543、SCAU544、SCAU545和SCAU546等5个菌株能在8%Na Cl的培养基上生长。【结论】川西高寒山区野生药用黄芪根瘤菌具有丰富的遗传多样性。多数菌株对高盐、高温、低温及酸碱环境有较强的耐受能力。

[ Objective ] The genetic diversity of the rhizobial bacteria nodulating medicinal plants of Astragalus grown wildly in the west of Si- chuan was evaluated to provide a basis for strain screening. [ Method] The pure culture method was used for isolating the rhizobial strains from the nodules of 10 spieces plants of Astragalus grown wildly in the west of Sichuan, and BOXAIR-PCR, 16S rDNA sequence analysis, 16S rDNA PCR- RFLP and Principal Component Analysis （PCA） were used for the determination of the genetic diversity and phylogenetic relationships. The growth performance under different salt concentrations in the range of pH and temperatures were tested. [ Result] A total of 35 rhizobial strains were isolated. Six clusters were fondled based on the BOXAIR-PCR analysis ; these strains were classified into 4 clus- ters in 16S rDNA PCR-RFLP, except SCAU549, SCAU568, and SCAU596 formed single cluster, respectively. The 16S rDNA Simpson ge- netic diversity index was D -0. 916. 16S rDNA sequence analysis revealed that these strains tested were closely related to Rhizobium （20/ 35strains ）, Ochrobactmm （3/35 strains） and Rhizobium-Agrobacterium （12/35 strains）, respectively. 27/35 isolates performed good growth in the temperature range of 4 ℃ to 45 ℃. All of 35 strains could grow at 28 ℃ after treatment in 60 ℃ for 10 minutes. Among the strains tested, 28 strains could grow in the pH range of 4.0 to 10, and 5 strains could grow in pH 4 to pH 8. Strains SCAU533 and SCAU536 could only grow in pH6 to pH 8. 18 strains could grow in YMA containing 4 % to 7 % NaC1, while SCAU542, SCAU543, SCAU544, SCAU545, and SCAU546 could tolerant 8 % NaC1. [Conclusion]This study revealed the high genetic diver- sity of the rhizobia isolated from medicinal plants of Astragalus in the west of Sichuan. Most of the strains tested were adapted to high salt, different range of temperatures and pH.