青藏高原东麓高山豆［Tibetia himalaica(Baker)H.P.Tsui］根瘤菌的遗传多样性

Genetic diversity of rhizobial bacteria nodulating Tibetia Himalaica in eastern Qinghai-Tibet plateau

摘要:

摘要:【目的】分离纯化青藏高原东麓(四川甘孜藏族自治州)高山豆根瘤菌，揭示其遗传多样性。【方法】采用纯培养法从该地区高山豆植物根瘤中分离纯化根瘤菌;通过BOXAIR、16S rDNA-RFLP 及PCA(PrincipalComponent Analysis)来分析高山豆根瘤菌的遗传多样性;通过16S rDNA序列同源性确定菌株的系统发育地位;通过测定菌株的耐盐性、初始pH 生长范围及生长温度范围来分析高山豆根瘤菌的抗逆性。【结果】从8个县12个采样点共分离纯化出22 个菌株。22个菌株在16S rDNA PCR-RFLP分析中聚成4 个遗传群，在BOX-PCR分析中则聚成9个遗传群。高山豆根瘤菌16S rDNASimpson遗传多样性指数D=0.872。22个菌株分别属于Rhizobium(11/22 株)、Mesorhizobium(4/22 株)、Rhizobium-Agrobacterium(7/22株)3个属。生理性状测定试验表明，所有菌株均能在1% NaCl的YMA培养基上生长，大多数(15/22 株)菌株能在4% NaCl的YMA 培养基上生长，其中，SCAU679、SCAU694、SCAU706等3个菌株能在7% NaCl的培养基上生长，SCAU689能在8% NaCl的培养基上生长;15/22的菌株能在pH4-11的培养基上生长;16 /22的菌株能在4－45℃条件下生长，所有菌株能在60℃(处理10 min后置28℃)条件下生长。【结论】青藏高原东麓(四川甘孜州)高山豆根瘤菌具有丰富的遗传多样性。大多数菌株对高盐、高温、低温及过酸过碱环境均具有很强的耐受能力。

Abstract:

Abstract:［Objective］Genetic diversity of the rhizobial bacteria nodulating Tibetia himalaica in the eastern part of Qinghai-Tibet Plateau (Ganzi State，Sichuan) were evaluated．［Methods］The pure culture method was used for isolating the rhiobial strains from the nodules． BOXAIR，16S rDNA sequences，16S rDNA PCR-RFLP and Principal Component Analysis (PCA) were used to determine the genetic diversity and phylogenetic relationships．The growth in different salt contents，pH and temperatures were tested．［Results］In total 22 strains were isolated from 12 samples in 8 counties．The strains tested were classified into 4 clusters in 16S rDNA PCR-RFLP，and 8 clusters were formed in BOXAIR． The 16S rDNA Simpson genetic diversity index was D=0.872. The strains tested were closely related to Rhizobium (11/22 strains)，Mesorhizobium (4 /22 strains) and Rhizobium-Agrobacterium (7 /22 strains)．All strains could regularly grow in YMA medium amended with 1 % NaCl，and 15/22 strains could grow in 4 % NaCl． SCAU679，SCAU694and SCAU706 could adapt to 7% NaCl，while SCAU689 could tolerant 8% NaCl．Among the strains tested，15 strains could grow in the pH range from 4.0 to 11; 16 isolates grew well from 4 to 45℃，and all of 22 strains could grow at 28 ℃ after treatment at 60℃ for 10 min．［Conclusion］This study revealed the high genetic diversity of the rhizobia isolated from Tibetia himalaica in the eastern part of Qinghai-Tibet Plateau． The strains tested were adapted to high salt，different range of temperatures and pH．