| 摘要: |
| 【目的】对吉兰泰盐湖土壤嗜盐菌进行分离鉴定及生物学特征和系统发育分析，探索利用菌株合成四氢嘧啶的可能性。【方法】从内蒙古吉兰泰盐湖采集土壤样本，通过0,1%，2%，3%，4%，5%，6%，8%，10%，12%，14%，16%，18%，20%（质量分数）NaCl梯度耐盐试验，对分离放线菌进行分类统计。对分离得到的2株边界极端嗜盐菌JG5、JG9进行培养特征观察和生物学特性研究，基于16S rRNA基因序列构建系统发育树。利用四氢嘧啶基因簇中最为保守的ectB基因，合成简并引物，PCR扩增筛选含有四氢嘧啶基因簇的菌株。体积分数80%乙醇法抽提胞内四氢嘧啶，薄层层析（TLC）定性检测菌株胞内四氢嘧啶的存在情况，高效液相色谱（HPLC）法定量检测不同NaCl处理（质量分数为0,5%，10%，15%）JG5和JG9的胞内四氢嘧啶积累量。【结果】从吉兰泰盐湖土壤中分离得到嗜盐放线菌28株，其中主要为中度嗜盐放线菌，占总数的60.7%；弱嗜盐菌次之，占21.4%；非嗜盐菌3株，占10.7%；边界极端嗜盐菌最少，有2株，分别为JG5和JG9，占7.2%。PCR结果显示，28株中有17株含四氢嘧啶基因簇，占60.7%。生理生化特征研究结果显示，2株边界极端嗜盐菌JG5、JG9能利用的碳源种类丰富，也可利用多种氨基酸作为氮源，但JG9不能利用柠檬酸钠作为碳源，JG5和JG9均不能利用谷氨酸作为氮源；JG5和JG9都能使硝酸盐还原、明胶分解、牛奶凝固与胨化，都能产生H2S，能降解纤维素，两者都不能分解尿素。系统发育树表明，2株极端嗜盐菌皆属于链霉菌属（Streptomyces)的密旋链霉菌（Streptomyces pactum），且亲缘关系较近。薄层层析结果表明，17株中有11株菌株可产四氢嘧啶。高效液相色谱分析结果显示，NaCl质量分数为10%时，JG5胞内四氢嘧啶积累量达最大值，为80.35 mg/L；NaCl质量分数为5%时，JG9积累量达到最大值，为97.89 mg/L。【结论】分离菌株大多含有四氢嘧啶基因簇，部分能够生产四氢嘧啶。2株边界极端嗜盐菌JG5和JG9在不同盐环境下，胞内均能产生四氢嘧啶。 |
| 关键词: 嗜盐菌 生物学特征 系统发育 四氢嘧啶 吉兰泰盐湖 |
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| 基金项目:中央高校基本科研业务费专项(Z109021426，Z109021432） |
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| Isolation and ectoine-producing characteristics of halophiles from soil of Jilantai Saline Lake |
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HONG Yu,GAO Bo,YAN Dong,et al
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| Abstract: |
| 【Objective】Isolation,identification,biological characteristics and phylogenetic analysis of halophiles from Jilantai Saline Lake soil were conducted to explore the feasibility of producing ectoine using the isolated strain.【Method】Soil samples were collected from Jilantai Salt Lake,and classified by NaCl test with mass fractions of 0,1%,2%,3%,4%,5%,6%,8%,10%,12%,14%,16%,18%,and 20%.Two strains of borderline halophilic bacteria of JG5 and JG9 were isolated and cultured.The phylogenetic tree was constructed based on 16S rRNA gene sequence.Using the most conserved ectB gene in the ectoine gene cluster,degenerate primers were synthesized to screen the strains containing ectoine gene cluster.The intracellular ectoine was extracted by 80% (volume fraction) ethanol.The existence of intracellular ectoine was qualitatively detected by TLC.The accumulation of intracellular ectoine in JG5 and JG9 at different concentrations of NaCl (0,5%,10%,and 15%,mass fraction) was quantitatively determined by HPLC.【Result】A total of 28 halophilic actinomycetes were isolated,with moderately halophilic actinomycetes of 60.7%,weak halophilic bacteria of 21.4%,and non-halophilic bacteria of 10.7%.The borderline extreme halophilic bacteria were the least (7.2%) with two strains of JG5 and JG9.PCR showed that there were 17 strains containing ectoine gene cluster,accounting for 60.7% of the total.Physiological and biochemical studies showed that the two strains could use abundant carbon sources and use many amino acids as nitrogen sources.JG9 cannot use sodium citrate as carbon source,while neither JG5 nor JG9 can use glutamate as nitrogen source.Both JG5 and JG9 can reduce nitrate,decompose gelatin,and coagulate milk and peptone.Both of them can produce H2S and degrade cellulose,while neither can decompose urea.Phylogenetic analysis of JG5 and JG9 showed that the two strains belonged to Streptomyces pactum,and the relationship between JG5 and JG9 was close.TLC showed that 11 strains can produce intracellular ectoine.HPLC showed that the intracellular accumulation of ectoine of strain JG5 reached the maximum concentration of 80.35 mg/L in 10% NaCl,and intracellular accumulation of ectoine of strain JG9 reached the maximum concentration of 97.89 mg/L in 5% NaCl.【Conclusion】Most of the isolated strains contained ectoine gene cluster,and some could produce intracellular ectoine.Two strains of extreme halophilic bacteria,JG5 and JG9,could produce intracellular ectoine in different salt environments. |
| Key words: halophiles biochemical characteristics phylogenetic ectoine Jilantai Saline lake |
