新疆达坂盐湖沉积土壤嗜盐细菌的多样性分析及钠离子输出相关基因的克隆
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摘要
嗜盐菌由于具有广泛的环境适应多样性、系统发育多样性、独特的耐盐机制和潜在应用价值,近年来受到科学界的广泛关注。本研究以新疆达坂盐湖沉积土壤为实验材料,对新疆达坂盐湖的嗜盐细菌进行多样性分析,并从中克隆获得1个新的钠离子输出相关基因。主要研究结论如下：
以选择性富集培养获得的嗜盐细菌基因组DNA为模板,扩增16S rRNA基因,在此基础上构建嗜盐细菌的16S rRNA基因文库,随机挑选文库中的100个阳性克隆子进行群落结构多样性分析。16S rRNA基因序列分析结果表明：100个克隆分属于细菌域9个属的27个种,其中芽孢杆菌(Bacillus)为优势菌群(48%),喜盐芽孢杆菌(Halobacillus) (14%)、嗜盐单胞菌(Halomonas) (13%)为次优势菌群。分析的阳性克隆子中,10个克隆子与GenBank中已报道16S rRNA基因序列的相似性在88.80%到96.90%之间,可能代表新属或新种。研究结果表明,新疆达坂盐湖沉积土壤的富集培养物中存在种类较为丰富的嗜盐细菌。
以大肠杆菌Na+/H+逆向转运蛋白基因缺陷株Escherichia coli KNabc为宿主,通过功能互补方法,从经Sau3AI部分酶切回收的总基因组DNA中筛选到1个新的钠离子输出相关编码基因smrL。序列分析表明：smrL基因预测编码含104个氨基酸、具有3个推测跨膜区的蛋白,与Sphaerobacter thermophilus中的药物/H+逆向转运蛋白SugE之间存在59%的同源性。smrL是从富集培养物的总基因组DNA中克隆得到的第一个钠离子输出相关基因。
携带smrL基因的大肠杆菌KNabc重组子,分别在含有不同浓度NaCl和LiCl的培养基中进行盐耐受性实验,以及在pH7.0-8.0的培养基中进行碱耐受性实验。研究结果表明,smrL能增强KNabc菌株对NaCl和碱性pH的抗性,但是没有LiCl抗性。
Halophiles living in high-salt environment, due to a wide diversity of environmental adaptation, phylogenetic diversity, unique salt tolerance mechanisms and potential applica-tions in recent years widespread attention by the scientific community. In this study, Daban Salt Lake in Xinjiang as materials deposited soil in order to obtain directional halophilic microorganisms enriched total DNA as a template of the Daban salt lake in Xinjiang diversity of halophilic bacteria, and a new sodium ion output related genes were then cloned and identified. The main conclusions were as follows:
The total DNA isolated from enriched culture of sedimentary soil samples from Daban Salt Lake in Xinjiang was used as PCR template to amplify 16S rRNA gene and the library carrying 16S rRNA genes of halophilic bacteria was constructed,100 clones of the library were selected randomly and sequenced for molecular systematic analysis. Phylogenetic analysis indicated that 100 selected clones were clustered into twenty-seven species of nine genera, a lineage of the domain Bacteria. Among them, Bacillus was the most dominant genus as represents 48% of the clones, followed by Halobacillus (14%) and Halomonas (13%). Furthermore, ten clones may be potential novel species or genera. The results show that the halophilic bacteria population diversity is abundant in enriched culture of sedimentary soil from Daban Salt Lake in Xinjiang.
A novel gene encoding an Na+/H+ antiporter gene was cloned from total DNA of partially digested with Sau3AI of enriched culture of sedimentary soil samples by functional complementation and expressed in the antiporter-deficient Escherichia coli strain KNabc. The gene was sequenced and designated smrL. The deduced amino acid of SmrL consists of 104, which was 59% identical to the SugE drug/H+ antiporter of Sphaerobacter thermophilus. The hydropathy profile was characteristic of a membrane protein with 3 putative transmembrane domain. smrL was the first Na+-transporting gene cloned from genomic DNA of enriched culture.
The effects of NaCl and LiCl concentrations and pH7.0-8.0 conditions on the growth of E. coli KNabc cells carrying smrL gene were tested, respectively. The results show that smrL gene confer the ability upon E. coli KNabc cells to grow under NaCl and alkaline pH conditions, but no the ability of LiCl resistance.
以选择性富集培养获得的嗜盐细菌基因组DNA为模板,扩增16S rRNA基因,在此基础上构建嗜盐细菌的16S rRNA基因文库,随机挑选文库中的100个阳性克隆子进行群落结构多样性分析。16S rRNA基因序列分析结果表明：100个克隆分属于细菌域9个属的27个种,其中芽孢杆菌(Bacillus)为优势菌群(48%),喜盐芽孢杆菌(Halobacillus) (14%)、嗜盐单胞菌(Halomonas) (13%)为次优势菌群。分析的阳性克隆子中,10个克隆子与GenBank中已报道16S rRNA基因序列的相似性在88.80%到96.90%之间,可能代表新属或新种。研究结果表明,新疆达坂盐湖沉积土壤的富集培养物中存在种类较为丰富的嗜盐细菌。
以大肠杆菌Na+/H+逆向转运蛋白基因缺陷株Escherichia coli KNabc为宿主,通过功能互补方法,从经Sau3AI部分酶切回收的总基因组DNA中筛选到1个新的钠离子输出相关编码基因smrL。序列分析表明：smrL基因预测编码含104个氨基酸、具有3个推测跨膜区的蛋白,与Sphaerobacter thermophilus中的药物/H+逆向转运蛋白SugE之间存在59%的同源性。smrL是从富集培养物的总基因组DNA中克隆得到的第一个钠离子输出相关基因。
携带smrL基因的大肠杆菌KNabc重组子,分别在含有不同浓度NaCl和LiCl的培养基中进行盐耐受性实验,以及在pH7.0-8.0的培养基中进行碱耐受性实验。研究结果表明,smrL能增强KNabc菌株对NaCl和碱性pH的抗性,但是没有LiCl抗性。
Halophiles living in high-salt environment, due to a wide diversity of environmental adaptation, phylogenetic diversity, unique salt tolerance mechanisms and potential applica-tions in recent years widespread attention by the scientific community. In this study, Daban Salt Lake in Xinjiang as materials deposited soil in order to obtain directional halophilic microorganisms enriched total DNA as a template of the Daban salt lake in Xinjiang diversity of halophilic bacteria, and a new sodium ion output related genes were then cloned and identified. The main conclusions were as follows:
The total DNA isolated from enriched culture of sedimentary soil samples from Daban Salt Lake in Xinjiang was used as PCR template to amplify 16S rRNA gene and the library carrying 16S rRNA genes of halophilic bacteria was constructed,100 clones of the library were selected randomly and sequenced for molecular systematic analysis. Phylogenetic analysis indicated that 100 selected clones were clustered into twenty-seven species of nine genera, a lineage of the domain Bacteria. Among them, Bacillus was the most dominant genus as represents 48% of the clones, followed by Halobacillus (14%) and Halomonas (13%). Furthermore, ten clones may be potential novel species or genera. The results show that the halophilic bacteria population diversity is abundant in enriched culture of sedimentary soil from Daban Salt Lake in Xinjiang.
A novel gene encoding an Na+/H+ antiporter gene was cloned from total DNA of partially digested with Sau3AI of enriched culture of sedimentary soil samples by functional complementation and expressed in the antiporter-deficient Escherichia coli strain KNabc. The gene was sequenced and designated smrL. The deduced amino acid of SmrL consists of 104, which was 59% identical to the SugE drug/H+ antiporter of Sphaerobacter thermophilus. The hydropathy profile was characteristic of a membrane protein with 3 putative transmembrane domain. smrL was the first Na+-transporting gene cloned from genomic DNA of enriched culture.
The effects of NaCl and LiCl concentrations and pH7.0-8.0 conditions on the growth of E. coli KNabc cells carrying smrL gene were tested, respectively. The results show that smrL gene confer the ability upon E. coli KNabc cells to grow under NaCl and alkaline pH conditions, but no the ability of LiCl resistance.
引文
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