Cloning, expression of proU of Lactobacillus plantarum ST-Ⅲ
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摘要: 分离自泡菜的植物乳杆菌ST-Ⅲ能够在盐浓度高达8%的环境下存活,其特有的质粒上存在一个与转运相容性溶质相关的proU基因簇,系统发育分析发现,proU基因簇与耐盐能力较高的戊糖乳杆菌IG1相应基因的相似性达100%,因此推测proU可能与植物乳杆菌ST-Ⅲ的耐盐能力相关。将proU及其包含的基因proX、proW、proV分别克隆到质粒pNZ8148上,并电转化到耐盐能力较低的乳酸乳球菌NZ9000中。重组菌株在添加3%NaCl和1mmol/L甜菜碱的化学成分确定培养基（CDM）中培养,以原始菌株乳酸乳球菌NZ9000（pNZ8148）为对照,测定耐盐能力。结果表明,各个基因均能在重组菌中表达,表达量与对照菌株相比至少提高了106倍,且重组菌株的耐盐能力均优于对照菌株,因此,proU与植物乳杆菌ST-Ⅲ耐盐能力直接相关。Abstract: Lactobacillus plantarum ST-Ⅲ, isolated from kimchi, could grow in 8% NaCl environment. The gene cluster proU, which located in the plasmid, involved in a transport system for glycine betaine.Through phylogenetic analysis, the similarity of the proU to the corresponding gene of the L. peutosus IG1 is 100%. proU had been speculated to relate to salt tolerance of L. plantarum ST- Ⅲ. The proU, and its three open reading frames proX, proW, and proV were cloned into pNZ8148, resulting in the construction of Lactococcus lactis expression systems.Introduction of expression vectors in osmotic sensitive strain Lactococcus lactis NZ9000 were achieved by electrotransformation.To determine the ability of salt tolerance, recombinant strains were cultured in CDM with 3%NaCl and 1mmol /L betaine.The results showed that, the gene proU, proX, proW, proV in recombinant strains were expressed, and the expression level were at least 106 times higher than control strain.The salt tolerance ability of recombinant strains were more exceed than control strain.The results proved that proU was directly related to salt tolerant ability of L.plantarum ST-Ⅲ.
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Keywords:
- Lactic acid bacteria /
- Lactobacillus plantarum ST-Ⅲ /
- salt tolerant genes /
- proU
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