[植物乳杆菌H+- atp酶缺陷突变体的分离、鉴定及实时RT-PCR相对定量基因表达]。

微生物学报 Pub Date : 2017-02-04

Xiang Zhang, Hui Fang, Dongfang Xie, Yuecheng Lin, Yingyan Tao, Hongpeng Wang, Jinyan Gong, Qing Ge, Guorong Pan, Jun Huang, Yuru You

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引用次数: 0

摘要

目的:分离具有较低H+- atp酶活性的植物乳杆菌酸敏感突变体，研究H+- atp酶在植物乳杆菌中的调控机制。方法:用新霉素分离植物乳杆菌酸敏感突变体，测定野生型和突变体的H+- atp酶活性和乳酸产量。从野生型ZUST和两个突变体(ZUST-1、ZUST-2)中提取基因组DNA，作为PCR模板。对菌株的H+- atp酶基因进行扩增，并对扩增产物进行测序。分析了H+- atp酶的序列相似性。采用Real-time RT-PCR评价H+-ATPase基因表达的相对定量。结果:突变体在MRS培养液中生长，细胞生物量和产酸量均低于野生型。突变体ZUST-1和ZUST-2的H+- atp酶活性分别比野生型低10.1%和28.8%。结果表明，突变体ZUST-1和ZUST-2的atpA基因存在22个突变，ZUST-2的atpC基因存在6个突变。突变体ZUST-1和ZUST-2的atpA基因表达量在指数期比野生型低41.1%和35.7%，在固定期比野生型低43.6%和14.2%。ZUST-1在指数期的atpC基因表达量与野生型相近，在固定期的atpC基因表达量比野生型高30%，ZUST-2的atpC基因不表达。结论:除ZUST-2 atpC基因未表达外，H+-ATPase活性较低的突变体在固定期均上调了H+-ATPase基因的表达。H+-ATPase活性与atpA和atpC基因表达差异有重要联系。这项研究的结果将为进一步了解H+- atp酶缺陷突变的机制铺平道路。

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[Isolation, characterization and real-time RT-PCR for relative quantification of gene expression in H+-ATPase-defective mutants from Lactobacillus plantarum].

Objective: The aim of this study was to isolate Lactobacillus plantarum acid-sensitive mutants with lower H+-ATPase activity, and to study the mechanism of H+-ATPase regulation in Lactobacillus plantarum.

Methods: We used neomycin to isolate acid-sensitive mutants of L. plantarum, and measured H+-ATPase activity and lactic acid production of wild-type and mutants. Genomic DNA was extracted from the wild-type ZUST and two mutants (ZUST-1, ZUST-2), and used as PCR templates. H+-ATPase genes of the strain were amplified, and the PCR products were sequenced. Sequence similarity of H+-ATPase was analyzed. Real-time RT-PCR was used to evaluate the relative quantification of the H+-ATPase genes expression.

Results: The growth of the mutants was characterized in MRS broth, which revealed that their cell biomass and acid production were lower than that of the wild-type. H+-ATPase activity of the mutants ZUST-1 and ZUST-2 was 10.1% and 28.8% lower than that of the wild-type. Results showed that atpA gene of the mutants ZUST-1 and ZUST-2 existed 22 mutations by alignment of the wild-type sequence, and atpC gene of ZUST-2 existed 6 mutations. Mutants ZUST-1 and ZUST-2 atpA gene expression were 41.1% and 35.7% lower than that of the wild-type in exponential phase, 43.6% and 14.2% in stationary phase, respectively. The atpC gene expression of ZUST-1 was similar to that of the wild-type in exponential phase, and was 30% higher than that of the wild-type in stationary phase, and ZUST-2 atpC gene was not expressed.

Conclusion: The mutants with lower H+-ATPase activity were found to up-regulate the expression of H+-ATPase genes in stationary phase, except ZUST-2 atpC gene was not expressed. H+-ATPase activity has an important connection with the difference in gene expression of atpA and atpC. The results of this study will pave the way for gaining further insights into the mechanism of the H+-ATPase-defective mutants.

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微生物学报

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期刊介绍： Acta Microbiologica Sinica(AMS) is a peer-reviewed monthly (one volume per year)international journal,founded in 1953.It covers a wide range of topics in the areas of general and applied microbiology.The journal publishes original papers,reviews in microbiological science,and short communications describing unusual observations. Acta Microbiologica Sinica has been indexed in Index Copernicus (IC),Chemical Abstract (CA),Excerpt Medica Database (EMBASE),AJ of Viniti (Russia),Biological Abstracts (BA),Chinese Science Citation Database (CSCD),China National Knowledge Infrastructure(CNKI),Institute of Scientific and Technical Information of China(ISTIC),Chinese Journal Citation Report(CJCR),Chinese Biological Abstracts,Chinese Pharmaceutical Abstracts,Chinese Medical Abstracts and Chinese Science Abstracts.