低H+-ATPase活性植物乳杆菌突变菌筛选及基因表达的相对定量分析

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

摘要:

[目的]筛选H⁺-ATPase活性降低的植物乳杆菌突变菌，比较其与亲本菌基因表达水平的差异，进一步探索H⁺-ATPase的调控机制。[方法]利用硫酸新霉素诱变、筛选突变菌，并对亲本菌（ZUST）和突变菌（ZUST-1、ZUST-2）进行生长、产酸能力及H⁺-ATPase活性的测定。分别提取亲本菌和突变菌的基因组DNA，扩增H⁺-ATPase全部编码基因并测序。通过荧光定量PCR对H⁺-ATPase全部编码基因进行相对定量分析。[结果]突变菌的生长和产酸能力均低于亲本菌，突变菌ZUST-1和ZUST-2的H⁺-ATPase活性比亲本菌分别降低了10.1%和28.8%。突变菌ZUST-1和ZUST-2的atpA基因均有22个位点发生突变，而ZUST-2的atpC基因有6个位点发生突变。突变菌ZUST-1和ZUST-2的atpA在对数期基因表达水平分别比亲本菌ZUST下调了41.1%和35.7%，在稳定期分别下调了43.6%和14.2%；ZUST-1的atpC基因在对数期的表达水平比ZUST略高，在稳定期比ZUST上调了30%，而ZUST-2的atpC基因未表达。[结论]突变菌H⁺-ATPase活性减弱会导致其全部编码基因在稳定期表达水平上调（除ZUST-2的atpC不表达外），而且atpA和atpC基因突变导致的基因表达水平的差异是影响H⁺-ATPase活性的主要因素，此研究结果为进一步研究植物乳杆菌中H⁺-ATPase的调控机制奠定了基础。

Abstract:

[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.