Transcriptome analysis of Lactobacillus paracasei SMN-LBK under ethanol stress
Transcriptome analysis of Lactobacillus paracasei SMN-LBK under ethanol stress
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DOI：
10.3168/jds.2019-16955
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发表时间：
2020-09-01
影响因子：
3.5
通讯作者：
Li, Kaixiong
中科院分区：
文献类型：
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作者：
Guo, Jinfeng;Li, Xu;Li, Kaixiong
副干酪乳杆菌SMN-LBK（序列号：CCTCC M 2017429）是乳酸菌中的乙醇耐药乳酸菌（LAB）。然而，副casei SMN-LBK抗乙醇胁迫的机制尚不清楚。因此，我们通过RNA测序对副casei L. SX10（10%乙醇胁迫下的L. paracasei SMN-LBK，简称SX10）和副casei L. SMN-LBK（简称S10）进行了转录组分析。我们使用实时定量PCR （RT-qPCR）来验证转录数据的准确性。转录组数据显示，与S10组相比，SX10组315个基因表达上调，332个基因表达下调。PFK、LDH、GPDH和GK基因上调，分别为1.10、0.30、0.56和1.512倍的对数(2)倍变化。基因本体富集分析显示，核糖体、核糖核蛋白复合物、非膜结合细胞器和细胞内非膜结合细胞器显著富集。使用京都基因和基因组百科数据库进行分析，发现与核糖体功能、丙酮酸代谢、氨基酸生物合成、脂肪酸生物合成、脂肪酸代谢、atp结合盒（ABC）转运体、糖酵解和甘油磷脂代谢相关的差异基因。RT-qPCR结果与转录组结果一致。乳球菌NZ9000是一种典型的宿主细菌。我们通过过表达PFK和GK来验证副干酪乳球菌SX10耐药基因在乳酸乳球菌NZ9000中的功能。利用十二烷基硫酸钠(SDS)-PAGE电泳技术，这些抗性基因在乳酸乳球菌NZ9000中成功表达。测定了乙醇胁迫下重组菌株的存活率和关键酶活性。乳酸乳球菌NZ9000-pNZ8148- pfk和乳酸乳球菌NZ9000-pNZ8148- gk在10%乙醇胁迫下的存活率分别是对照乳酸乳球菌NZ9000-pNZ8148的3.43和3.80倍。综上所述，PFK和GK对乳酸菌的乙醇耐受性有重要影响，可以提高乳酸乳球菌NZ9000的乙醇耐受性。因此，PFK和GK被确定为副干酪乳杆菌SX10高乙醇耐受性的关键基因。我们的研究结果为进一步研究对差异表达基因进行系统分析并确定其在LAB乙醇耐受机制中的潜在功能提供了新的见解。
Lactobacillus paracasei SMN-LBK (serial number: CCTCC M 2017429) is an ethanol-resistant lactic acid bacteria (LAB) in kumiss. However, the anti-ethanol stress mechanism of L. paracasei SMN-LBK remains unclear. Hence, we performed a transcriptome analysis between L. paracasei SX10 (L. paracasei SMN-LBK un-der 10% ethanol stress strain, abbreviated as SX10) and L. paracasei SMN-LBK (abbreviated as S10) by RNA sequencing. We performed real-time quantitative PCR (RT-qPCR) to verify the accuracy of the transcription data. The transcriptome data revealed that 315 genes exhibited upregulated expression, and 332 genes were downregulated in the SX10 compared with the S10 group. The PFK, LDH, GPDH, and GK genes were upregulated, with a log(2)-fold change of 1.10, 0.30, 0.56, and 1.512, respectively. A gene ontology enrichment analysis revealed significant enrichment of ribosomes, ribonucleoprotein complex, non-membrane-bounded organelles, and intracellular non-membrane-bound organelles. Analysis using the Kyoto Encyclopedia of Genes and Genomes database revealed differential genes associated with ribosome function, pyruvate metabolism, biosynthesis of amino acids, fatty acid bio-synthesis, fatty acid metabolism, ATP-binding cassette (ABC) transporter, glycolysis, and glycerophospholipid metabolism. The RT-qPCR results were consistent with the transcriptome results. Lactococcus lactis NZ9000 is a typical host bacterium. We performed PFK and GK overexpression to verify the function of the L. paracasei SX10 resistance gene in Lactococcus lactis NZ9000. Us-ing sodium dodecyl sulfate (SDS)-PAGE electrophore-sis, these resistance genes were successfully expressed in Lactococcus lactis NZ9000. The survival rate and key enzyme activity of the recombinant strains were deter - mined under ethanol stress. The survival rate of Lactococcus lactis NZ9000-pNZ8148-PFK and Lactococcus lactis NZ9000-pNZ8148-GK under 10% ethanol stress were 3.43-and 3.80-fold higher compared with the Lactococcus lactis NZ9000-pNZ8148 control, respectively. These results indicate that PFK and GK are important for the ethanol tolerance of LAB and can increase the ethanol tolerance of Lactococcus lactis NZ9000. Hence, PFK and GK were identified as key genes of L. paracasei SX10 with a high ethanol tolerance. Our results provide novel insight for further studies to perform a systematic analysis of the differentially expressed genes and to determine their potential functions in the ethanol tolerance mechanism of LAB.