低温和低pH胁迫下青贮乳酸菌OL77的内参基因筛选及CspP基因的表达模式分析

Screening of internal reference genes of Lactobacillus strain OL77 and determination of CspP expression patterns under low-temperature and low-pH stress

为了明确低温青贮中筛选出的优异耐低温乳酸菌菌株戊糖片球菌OL77在低温和低pH胁迫下的基因表达情况,探讨其耐低温机理,本研究通过同源克隆获得了OL77的CspP基因,运用GeNorm,NormFinder,BestKeeper和RefFinder程序,基于SYBR-Green的实时荧光定量PCR(RT-qPCR)筛选OL77在低温和低pH胁迫条件下表达稳定的内参基因,并利用其研究了CspP基因的低温表达模式。结果表明:OL77在受到外界低温和低pH胁迫时表达较稳定的内参基因为Ldh、tufA和6PGDH,而GAPDH和GyrA是表达较不稳定的内参基因。采用同源克隆获得OL77的CspP基因片段,以Ldh和6PGDH为内参基因,对25、15和5℃下培养0、1、3、6、12、24 h的OL77中CspP基因的相对表达量进行分析,发现低温条件下CspP基因瞬时表达量激增,在5℃下上调86倍,这可能是OL77耐低温的主要原因,OL77对低温的适应性部分来自其较强的冷休克蛋白表达能力。CspP基因可用作OL77低温适应性的指示基因。

The aim of this study was to explore aspects of gene expression under low temperature and low pH in the cold-tolerant bacterium Pediococcus pentosaceus OL77,which was selected during low-temperature ensiling.To investigate the cold tolerance mechanism of P.pentosaceus OL77,we obtained its CspP gene by homologous cloning.GeNorm,NormFinder,BestKeeper,and RefFinder procedures were used to select internal reference genes with stable expression in OL77 under low-temperature and low-pH stress as detected using SYBR-Green real-time fluorescence quantitative PCR(RT-qPCR).The selected internal reference genes were then used to analyze the transcriptional profile of CspP under low temperature.It was found that the relatively stably expressed internal reference genes of OL77 under low-temperature and low-pH stress were Ldh,tufa,and 6PGDH,while GAPDH and GyrA were not stably expressed.A gene fragment of CspP was obtained by homologous cloning,and its relative transcript levels in OL77 at 0,1,3,6,12 and 24 h of treatment at 25,15 and 5℃were analyzed using Ldh and 6PGDH as internal reference genes.The transient expression of CspP increased sharply under low temperature.Its transcript level was increased by 86 times at 5℃compared with that at 25℃,suggesting that it plays roles in the cold tolerance of OL77.The cold tolerance of OL77 may result from increased expression of a cold shock protein at low temperature.Our findings suggest that CspP could be used as a cold tolerance indicator gene for OL77.