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酸冷胁迫下保加利亚乳杆菌定量PCR内参基因的筛选
孙永胜;许沙;王宇航;魏新燕;李晨;康红彦;田洪涛
作者单位
孙永胜;许沙;王宇航;魏新燕;李晨;康红彦;田洪涛 河北农业大学食品科技学院国家北方山区农业工程技术研究中心河北新希望天香乳业有限公司河北省益生功能性乳制品技术创新中心 
摘要:
实时荧光定量PCR(qRT-PCR)技术具有灵敏度高、特异性强、重复性好、定量准确等优点,被广泛应用于目的基因表达的定量分析,而筛选表达水平稳定的内参基因是提高qRT-PCR结果准确性的重要前提。保加利亚乳杆菌既是酸奶发酵剂的常用菌种,又是引起酸奶后酸化的主要菌种。为了筛选保加利亚乳杆菌后酸化相关功能基因表达分析的qRT-PCR内参基因,以保加利亚乳杆菌的模式菌株ATCC 11842为试验菌株,根据前期ATCC11842菌株在后酸化不同条件下转录组学测序结果,选择5个候选内参基因(16SrRNA、rpoB、ldh、rodA、recA),通过qRT-PCR技术研究候选内参基因在酸胁迫和酸冷胁迫下的表达量水平,即Ct值变化。采用3款软件geNorm、NormFinder和Bestkeeper分析比较候选内参基因在酸胁迫和酸冷胁迫下qRT-PCR的表达稳定性,并筛选获得qRT-PCR的最适内参基因。运用筛选到的最适内参基因,分析ATCC 11842菌株的3个目的基因(poxI、Ldb1301、dnaJ)在酸胁迫和酸冷胁迫下qRT-PCR的相对表达量,验证筛选的内参基因的可靠性。结果表明:同一候选内参基因Ct值在酸胁迫和酸冷胁迫下,rpoB和recA表达量变化最小。比较3个软件分析结果,一致得出:rpoB和recA是在酸胁迫和酸冷胁迫下表达最稳定的2个基因,即筛选出的qRT-PCR最适内参基因为rpoB和recA;3个目的基因(poxI、Ldb1301、dnaJ)在酸胁迫和酸冷胁迫下qRT-PCR的相对表达量的分析结果与前期转录组学测序结果一致,进一步证实本研究筛选的2个内参基因rpoB和recA的可靠性。本文为利用qRT-PCR技术研究保加利亚乳杆菌后酸化功能基因表达以及揭示后酸化机制及定向选育弱后酸化菌株提供了依据;也为采用qRT-PCR技术研究其它益生乳酸菌引起酸奶后酸化或不同条件下功能基因表达提供借鉴。
关键词:  保加利亚乳杆菌  后酸化  酸胁迫与酸冷胁迫  实时荧光定量PCR  内参基因
DOI:
分类号:
基金项目:国家自然科学基金项目(31301520);河北省农业科技成果转化项目(16822807D);河北省重点研发计划项目(19227134D)
Screening of Internal Reference Genes for Quantitative PCR of Lactobacillus bulgaricus under Acid and Cold Stress
Abstract:
Real-time quantitative PCR(qRT-PCR) technology has the advantages of high sensitivity, strong specificity, good repeatability, and accurate quantification. It is widely used in quantitative analysis of target gene expression, and screening of internal reference genes with stable expression levels is improved an important prerequisite for the accuracy of qRT-PCR results, Lactobacillus bulgaricus is not only a common strain of yogurt starter, but also the main strain that causes acidification after yogurt. In order to select the qRT-PCR internal reference gene for expression analysis of functional genes related to acidification after Lactobacillus bulgaricus, the model strain ATCC 11842 of Lactobacillus bulgaricus was used as the test strain. Five candidate internal reference genes (16SrRNA, rpoB, ldh, rodA, recA), using qRT-PCR technology, studied the expression level of the candidate internal reference gene under acid stress and acid cold stress, that is, Ct value changes; using three software geNorm, NormFinder and Bestkeeper, analyzed and compared the expression stability of qRT-PCR of candidate internal reference genes under acid stress and acid cold stress, and screened the most suitable internal reference genes for qRT-PCR; using the optimal internal reference genes obtained by screening, analysis the relative expression levels of qRT-PCR of three target genes(poxI, Ldb1301, dnaJ) of ATCC 11842 strain under acid stress and acid cold stress were verified, and the reliability of the internal reference genes obtained by screening was verified. The results show that the Ct value of the same candidate internal reference gene under acid stress and acid cold stress has the smallest change in the expression of rpoB and recA; the results of the analysis and comparison of the three softwares are consistent: rpoB and recA are the most expressed under acid stress and acid cold stress two stable genes, the most suitable qRT-PCR selected internal reference genes are rpoB and recA; the analysis of the relative expression of qRT-PCR under three acid genes(poxI, Ldb1301, dnaJ) under acid stress and acid cold stress the results were consistent with the results of the previous transcriptomics sequencing analysis, which further confirmed the reliability of the two internal reference genes rpoB and recA obtained in this study. This article provides a reliable basis for in-depth research on the expression of post-acidification function genes of Lactobacillus bulgaricus using qRT-PCR technology, and further reveals the post-acidification mechanism and selective breeding of weak post-acidification strains; functional gene expression under acidification or under different conditions provides guidance.
Key words:  Lactobacillus bulgaricus  post-acidification  acid stress and acid cold stress  real-time quantitative PCR  reference gene

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