外源AHLs培养下荧光假单胞菌qPCR内参基因的筛选
Screening of Reference Genes for qPCR of Pseudomonas fluorescens under Exogenous AHLs Culture
崔方超 1王芸婷 1王当丰 1檀茜倩 1李秋莹 1励建荣 1李婷婷2
作者信息
- 1. 渤海大学食品科学与工程学院 生鲜农产品贮藏加工及安全控制技术国家地方联合工程研究中心中国轻工业海水鱼加工重点实验室 辽宁锦州 121013
- 2. 大连民族大学生命科学学院 辽宁大连 116600
- 折叠
摘要
目的:荧光假单胞菌是冷藏食品的优势腐败菌,其致腐基因受到群体感应系统的调控.为了准确定量腐败基因的表达,研究群体感应调控食品腐败的机制,需筛选荧光假单胞菌的内参基因.方法:以荧光假单胞菌PF-08为研究对象,选择8个常见的内参基因(dsbA、carA、rpsL、gyrB、atpD、rpoD、gltA、16S rRNA),添加不同类型群体感应信号分子培养后,利用实时荧光定量PCR(qPCR)技术检测其基因表达量,采用geNorm、Normfinder、BestKeeper和RefFinder综合评估候选内参基因的表达稳定性,筛选最适内参基因.结果:在不同类型外源信号分子培养下,荧光假单胞菌中Ct值变化程度最小的是gltA,16S rRNA的Ct值过低;geNorm分析表达最稳定的内参基因是atpD和rpoD,且二者联用能准确定量目的基因表达水平;Normfinder和BestKeeper的分析结果都显示gltA是最稳定的内参基因;进一步用RefFinder综合评估,内参基因中表达较稳定的是rpoD和gltA.结论:rpoD和gltA在荧光假单胞菌经不同QS信号分子处理后均稳定表达,可用于后续荧光假单胞菌腐败基因的表达研究,也可为研究其它腐败菌的QS相关基因表达提供内参基因参考.
Abstract
Objective:Pseudomonas flluorescens is the dominant spoilage organism in frozen foods,and its spoilage-caus-ing genes are regulated by quorum sensing system.In order to accurately quantify the expression of spoilage genes and thus investigate the mechanism of quorum sensing regulation in food spoilage,it is required to screen reference genes of Pseudomonas fluorescens.Methods:Using Pseudomonas fluorescens PF-08 as the research object,eight common reference genes(dsbA,carA,rpsL,gyrB,atpD,rpoD,gltA,16S rRNA)were selected and their gene expression was measured by quantitative real-time PCR(qPCR)after incubation with different types of quorum sensing signal molecules.geNorm,Normfinder,BestKeeper and RefFinder were used to evaluate the expression stability of the candidate reference genes and to screen out the most appropriate reference genes.Results:Under different types of exogenous signal molecules incuba-tion,the least change in Ct value of Pseudomonas fluorescens was gltA and the Ct value of 16S rRNA was too low.The most stable reference genes by geNorm analysis were atpD and rpoD and the combination of the two could more accu-rately quantify the expression levels of target genes.gltA was the most stable reference gene analyzed by Normfinder and BestKeeper.Further comprehensive evaluation with RefFinder showed rpoD and gltA were the most stable reference genes.Conclusion:Both rpoD and gltA were stably expressed in Pseudomonas fluorescens after incubation with different QS sig-nal molecules.They could be used for the subsequent study of Pseudomonas fluorescens spoilage gene expression,and could also provide reference genes for studying the expression of QS-related genes in other spoilage bacteria.
关键词
荧光假单胞菌/群体感应/实时荧光定量PCR/内参基因Key words
Pseudomonas fluorescens/quorum sensing/quantitative real-time PCR/reference gene引用本文复制引用
基金项目
国家自然科学基金重点项目(U20A2067)
辽宁省自然科学基金博士科研启动基金计划(2022-BS-301)
出版年
2024
NETL
NSTL
万方数据