基于金针菇(Flammulina filiformis)基因组和转录组的测序组装结果,选取13个内参基因(α-TUB、β-TUB、PGM、RPL19C、RPL6、CYC、18S rRNA、Vha68、ACT、GPD、PAS、TEF和VSN),通过设计跨内含子引物,分别对金针菇不同菌株(Fy331、Fw12、...基于金针菇(Flammulina filiformis)基因组和转录组的测序组装结果,选取13个内参基因(α-TUB、β-TUB、PGM、RPL19C、RPL6、CYC、18S rRNA、Vha68、ACT、GPD、PAS、TEF和VSN),通过设计跨内含子引物,分别对金针菇不同菌株(Fy331、Fw12、川金10、Fv296、金黄11和福建白金)菌丝和Fy331菌株不同发育阶段(菌丝、原基、幼嫩子实体和成熟子实体)样品进行实时荧光定量PCR(quantitative real time polymerase chain reaction,qRT-PCR)检测,并采用ΔCt算法和NormFinder、BestKeeper、GeNorm等软件对检测结果进行表达稳定性分析,以期筛选出在不同菌株菌丝和不同发育阶段中稳定表达的内参基因。结果表明:Vha68、TEF和β-TUB基因在不同菌株菌丝中表达稳定;Vha68、ACT和GPD基因在不同发育阶段样品中表达稳定。综合评价与qRT-PCR验证分析结果显示,Vha68和TEF、Vha68和ACT分别适合作为金针菇不同菌株菌丝、不同发育阶段qRT-PCR检测基因表达的最佳内参基因。展开更多
Quantitative real-time PCR(qRT-PCR)is widely used for investigating gene expression patterns and has many advantages,including its high sensitivity,fidelity,and specificity.Selecting a satisfactory internal reference ...Quantitative real-time PCR(qRT-PCR)is widely used for investigating gene expression patterns and has many advantages,including its high sensitivity,fidelity,and specificity.Selecting a satisfactory internal reference gene is crucial for obtaining precise gene expression results in qRT-PCR analyses.In this study,the transcriptomic data of 2 potato varieties were screened for housekeeping genes with stable expression patterns.A total of 77 putative genes were selected,which were highly and stably expressed.Then,qRT-PCR analyses were performed to examine the expression levels of these 77 candidate reference genes in various potato tissues,including leaves,flowers,stolons,and tubers.Gene expression was represented by analyzing the Ct values at given threshold.Through geNorm and NormFinder program analyses,10 candidate genes with the most stable expression patterns were obtained,including RPL19,RPS15,RPS9,EF1α,TrxP1,RPS8,NTF,CAM,AACM,and RPS28.Moreover,through the comprehensive analyses of 4 statistical algorithms(i.e.,geNorm,NormFinder,BestKeeper,and RefFinder),results indicated that the most appropriate internal reference genes were RPL19 and EF1α.The obtained stable reference genes will contribute to future qRT-PCR analyses on potato tissue-related gene expression.展开更多
The Emerald ash borer （EAB, Agrilus planipennis Fairmaire） an exotic invasive insect pest has killed millions of ash trees （Fraxinus spp.） across North America and threatens billions more. We validated six A. plan...The Emerald ash borer （EAB, Agrilus planipennis Fairmaire） an exotic invasive insect pest has killed millions of ash trees （Fraxinus spp.） across North America and threatens billions more. We validated six A. planipennis reference genes （actin, ACT; beta tubulin, E-TUB; glyceraldehyde-3-phosphate dehydrogenase, GAPDH ; ribosomal protein, RPL7; translation elongation factor 1~, TEF-la; and ubiquitin, UBQ） using geNorm, Normfinder and BestKeeper for accurate determination of target messenger RNA levels in gene expression studies. The stability of the six reference genes was evaluated in different larval tissues, developmental stages and two treatments ofA. planipennis using quantitative real-time polymerase chain reaction. Although there was no consistent ranking observed among the reference genes across the samples, the overall analysis revealed TEF-la as the most stable reference gene. GAPDH and ACT showed least stability for all the samples studied. We conclude that TEF-I~ is the most appropriate reference gene for gene expression studies in A. planipennis. Results obtained can be applicable for transcript profiling in other invasive insect pests. Further, these validated reference genes could also serve as the basis for selection of candidate reference genes in any given insect system post-validation.展开更多
文摘基于金针菇(Flammulina filiformis)基因组和转录组的测序组装结果,选取13个内参基因(α-TUB、β-TUB、PGM、RPL19C、RPL6、CYC、18S rRNA、Vha68、ACT、GPD、PAS、TEF和VSN),通过设计跨内含子引物,分别对金针菇不同菌株(Fy331、Fw12、川金10、Fv296、金黄11和福建白金)菌丝和Fy331菌株不同发育阶段(菌丝、原基、幼嫩子实体和成熟子实体)样品进行实时荧光定量PCR(quantitative real time polymerase chain reaction,qRT-PCR)检测,并采用ΔCt算法和NormFinder、BestKeeper、GeNorm等软件对检测结果进行表达稳定性分析,以期筛选出在不同菌株菌丝和不同发育阶段中稳定表达的内参基因。结果表明:Vha68、TEF和β-TUB基因在不同菌株菌丝中表达稳定;Vha68、ACT和GPD基因在不同发育阶段样品中表达稳定。综合评价与qRT-PCR验证分析结果显示,Vha68和TEF、Vha68和ACT分别适合作为金针菇不同菌株菌丝、不同发育阶段qRT-PCR检测基因表达的最佳内参基因。
文摘Quantitative real-time PCR(qRT-PCR)is widely used for investigating gene expression patterns and has many advantages,including its high sensitivity,fidelity,and specificity.Selecting a satisfactory internal reference gene is crucial for obtaining precise gene expression results in qRT-PCR analyses.In this study,the transcriptomic data of 2 potato varieties were screened for housekeeping genes with stable expression patterns.A total of 77 putative genes were selected,which were highly and stably expressed.Then,qRT-PCR analyses were performed to examine the expression levels of these 77 candidate reference genes in various potato tissues,including leaves,flowers,stolons,and tubers.Gene expression was represented by analyzing the Ct values at given threshold.Through geNorm and NormFinder program analyses,10 candidate genes with the most stable expression patterns were obtained,including RPL19,RPS15,RPS9,EF1α,TrxP1,RPS8,NTF,CAM,AACM,and RPS28.Moreover,through the comprehensive analyses of 4 statistical algorithms(i.e.,geNorm,NormFinder,BestKeeper,and RefFinder),results indicated that the most appropriate internal reference genes were RPL19 and EF1α.The obtained stable reference genes will contribute to future qRT-PCR analyses on potato tissue-related gene expression.
文摘The Emerald ash borer （EAB, Agrilus planipennis Fairmaire） an exotic invasive insect pest has killed millions of ash trees （Fraxinus spp.） across North America and threatens billions more. We validated six A. planipennis reference genes （actin, ACT; beta tubulin, E-TUB; glyceraldehyde-3-phosphate dehydrogenase, GAPDH ; ribosomal protein, RPL7; translation elongation factor 1~, TEF-la; and ubiquitin, UBQ） using geNorm, Normfinder and BestKeeper for accurate determination of target messenger RNA levels in gene expression studies. The stability of the six reference genes was evaluated in different larval tissues, developmental stages and two treatments ofA. planipennis using quantitative real-time polymerase chain reaction. Although there was no consistent ranking observed among the reference genes across the samples, the overall analysis revealed TEF-la as the most stable reference gene. GAPDH and ACT showed least stability for all the samples studied. We conclude that TEF-I~ is the most appropriate reference gene for gene expression studies in A. planipennis. Results obtained can be applicable for transcript profiling in other invasive insect pests. Further, these validated reference genes could also serve as the basis for selection of candidate reference genes in any given insect system post-validation.
