红苞凤梨实时荧光定量PCR分析中内参基因的筛选

为筛选表达稳定的内参基因,以红苞凤梨(Ananas comosus var.bracteatus)不同发育时期的全绿、全白苗为材料,对10个组成型表达基因EF1、UBQ、ACT、GADPH、Histone、TUA、TUB、18S、elf-5A、α-tubulin进行筛选,并分析PetF基因的表达模式。结果表明,10个候选内参基因在红苞凤梨全绿、全白苗不同生长阶段中的表达稳定性不同。红苞凤梨不同生长时期以Histone和α-tubulin为最适内参基因,而全绿苗和全白苗的对比分析以18S、EF1和α-tubulin为最理想的内参组合。PetF基因在红苞凤梨发育过程及绿、白苗对比分析中的表达水平变化趋势一致,因此,所选的内参基因是合适的。     

大灰象甲实时定量PCR内参基因的筛选

【目的】筛选出适合分析大灰象甲Sympiezomias velatus成虫不同组织中基因表达水平的内参基因。【方法】利用转录组测序技术获得大灰象甲管家基因序列作为候选内参基因,采用实时荧光定量PCR(qRT-PCR)技术分析候选基因在大灰象甲雌雄成虫触角、头、胸、腹和足中的表达量;并利用geNorm, NormFinder和BestKeeper软件及在线工具RefFinder评价候选基因的表达稳定性。以大灰象甲气味结合蛋白1(odorant bindng protein 1, OBP1)基因为目标基因验证候选基因在大灰象甲成虫不同组织中的表达稳定性。【结果】基于大灰象甲转录组数据首次鉴定得到β-肌动蛋白基因(ACT)、3-磷酸甘油醛脱氢酶基因(GAPDH)、18S核糖体RNA基因(18S rRNA)、60S核糖体蛋白L12基因(RPL12)、60S核糖体蛋白L32基因(RPL32)、40S核糖体蛋白S20基因(RPS20)、延伸因子2基因(EF2)、α-微管蛋白基因(TUA)和β-微管蛋白基因(TUB)共9个管家基因序列。geNorm分析结果显示,RPL12和RPS20是最稳定表达的内参基因,而BestKeeper和NormFinder分析结果显示最稳定表达的内参基因分别是TUA和TUB。综合各分析方法得出9个候选基因中TUB,TUA,RPS20和RPL12是最稳定表达的内参基因,而18S rRNA,ACT和GAPDH这3个广泛应用的内参基因则表现出最低的表达稳定性。最后以OBP1为目标基因对稳定性不同的4个候选基因进行稳定性验证,发现以TUB和RPL12为内参基因,OBP1在成虫不同组织之间的表达模式基本一致;而以RPL32为内参基因,表达模式与应用TUB作为内参基因时稍有不同,使用18S rRNA作为内参基因得到的OBP1表达模式则与应用TUB作为内参基因时的完全不一致。【结论】TUB,TUA,RPS20和RPL12可以作为分析大灰象甲成虫不同组织中基因表达水平的内参基因,为后续基因表达研究奠定了基础。     

孟氏隐唇瓢虫qRT-PCR分析中内参基因的筛选

选择合适的内参基因是qRT-PCR研究的关键。本文以孟氏隐唇瓢虫Cryptolaemus montrouzieri Mulsant为研究材料,利用qRT-PCR技术,对孟氏隐唇瓢虫4个候选内参基因Actin、RPS23、GAPDH和β-tubulin的mRNA的表达量进行了分析,并用Ge Norm、Norm Finder和Best Keeper软件分析它们在孟氏隐唇瓢虫不同发育阶段及成虫不同组织中的表达稳定性。结果表明,以成虫不同组织为材料时,综合三种软件分析结果显示4个候选基因表达稳定性平均等级值排名为RPS23(rank=1)β-tubulin(rank=2.3)GAPDH(rank=3)Actin(rank=3.7),以不同发育时期虫体为材料时,综合分析结果显示4个候选内参基因表达稳定性平均等级值排名为RPS23(rank=1.7)Actin(rank=2)GAPDH(rank=2.7)β-tubulin(rank=3.7)。综合分析在瓢虫不同发育阶段及成虫不同组织两种处理下,三种软件的评价效果,4个候选基因表达稳定性等级值的总平均排名为RPS23(rank=1.3)Actin(rank=2.8)=GAPDH(rank=2.8)β-tubulin(rank=3)。RPS23在瓢虫不同发育阶段及成虫不同组织中均显示出较高的表达稳定性及与其它基因之间极大的相关性,可以确定为孟氏隐唇虫不同发育阶段及成虫不同组织基因表达分析中一个稳定表达的基因,可作为单个内参基因或者其它内参基因的协同基因,本实验为开展孟氏隐唇瓢虫功能基因表达分析奠定了方法学基础。     

微孢子感染东亚飞蝗实时定量PCR内参基因的筛选

【目的】筛选出微孢子Paranosema locustae感染条件下东亚飞蝗Locusta migratoria实时定量PCR最适内参基因。【方法】本研究应用实时荧光定量PCR技术测定东亚飞蝗甘油醛-3-磷酸脱氢酶基因(Glyceralde-hyde-3-phosphate dehydrogenase,GAPDH)、18s核糖体RNA(18s RNA)、微管蛋白基因(Tubulin,TUB)、肌动蛋白基因(Actin,ACT)和延伸因子1基因(Elongation factor1,EF1)5个候选基因在3个微孢子浓度(1×10~4、1×10~6和1×10~8个孢子/头)感染12 d后的表达稳定性;应用ge Norm、Bestkeeper和Normfinder 3个软件综合分析5个内参基因的稳定性。【结果】ge Norm软件对5个内参基因在不同微孢子虫浓度感染下稳定性分析结果表明:18S基因稳定性最低,平均表达稳定性值M值最高为0.658 4,TUB基因稳定性最高,M值最低为0.278 4。Norm Finder软件分析在不同微孢子虫浓度感染下的5个内参基因的稳定值分别为0.152(EF1)、0.181(ACT)、0.212(TUB)、0.329(GAPDH)和0.395(18S),可知内参基因稳定性顺序为:EF1>ACT>TUB>GAPDH>18S。由Best Keeper软件分析表明5个候选内参基因的SD值均小于1.0,表达均较稳定。根据其变异系数值CV的大小可知,ACT基因最为稳定。【结论】综合3种软件分析结果,在不同微孢子虫浓度感染下,可选择的较为稳定的内参基因ACT、EF1和TUB基因作为相对定量的参照基因。     

黄精实时荧光定量PCR内参基因的筛选

本研究采用实时荧光定量PCR(quantitative Real-t Time PCR,q RT-PCR)筛选适用于黄精(Polygonatum sibiricum)的内参基因。利用黄精转录组数据库,筛选8个候选内参基因,18S核糖体r RNA基因(18S-r RNA,18S)、肌动蛋白基因(Actin,ACT)、细胞色素基因(cytochrome,CYP)、3-磷酸甘油醛脱氢酶基因(GAPDH)、α-微管蛋白基因(α-Tubulin,TUA)、β-微管蛋白基因(β-Tubulin,TUB)、多聚泛素酶基因(ubiquitin 5,UBQ 5)和(ubiquitin 10,UBQ 10)。应用q RT-PCR技术检测这8个候选内参基因在黄精不同组织器官中(根,根茎,茎,叶,花和种子)的表达情况。利用Ge Norm、Norm Finder和Best Keeper 3种统计学软件综合评价8个内参基因的表达稳定性。研究结果表明,TUB在黄精不同组织部位中表达稳定性最好,运用q RT-PCR技术研究黄精器官基因表达时,可选用TUB作为内参基因。确定黄精q RT-PCR分析的合适内参基因,为后续相关基因表达研究奠定基础。     

菊叶薯蓣不同发育时期块茎内参基因的筛选

薯蓣皂苷元为重要的药物原料。为初步分析菊叶薯蓣(Dioscorea composita)块茎中薯蓣皂苷元合成关键基因的表达,筛选稳定表达的内参基因至关重要。根据已建立的菊叶薯蓣转录组数据库和已报道的传统内参基因,筛选出ubiquitin-conjugating enzyme E2(UBC7)、MMS ZWEI homologue 3(MMZ3)、tubulin beta-7(TUB7)、actin 1(ACT1)、glyceraldehyde-3-phosphate dehydrogenase C subunit 1(GAPC1)、elongation factor 1-alpha(EF-1α2)、cyclophilin 5(CYP5)、histone H2A2(H2A2)共8个候选内参基因,利用实时荧光定量PCR(qPCR)技术,结合Ge Norm、Norm Finder和Best Keeper三个统计学软件进行分析。结果表明,在菊叶薯蓣块茎不同发育过程中,TUB7和H2A2表达最稳定,为最佳内参基因。本研究为探究薯蓣皂苷元合成途径分子机理提供参考。     

红掌佛焰苞基因qRT-PCR分析中内参基因的筛选

为筛选红掌(Anthurium andraeanumLinden)中稳定表达、可用于佛焰苞中实时荧光定量PCR分析(qRT-PCR)的内参基因,对5个组成型表达基因EF1-a、UBQ7、ACTB、GADPH、His3进行表达稳定性分析,并利用所筛选的内参基因研究红掌的二氢黄酮醇还原酶基因(dfr)的表达水平。结果表明,5种内参基因在不同品种间的表达稳定性不同。据内参基因标准化因子的配对差异分析(Vn/n+1),判定内参基因的最适数目为2,ACTB和UBQ7在红掌不同品种及佛焰苞发育不同阶段中表达均稳定,是理想的内参基因。dfr在不同品种的佛焰苞及佛焰苞发育过程中均有表达,且dfr表达水平的变化趋势一致,因此,所选内参基因是合适的。     

实时荧光定量PCR分析中毛果杨内参基因的筛选和验证

实时荧光定量PCR(qRT-PCR)技术具有高灵敏性、高保真性和高特异性, 被广泛应用于基因表达的分析。在数据处理过程中, 选用稳定表达的基因作为内参基因对准确分析实验结果非常关键。以毛果杨(Populus trichocarpa)的不同组织以及锌胁迫下的组培苗为材料, 使用荧光定量PCR方法分析了TUA8、TUB6、ubiquitin、GAPDH、actin、18S rRNA和EF1α 7个看家基因的表达情况。通过geNorm、NormFinder和BestKeeper 3个程序的综合分析, 发现actin、ubiquitin、EF1α和18S rRNA的稳定性较好, 可用作毛果杨基因表达研究的内参基因; 而TUB6在不同组织中稳定性最差; GAPDH在锌胁迫下的组织中稳定性最差, 因此不适宜作为内参基因。毛果杨NAC基因的表达分析, 进一步验证了上述结果。该研究对采用qRT-PCR方法分析毛果杨基因表达过程中内参基因的选择具有指导作用, 同时对揭示NAC基因的功能也有一定的意义。     

光裸星虫(Sipunculus nudus)不同发育时期卵细胞内参基因的筛选

内参基因的选择对功能基因表达量的归一化处理尤为重要。为了筛选出光裸星虫不同发育时期卵子的最适内参基因,利用qRT-PCR测定了甘油醛-3-磷酸脱氢酶(GAPDH)、肽基脯氨酰顺反异构酶A(PPIA)、60S核糖体蛋白L10(60S-L10)、铁蛋白(Ferritin)、β-肌动蛋白(β-actin)、泛素C(UBC)、真核生物翻译起始因子(eIF)、NADH脱氢酶(NDH)、28S核糖体RNA(28S)、TATA盒结合蛋白(TBP)、18S核糖体RNA(18S)和琥珀酸脱氢酶A亚基(SDHA)共12个候选内参基因的表达水平,并通过4个程序(geNorm,NormFinder,BestKeeper以及RefFinder)综合分析了各基因的表达稳定性。结果显示:(1)12个候选内参基因均能获得特异性扩增产物,但表达情况各异;(2)对候选内参基因进行综合打分,得到候选内参基因稳定性排名为18S>GAPDH>28S>β-actin>UBC>e IF>NDH|TBP>PPIA|Ferritin>60S-L10>SDHA。18S和GAPDH稳定性较好,可作为不同发育时期卵细胞基因表达研究的单内参基因,或最优组合内参基因。     

Identification and Validation of Reference Genes for Quantification of Target Gene Expression with Quantitative Real-time PCR for Tall Fescue under Four Abiotic Stresses

Tall fescue (Festuca arundinacea Schreb.) is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes (ACT, TUB, EF1a, GAPDH, SAND, CACS, F-box, PEPKR1 and TIP41) was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of SAND and TUB or TIP41 and TUB were most stably expressed in salt-treated roots or leaves. The combinations of GAPDH with TIP41 or TUB were stable in roots and leaves under drought stress. TIP41 and PEPKR1 exhibited stable expression in cold-treated roots, and the combination of F-box, TIP41 and TUB was also stable in cold-treated leaves. CACS and TUB were the two most stable reference genes in heat-stressed roots. TIP41 combined with TUB and ACT was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) assays of the target gene FaWRKY1 using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species.     

Selection and Validation of Reference Genes for Normalization of RT-qPCR Analysis in Developing or Abiotic-Stressed Tissues of Loquat (<i>Eriobotrya japonica</i>)

Loquat (Eriobotrya japonica Lindl.) is a subtropical evergreen fruit tree that produces fruits with abundant nutrients and medicinal components. Confirming suitable reference genes for a set of loquat samples before qRT-PCR experiments is essential for the accurate quantification of gene expression. In this study, eight candidate reference genes were selected from our previously published RNA-seq data, and primers for each candidate reference gene were designed and evaluated. The Cq values of the candidate reference genes were calculated by RT-qPCR in 31 different loquat samples, including 12 subgroups of developing or abiotic-stressed tissues. Different combinations of stable reference genes were screened according to a comprehensive rank, which was synthesized from the results of four algorithms, including the geNorm, NormFinder, BestKeeper and ΔCt methods. The screened reference genes were verified by normalizing EjLGA1 in each subgroup. The obtained suitable combinations of reference genes for accurate normalization were GAPDH, EF1α and ACT for floral development; GAPDH, UBCE and ACT for fruit setting; EF1α, GAPDH and eIF2B for fruit ripening; ACT, EF1α and UBCE for leaves under heat stress; eIF2B, UBCE and EF1α for leaves under freezing stress; EF1α, TUA and UBCE for leaves under salt stress; ACT, EF1α and eIF2B for immature pulp under freezing stress; ACT, UBCE and eIF2B for immature seeds under freezing stress; EF1α, eIF2B and UBCE for both immature pulp and seeds under freezing stress; UBCE, TUB and TUA for red-fleshed fruits under cold-storage stress; eIF2B, RPS3 and TUB for white-fleshed fruits under cold-storage stress; and eIF2B, UBCE and RPS3 for both red- and white-fleshed fruits under cold-storage stress. This study obtained different combinations of stable reference genes for accurate normalization in twelve subgroups of developing or abiotic-stressed tissues in loquat. To our knowledge, this is the first report to obtain stable reference genes for normalizing gene expression of abiotic-stressed tissues in E. japonica. The use of the three most stable reference genes could increase the reliability of future quantification experiments.     

Selection of Stable Reference Genes for Quantitative Real-Time PCR on Herbaceous Peony (<i>Paeonia lactiflora</i> Pall.) in Response to Drought Stress

Herbaceous peony (Paeonia lactiflora Pall.), as a high-end cut flower in the international market, has high ornamental and medicinal values. But in Northern China, drought is a major environmental factor influencing the growth and development of P. lactiflora. Quantitative real-time polymerase chain reaction (qRT-PCR) can evaluate gene expression levels under different stress conditions, and stable internal reference is the key for qRT-PCR. At present, there is no systematic screening of internal reference for correcting gene expressions of P. lactiflora in response to drought stress. In this study, 10 candidate genes [ubiquitin (UBQ2), UBQ1, elongation factor 1-α (EF-1α), Histidine (His), eukaryotic initiation factor (eIF), tubulin (TUB), actin (ACT), UBQ3, ACT2, RNA polymerase II (RNA Pol II)] were chosen, and 4 analysis methods were used to compare the stabilities for these 10 genes coping with drought stress. Due to the difference of operation methods, the results of different analysis were distinct, and the final comprehensive analysis indicated that EF-1α was a relatively stable internal reference gene for P. lactiflora under drought stress. Also, UBQ1 and UBQ2 were the best reference gene combination according to GeNorm analysis. This study will lay a foundation for screening the key genes of P. lactiflora in response to drought stress.     

Reference gene selection for normalizing gene expression using quantitative real-time PCR in Haemaphysalis longicornis

The Asian longhorned tick, Haemaphysalis longicornis, the dominant species of Ixodidae in Korea, has a wide distribution in East Asia, far-East Russia, and Western Pacific countries, and has recently been discovered in the Eastern states of the United States of America. H. longicornis transmits various pathogens, including Babesia ovate, Rickettsia japonica, and severe fever with thrombocytopenia syndrome virus (SFTSV). Considering its medical importance, in order to understand the physiology of H. longicornis, it is crucial to determine the expression of the genes of interest. Although quantitative real-time PCR (qRT-PCR) has been widely used to analyze gene expression, stably-expressed internal reference genes across samples of different conditions should be selected for the accurate normalization of target gene expression levels. Therefore, in this study, we investigated the expression levels of five candidate reference genes, namely ACT, RPP0, RPL23, TUB, and GAPDH, in H. longicornis under different conditions, including different collection months, developmental stages, and SFTSV infection status. Using four software programs, namely, NormFinder, BestKeeper, geNorm, and RefFinder, their expression stabilities were evaluated. Subsequently, a single gene between RPL23 and RPP0 was validated, which was found to be most stable reference gene after comparing the expression levels of HSP70 determined using different normalization methods.     

Selection of reliable reference genes for quantitative real-time polymerase chain reaction studies in maize grains

Key message

The stability of candidate reference genes was evaluated in maize landrace varieties and during multiple grain developmental stages to evaluate the expression of carotenoid-related genes by RT-qPCR for application to maize biofortification.

Abstract

Vitamin A deficiency affects millions of children worldwide; therefore, increasing the content of vitamin A precursors in maize grains is of interest. The study of the expression of genes involved in the carotenoid biosynthetic pathway in maize grains has provided useful information for metabolic engineering approaches. However, reliable results using real-time quantitative polymerase chain reaction (RT-qPCR) experiments are dependent on the use of the appropriate reference genes. In this study, we utilized geNorm and NormFinder softwares to identify the most stably expressed candidate reference genes in samples from seven stages of grain development and from eight landrace varieties. The results of the analysis performed using geNorm indicated that tubulin (TUB) and actin (ACT) were the most suitable reference genes among all experimental conditions, while glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) showed the least stability. The same result was obtained with the NormFinder software. The minimum number of genes required in each experimental condition to normalize the gene expression data was also determined by geNorm. The expression of phytoene synthase gene (PSY1), the first enzyme in the carotenoid biosynthetic pathway, was overestimated when the least stable candidate gene (GAPDH) was used as the internal control instead of the most stable gene pair (ACT + TUB), thus highlighting the importance of validating reference genes before conducting a RT-qPCR experiment to obtain accurate results. This study is the first survey of the stability of genes for use as reference genes to normalize RT-qPCR data from maize landraces during multiple stages of grain development.