Selection of Reference Genes for qRT-PCR Analysis in Medicinal Plant Glycyrrhiza under Abiotic Stresses and Hormonal Treatments

Best known as licorice, Glycyrrhiza Linn., a genus of herbaceous perennial legume, has been used as a traditional herbal medicine in Asia and a flavoring agent for tobacco and food industry in Europe and America. Abiotic stresses and hormonal treatments can significantly impact the development and metabolism of secondary metabolites in Glycyrrhiza. To better understand the biosynthesis of the trace-amount bioactive compounds, we first screened for the suitable reference genes for quantitative real-time reverse transcription PCR (qRT-PCR) analysis in Glycyrrhiza. The expression profiles of 14 candidate reference genes, including Actin1 (ACT), Clathrin complex AP1 (CAC), Cyclophilin (CYP), Heat-shock protein 40 (DNAJ), Dehydration responsive element binding gene (DREB), Translation elongation factor1 (EF1), Ras related protein (RAN), Translation initiation factor (TIF1), β-Tubulin (TUB), Ubiquitin-conjugating enzyme E2 (UBC2), ATP binding-box transpoter 2 (ABCC2), COP9 signal compex subunit 3 (COPS3), Citrate synthase (CS), and R3H domain protein 2 (R3HDM2) from two congeneric species, Glycyrrhiza uralensis F. and Glycyrrhiza inflata B., were examined under abiotic stresses (osmotic and salinity) and hormonal treatments (Abscisic acid (ABA) and methyl jasmonic acid (MeJA)) using a panel of software, including geNorm, NormFinder, BestKeeper, and Delta CT. The overall stability, however, was provided by RefFinder, a comprehensive ranking system integrating inputs from all four algorithms. In G. uralensis, the most stable reference genes under osmotic stress, salt stress, ABA treatment, and MeJA treatment were TIF1, DNAJ, CS, and ABCC2 for leaves and DNAJ, DREB, CAC, and CAC for roots, respectively. In comparison, the top ranked genes were TUB, CAC, UBC2, and RAN for leaves and TIF1, ABCC2, CAC, and UBC2 for roots, respectively, under stress and hormonal treatments in G. inflata. ACT and TIF1, on the other hand, were the least stable genes under the most experimental conditions in the two congeneric species. Finally, our survey of the reference genes in legume shows that EF, ACT, UBC2, and TUB were the top choices for the abiotic stresses while EF, UBC2, CAC, and ABCC2 were recommended for the hormonal treatments in Leguminosae. Our combined results provide reliable normalizers for accurate gene quantifications in Glycyrrhiza species, which will allow us to exploit its medicinal potential in general and antiviral activities in particular.

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Selective Interaction of Sugarcane eIF4E with VPgs from Sugarcane Mosaic Pathogens

Viruses ◽

10.3390/v13030518 ◽

2021 ◽

Vol 13 (3) ◽

pp. 518

Author(s):

Zongtao Yang ◽

Meng Dong ◽

Guangyuan Cheng ◽

Shuxian Liu ◽

Hai Zhang ◽

...

Keyword(s):

Mosaic Virus ◽

Expression Profiles ◽

Mosaic Disease ◽

Translation Initiation Factor ◽

Bimolecular Fluorescence Complementation ◽

Initiation Factor ◽

Pcr Analysis ◽

Protein Coding ◽

Sugarcane Cultivar ◽

Real Time Quantitative Pcr

Eukaryotic translation initiation factor 4E (eIF4E) plays a key role in the infection of potyviruses in susceptible plants by interacting with viral genome-linked protein (VPg). Sugarcane (Saccharum spp.) production is threatened by mosaic disease caused by Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), and Sugarcane streak mosaic virus (SCSMV). In this study, two eIF4Es and their isoform eIF(iso)4E and 4E-binding protein coding genes were cloned from sugarcane cultivar ROC22 and designated SceIF4Ea, SceIF4Eb, SceIF(iso)4E, and ScnCBP, respectively. Real-time quantitative PCR analysis showed different expression profiles of these four genes upon SCMV challenge. A subcellular localization assay showed that SceIF4Ea, SceIF4Eb, SceIF(iso)4E, and ScnCBP were distributed in the nucleus and cytoplasm. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that SceIF4Ea/b and SceIF(iso)4E were selectively employed by different sugarcane mosaic pathogens, i.e., SCMV-VPg interacted with SceIF4Ea/b and SceIF(iso)4E, SrMV-VPg interacted with both SceIF4Eb and SceIF(iso)4E, and SCSMV-VPg interacted only with SceIF(iso)4E. Intriguingly, the BiFC assays, but not the Y2H assays, showed that ScnCBP interacted with the VPgs of SCMV, SrMV, and SCSMV. Competitive interaction assays showed that SCMV-VPg, SrMV-VPg, and SCMV-VPg did not compete with each other to interact with SceIF(iso)4E, and SceIF(iso)4E competed with SceIF4Eb to interact with SrMV-VPg but not SCMV-VPg. This study sheds light on the molecular mechanism of sugarcane mosaic pathogen infection of sugarcane plants and benefits sugarcane breeding against the sugarcane mosaic disease.

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Comprehensive Analysis of TIFY Transcription Factors and Their Expression Profiles under Jasmonic Acid and Abiotic Stresses in Watermelon

International Journal of Genomics ◽

10.1155/2019/6813086 ◽

2019 ◽

Vol 2019 ◽

pp. 1-13 ◽

Cited By ~ 3

Author(s):

Youxin Yang ◽

Golam Jalal Ahammed ◽

Chunpeng Wan ◽

Haoju Liu ◽

Rongrong Chen ◽

...

Keyword(s):

Jasmonic Acid ◽

Gene Family ◽

Abiotic Stresses ◽

Expression Profiles ◽

Citrullus Lanatus ◽

Functional Characterization ◽

Duplication Event ◽

Pcr Analysis ◽

Preferential Expression ◽

Qrt Pcr

The TIFY gene family is plant-specific and encodes proteins involved in the regulation of multiple biological processes. Here, we identified 15 TIFY genes in the watermelon genome, which were divided into four subfamilies (eight JAZs, four ZMLs, two TIFYs, and one PPD) in the phylogenetic tree. The ClTIFY genes were unevenly located on eight chromosomes, and three segmental duplication events and one tandem duplication event were identified, suggesting that gene duplication plays a vital role in the expansion of the TIFY gene family in watermelon. Further analysis of the protein architectures, conserved domains, and gene structures provided additional clues for understanding the putative functions of the TIFY family members. Analysis of qRT-PCR and RNA-seq data revealed that the detected ClTIFY genes had preferential expression in specific tissues. qRT-PCR analysis revealed that nine selected TIFY genes were responsive to jasmonic acid (JA) and abiotic stresses including salt and drought. JA activated eight genes and suppressed one gene, among which ClJAZ1 and ClJAZ7 were the most significantly induced. Salt and drought stress activated nearly all the detected genes to different degrees. These results lay a foundation for further functional characterization of TIFY family genes in Citrullus lanatus.

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Selection of reliable reference genes for quantitative RT-PCR in garlic under salt stress

PeerJ ◽

10.7717/peerj.7319 ◽

2019 ◽

Vol 7 ◽

pp. e7319 ◽

Cited By ~ 2

Author(s):

Guanglong Wang ◽

Chang Tian ◽

Yunpeng Wang ◽

Faxiang Wan ◽

Laibao Hu ◽

...

Keyword(s):

Gene Expression ◽

Salt Stress ◽

Reference Genes ◽

Expression Profiles ◽

Accurate Determination ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Specific Reference ◽

Variable Expression ◽

Selection Of

Quantitative real-time reverse-transcriptase PCR (qRT-PCR) has been frequently used for detecting gene expression. To obtain reliable results, selection of suitable reference genes is a fundamental and necessary step. Garlic (Allium sativum), a member from Alliaceae family, has been used both as a food flavoring and as a traditional medicine. In the present study, garlic plants were exposed to salt stress (200 mM NaCl) for 0, 1, 4 and 12 h, and garlic roots, bulbs, and leaves were harvested for subsequent analysis. The expression stability of eight candidate reference genes, eukaryotic translation initiation factor 4α (eIF-4α), actin (ACTIN), tubulin β-7 (TUB7), TAP42-interacting protein of 41 kDa (TIP41), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), SAND family protein (SAND), elongation factor 1 alpha (EF-1α), and protein phosphatase 2A (PP2A) were evaluated by geNorm, NormFinder, and BestKeeper. All genes tested displayed variable expression profiles under salt stress. In the leaf and root group, ACTIN was the best reference gene for normalizing gene expression. In garlic clove, ACTIN and SAND were the least variable, and were suitable for gene expression studies under salt stress; these two genes also performed well in all samples tested. Based on our results, we recommend that it is essential to use specific reference genes in different situations to obtain accurate results. Using a combination of multiple stable reference genes, such as ACTIN and SAND, to normalize gene expression is encouraged. The results from the study will be beneficial for accurate determination of gene expression in garlic and other plants.

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Selection and Validation of Reference Genes for Quantitative Real-Time PCR Analysis of Development and Tissue-Dependent Flower Color Formation in Cymbidium lowianum

International Journal of Molecular Sciences ◽

10.3390/ijms23020738 ◽

2022 ◽

Vol 23 (2) ◽

pp. 738

Author(s):

Xiu-Mei Dong ◽

Wei Zhang ◽

Shi-Bao Zhang

Keyword(s):

Real Time ◽

Reference Gene ◽

Reference Genes ◽

Molecular Mechanisms ◽

Gene Selection ◽

Expression Profiles ◽

Flower Color ◽

Pcr Analysis ◽

Color Formation ◽

Qrt Pcr

The development and tissue-dependent color formation of the horticultural plant results in various color pattern flowers. Anthocyanins and carotenoids contribute to the red and yellow colors, respectively. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) is used to analyze the expression profiles of anthocyanin and carotenoids biosynthesis genes in Cymbidium lowianum (Rchb.f.) Rchb.f. Appropriate reference gene selection and validation are required before normalization of gene expression in qRT-PCR analysis. Thus, we firstly selected 12 candidate reference genes from transcriptome data, and used geNorm and Normfinder to evaluate their expression stability in lip (divided into abaxial and adaxial), petal, and sepal of the bud and flower of C. lowianum. Our results show that the two most stable reference genes in different tissues of C. lowianum bud and flower are EF1δ and 60S, the most unstable reference gene is 26S. The expression profiles of the CHS and BCH genes were similar to FPKM value profiles after normalization to the two most stable reference genes, EF1δ and 60S, with the upregulated CHS and BCH expression in flower stage, indicating that the ABP and CBP were activated across the stages of flower development. However, when the most unstable reference gene, 26S, was used to normalize the qRT-PCR data, the expression profiles of CHS and BCH differed from FPKM value profiles, indicating the necessity of selecting stable reference genes. Moreover, CHS and BCH expression was highest in the abaxial lip and adaxial lip, respectively, indicating that the ABP and CBP were activated in abaxial and adaxial lip, respectively, resulting in a presence of red or yellow segments in abaxial and adaxial lip. This study is the first to provide reference genes in C. lowianum, and also provide useful information for studies that aim to understand the molecular mechanisms of flower color formation in C. lowianum.

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Selection and Validation of Reference Genes For qRT-PCR Analysis of Rhopalosiphum padi (Hemiptera: Aphididae)

Frontiers in Physiology ◽

10.3389/fphys.2021.663338 ◽

2021 ◽

Vol 12 ◽

Author(s):

Mengyi Li ◽

Xinan Li ◽

Chao Wang ◽

Qiuchi Li ◽

Saige Zhu ◽

...

Keyword(s):

Reference Genes ◽

Developmental Stages ◽

Expression Profiles ◽

Rhopalosiphum Padi ◽

Pcr Analysis ◽

Future Research ◽

Wing Dimorphism ◽

Experimental Conditions ◽

Online Tool ◽

Qrt Pcr

Rhopalosiphum padi (L.) (Hemiptera: Aphididae) is an important cosmopolitan pest in cereal crops. Reference genes can significantly affect qRT-PCR results. Therefore, selecting appropriate reference genes is a key prerequisite for qRT-PCR analyses. This study was conducted to identify suitable qRT-PCR reference genes in R. padi. We systematically analyzed the expression profiles of 11 commonly used reference genes. The ΔCt method, the BestKeeper, NormFinder, geNorm algorithms, and the RefFinder online tool were used to evaluate the suitability of these genes under diverse experimental conditions. The data indicated that the most appropriate sets of reference genes were β-actin and GAPDH (for developmental stages), AK and TATA (for populations), RPS18 and RPL13 (for tissues), TATA and GAPDH (for wing dimorphism), EF-1α and RPS6 (for antibiotic treatments), GAPDH and β-actin (for insecticide treatments), GAPDH, TATA, RPS18 (for starvation-induced stress), TATA, RPS6, and AK (for temperatures), and TATA and GAPDH (for all conditions). Our study findings, which revealed the reference genes suitable for various experimental conditions, will facilitate the standardization of qRT-PCR programs, while also improving the accuracy of qRT-PCR analyses, with implications for future research on R. padi gene functions.

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Choice of a Stable Set of Reference Genes for qRT-PCR Analysis in Amblyomma maculatum (Acari: Ixodidae)

Journal of Medical Entomology ◽

10.1603/me12123 ◽

2012 ◽

Vol 49 (6) ◽

pp. 1339-1346 ◽

Cited By ~ 15

Author(s):

Rebecca Browning ◽

Steven Adamson ◽

Shahid Karim

Keyword(s):

Reference Genes ◽

Stable Set ◽

Pcr Analysis ◽

Amblyomma Maculatum ◽

Qrt Pcr

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Identification and validation of miRNA reference genes in poplar under pathogen stress

10.21203/rs.3.rs-225734/v1 ◽

2021 ◽

Author(s):

Lichun Zhang ◽

Xiaoqian Yang ◽

Yiyi Yin ◽

Jinxing Wang ◽

Yanwei Wang

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Small Rna ◽

Reference Genes ◽

Expression Profiles ◽

Small Rna Sequencing ◽

Rna Seq ◽

Internal Standards ◽

Qrt Pcr ◽

Canker Pathogen

Abstract Quantitative real time polymerase chain reaction (qRT-PCR) is a common method to analyze gene expression. Due to differences in RNA quantity, quality, and reverse transcription efficiency between qRT-PCR samples, reference genes are used as internal standards to normalize gene expression. However, few universal genes especially miRNAs have been identified as reference so far. Therefore, it is essential to identify reference genes that can be used across various experimental conditions, stress treatments, or tissues. In this study, 14 microRNAs (miRNAs) and 5.8S rRNA were assessed for expression stability in poplar trees infected with canker pathogen. Using three reference gene analysis programs, we found that miR156g and miR156a exhibited stable expression throughout the infection process. miR156g and miR156a were then tested as internal standards to measure the expression of miR1447 and miR171c, and the results were compared to small RNA sequencing (RNA-seq) data. We found that when miR156a was used as the reference gene, the expression of miR1447 and miR171c were consistent with the small RNA-seq expression profiles. Therefore, miR156a was the most stable miRNAs examined in this study, and could be used as a reference gene in poplar under canker pathogen stress, which should enable comprehensive comparisons of miRNAs expression and avoid the bias caused by different lenth between detected miRNAs and traditional referece genes. The present study has expanded the miRNA reference genes available for gene expression studies in trees under biotic stress.

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Validation of reference genes for qRT-PCR analysis in peel and flesh of six apple cultivars (Malus domestica) at diverse stages of fruit development

Scientia Horticulturae ◽

10.1016/j.scienta.2018.09.033 ◽

2019 ◽

Vol 244 ◽

pp. 165-171 ◽

Cited By ~ 5

Author(s):

Lifang Zhu ◽

Chengquan Yang ◽

Yaohua You ◽

Wei Liang ◽

Nannan Wang ◽

...

Keyword(s):

Malus Domestica ◽

Fruit Development ◽

Reference Genes ◽

Pcr Analysis ◽

Qrt Pcr ◽

Apple Cultivars

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Dysregulation of tRNA-derived small RNAs and their potential roles in lupus nephritis

Lupus ◽

10.1177/09612033211061482 ◽

2021 ◽

pp. 096120332110614

Author(s):

Yan Liang ◽

Ji Zhang ◽

Wenxian Qiu ◽

Bo Chen ◽

Ying Zhou ◽

...

Keyword(s):

Lupus Nephritis ◽

Signaling Pathway ◽

Small Rnas ◽

High Throughput Sequencing ◽

Target Genes ◽

Expression Profiles ◽

Mapk Signaling ◽

Pcr Analysis ◽

Clinical Indicators ◽

Qrt Pcr

Objective Lupus nephritis (LN) is a major end-organ complication of systemic lupus erythematosus (SLE), and the molecular mechanism of LN is not completely clear. Accumulating pieces of evidence indicate the potential vital role of tRNA-derived small RNAs (tsRNAs) in human diseases. Current study aimed to investigate the potential roles of tsRNAs in LN. Methods We herein employed high‐throughput sequencing to screen the expression profiles of tsRNAs in renal tissues of the LN and control groups. To validate the sequencing data, we performed quantitative real-time PCR (qRT-PCR) analysis. Correlational analysis of verified tsRNAs expression and clinical indicators was conducted using linear regression. The potential target genes were also predicted. The biological functions of tsRNAs were annotated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Results Our findings revealed that the expression profiles of tsRNAs were significantly altered in the kidney tissues from LN patients compared with control. Overall, 160 tsRNAs were significantly dysregulated in the LN group, of which 79 were upregulated, whereas 81 were downregulated. Subsequent qRT-PCR results confirmed the different expression of candidate tsRNAs. Correlation analysis results found that expression of verified tsRNAs were correlated to clinical indicators. The target prediction results revealed that verified tsRNAs might act on 712 target genes. Further bioinformatics analysis uncovered tsRNAs might participate in the pathogenesis of LN through several associated pathways, including cell adhesion molecules, MAPK signaling pathway, PI3K-Akt signaling pathway and B cell receptor signaling pathway. Conclusion This study provides a novel insight for studying the mechanism of LN.

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Comprehensive Analysis of the TIFY Gene Family and Its Expression Profiles under Phytohormone Treatment and Abiotic Stresses in Roots of Populus trichocarpa

Forests ◽

10.3390/f11030315 ◽

2020 ◽

Vol 11 (3) ◽

pp. 315

Author(s):

Hanzeng Wang ◽

Xue Leng ◽

Xuemei Xu ◽

Chenghao Li

Keyword(s):

Gene Family ◽

Abiotic Stresses ◽

Expression Profiles ◽

Populus Trichocarpa ◽

Expression Patterns ◽

Interaction Network ◽

Pcr Analysis ◽

Phylogenetic Tree Analysis ◽

Element Analysis ◽

Cis Element

The TIFY gene family is specific to land plants, exerting immense influence on plant growth and development as well as responses to biotic and abiotic stresses. Here, we identify 25 TIFY genes in the poplar (Populus trichocarpa) genome. Phylogenetic tree analysis revealed these PtrTIFY genes were divided into four subfamilies within two groups. Promoter cis-element analysis indicated most PtrTIFY genes possess stress- and phytohormone-related cis-elements. Quantitative real-time reverse transcription polymerase chain reaction (qRT–PCR) analysis showed that PtrTIFY genes displayed different expression patterns in roots under abscisic acid, methyl jasmonate, and salicylic acid treatments, and drought, heat, and cold stresses. The protein interaction network indicated that members of the PtrTIFY family may interact with COI1, MYC2/3, and NINJA. Our results provide important information and new insights into the evolution and functions of TIFY genes in P. trichocarpa.

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