Reference genes for qRT-PCR normalisation in different tissues, developmental stages, and stress conditions of <i>Hypericum perforatum</i>

Zhou, Wen; Wang, Shiqiang; Yang, Lei; Sun, Yan; Zhang, Qian; Li, Bin; Wang, Bin; Li, Lin; Wang, Donghao; Wang, Zhezhi

doi:10.7717/peerj.7133

Cited by 14 publications

(15 citation statements)

References 47 publications

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“…These samples were used to perform the transcriptome sequencing and to further detect the tissue-specific expression patterns of the HpERFs . The RNA-Seq data can be obtained from the Sequence Read Archive (SRA) database of NCBI with the accession numbers SRR8438983 (flower), SRR8438984 (leaf), SRR8438985 (stem), and SRR8438986 (root) [ 70 , 71 ]. The method of computing the transcript abundance of HpERFs was following the fragments per kilobase of exon model per million mapped reads (FPKM).…”

Section: Methodsmentioning

confidence: 99%

Genome-Wide Analysis and the Expression Pattern of the ERF Gene Family in Hypericum perforatum

Zhang

Zhou

et al. 2021

Plants

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Hypericum perforatum is a well-known medicinal herb currently used as a remedy for depression as it contains many high levels of secondary metabolites. The ethylene response factor (ERF) family encodes transcriptional regulators with multiple functions that play a vital role in the diverse developmental and physiological processes of plants, which can protect plants from various stresses by regulating the expression of genes. Although the function of several ERF genes from other plants has been further confirmed, H. perforatum is the first sequenced species in Malpighiales, and no information regarding the ERFs has been reported thus far. In this study, a total of 101 ERF genes were identified from H. perforatum. A systematic and thorough bioinformatic analysis of the ERF family was performed using the genomic database of H. perforatum. According to the phylogenetic tree analysis, HpERFs were further classified into 11 subfamilies. Gene ontology (GO) analysis suggested that most of the HpERFs likely participate in the biological processes of plants. The cis-elements were mainly divided into five categories, associated with the regulation of gene transcription, response to various stresses, and plant development. Further analysis of the expression patterns showed that the stress-responsive HpERFs responded to different treatments. This work systematically analyzed HpERFs using the genome sequences of H. perforatum. Our results provide a theoretical basis for further investigation of the function of stress-related ERFs in H. perforatum.

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Section: Methodsmentioning

confidence: 99%

Genome-Wide Analysis and the Expression Pattern of the ERF Gene Family in Hypericum perforatum

Zhang

Zhou

et al. 2021

Plants

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“…ACT2, TUB-β, and EF1-α were the most stable reference genes in different tissues, developmental stages, and stress conditions in H. perforatum. 2 Expression of ACT3 was stable in cucumber upon cold and heat stress. 34 ACT7 was stably expressed in Corchorus olitorius during drought and water stress.…”

Section: Nanoparticles Affect Expression Stability Of Housekeeping Genesmentioning

confidence: 99%

“…The forward and reverse primer sequences used in the amplification of these genes were derived from previous reports. 2,6 Primers were synthesized and their specificity was tested using PCR and qRT-PCR based on melting curve analysis. Amplification of specific fragments of the genes using qRT-PCR was carried out in a Lightcycler 480 real-time PCR system (Roche, Basel, Switzerland) using the SYBR Green-based PCR assay.…”

Section: Genes Primers and Qrt-pcr Conditionsmentioning

confidence: 99%

“…This technique is widely used for the quantification of gene expression, reflecting its accuracy across a relatively broad and dynamic range. 2 However, the reliability of the results depends on the reference genes used in the normalization step of qRT-PCR. A good reference gene should be consistently expressed under both control and experimental conditions.…”

Section: Introductionmentioning

confidence: 99%

“…Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), tubulin alpha (TUB-α), tubulin beta (TUB-β), translation elongation factor, ubiquitinconjugating enzyme (UBC), 18S ribosomal RNA (18SrRNA), mitogen-activated protein kinase 6 (MPK6), and 25S ribosomal RNA (25SrRNA) are common reference genes. 2,3 The stability of reference genes differs according to the experimental conditions and plant species. EF1-α has been found to be the most stable gene under heat stress, whereas the ADP-ribosylation factor 1 (ADP) gene is the most stable under salt stress in Hordeum brevisubulatum.…”

Section: Introductionmentioning

confidence: 99%

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<p>Nanoparticles Affect the Expression Stability of Housekeeping Genes in Plant Cells</p>

Selvakesavan¹,

Franklin²

2020

NSA

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We report on the expression stability of several housekeeping/reference genes that can be used in the normalization of target gene expression in quantitative real-time PCR (qRT-PCR) analysis of plant cells challenged with metal nanoparticles (NPs). Materials and Methods: Uniform cell suspension cultures of Hypericum perforatum were treated with 25 mg/l silver and gold NPs (14-15 nm in diameter). Cells were collected after 0.5, 4.0, and 12 h. The total RNA isolated from the cells was analyzed for the stability of ACT2, ACT3, ACT7, EF1-α, GAPDH, H2A, TUB-α, TUB-β, and 18S rRNA genes using qRT-PCR. The cycle threshold (Ct) values of the genes were analyzed using the geNorm, NormFinder, BestKeeper, and RefFinder statistical algorithms to rank gene stability. The stability of the top-ranked genes was validated by normalizing the expression of HYP1. Results: The expression of the tested housekeeping genes varied with treatment duration and NP types. EF1-α in gold NP treatment and TUB-α and EF1-α in silver NP treatment ranked among the top three positions. However, none of the genes retained their top ranking with time and across NP types. Conclusion: EF1-α can be used as a reference for treatment involving both silver and gold NPs in H. perforatum cells. TUB-α can be used only for silver NP-treated cells. The expression instability of most of the housekeeping genes highlights the importance of systematic standardization of reference genes for NP treatment conditions to draw proper conclusions on the target gene expression.

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Identification and characterization of glycosyltransferases catalyzing direct xanthone 4‐C‐glycosylation in Hypericum perforatum

2021

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Xanthones are compounds with a diphenyl ether skeleton mainly found in plants and often glycosylated at carbon atoms. Although many C‐glycosyltransferases (CGTs) participating in flavone C‐glycosylation have been identified, MiCGT from Mangifera indica, adding sugar to an open‐chain benzophenone skeleton, is the only identified xanthone biosynthesis‐related CGT. Here, we identified two CGTs from Hypericum perforatum that add sugar to the closed‐ring xanthone, but not benzophenone. These CGTs catalyze sugar transfer to the C‐4 position of norathyriol (1,3,6,7‐tetrahydroxyxanthone) to form isomangiferin (1,3,6,7‐tetrahydroxyxanthone 4‐C‐glucoside), a major xanthone C‐glucoside. This is the first study to report CGTs that mediate the direct C‐glycosylation of xanthone.

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Reference genes for qRT-PCR normalisation in different tissues, developmental stages, and stress conditions of Hypericum perforatum

Cited by 14 publications

References 47 publications

Genome-Wide Analysis and the Expression Pattern of the ERF Gene Family in Hypericum perforatum

Genome-Wide Analysis and the Expression Pattern of the ERF Gene Family in Hypericum perforatum

<p>Nanoparticles Affect the Expression Stability of Housekeeping Genes in Plant Cells</p>

Identification and characterization of glycosyltransferases catalyzing direct xanthone 4‐C‐glycosylation in Hypericum perforatum

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