Selection of reference genes for quantitative real-time PCR analysis in Lathyrus sativus L. under different development stages and drought stress

Zhang, Ying; Liu, Fengjuan; Guo, Huajun; Ma, Hui; Chen, Hong; Song, Yaoyao; Chen, Peng; Xu, Quanxi

doi:10.1007/s10722-022-01374-x

Cited by 6 publications

(12 citation statements)

References 34 publications

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“…On the other hand, the expression level of LsbHLHR86 was consistent with previous reports, while the expression level of the CsbHLH86 gene in cucumber showed significant downregulation after exposure to 100 mM NaCl for 24 h ( Li et al, 2020) . Interestingly, our results revealed an opposite pattern for genes LsbHLHD4 and LsbHLHR14 compared to other studies that did not show significant upregulation clearly ( Xu et al, 2022 ; Zhang et al, 2022) . Overall, these results provide a solid foundation for further studies on the role of the bHLH gene family in grass pea.…”

Section: Discussioncontrasting

confidence: 99%

“…The amplification program consisted of initial denaturation at 95°C for 60 s, followed by 40 cycles of denaturation at 95°C for 3 s and annealing at 60°C for 40 s. A melting curve analysis was performed from 65°C to 95°C to verify the specificity of the qPCR products. The genes ABCT and Elf1b were used as internal reference genes for normalization ( Zhang et al, 2022) . All reactions were conducted in triplicate, and relative expression levels were calculated using the 2 −△△ Ct method ( Schmittgen and Livak, 2008) .…”

Section: Methodsmentioning

confidence: 99%

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Genome-wide identification, characterization, and validation of the bHLH transcription factors in grass pea

et al. 2023

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Background: The basic helix-loop-helix (bHLH) transcription factor is a vital component in plant biology, with a significant impact on various aspects of plant growth, cell development, and physiological processes. Grass pea is a vital agricultural crop that plays a crucial role in food security. However, the lack of genomic information presents a major challenge to its improvement and development. This highlights the urgency for deeper investigation into the function of bHLH genes in grass pea to improve our understanding of this important crop.Results: The identification of bHLH genes in grass pea was performed on a genome-wide scale using genomic and transcriptomic screening. A total of 122 genes were identified as having conserved bHLH domains and were functionally and fully annotated. The LsbHLH proteins could be classified into 18 subfamilies. There were variations in intron-exon distribution, with some genes lacking introns. The cis-element and gene enrichment analyses showed that the LsbHLHs were involved in various plant functions, including response to phytohormones, flower and fruit development, and anthocyanin synthesis. A total of 28 LsbHLHs were found to have cis-elements associated with light response and endosperm expression biosynthesis. Ten conserved motifs were identified across the LsbHLH proteins. The protein-protein interaction analysis showed that all LsbHLH proteins interacted with each other, and nine of them displayed high levels of interaction. RNA-seq analysis of four Sequence Read Archive (SRA) experiments showed high expression levels of LsbHLHs across a range of environmental conditions. Seven highly expressed genes were selected for qPCR validation, and their expression patterns in response to salt stress showed that LsbHLHD4, LsbHLHD5, LsbHLHR6, LsbHLHD8, LsbHLHR14, LsbHLHR68, and LsbHLHR86 were all expressed in response to salt stress.Conclusion: The study provides an overview of the bHLH family in the grass pea genome and sheds light on the molecular mechanisms underlying the growth and evolution of this crop. The report covers the diversity in gene structure, expression patterns, and potential roles in regulating plant growth and response to environmental stress factors in grass pea. The identified candidate LsbHLHs could be utilized as a tool to enhance the resilience and adaptation of grass pea to environmental stress.

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Section: Discussioncontrasting

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Genome-wide identification, characterization, and validation of the bHLH transcription factors in grass pea

et al. 2023

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“…To ensure gene expression studies’ steady and accuracy, these benchmark genes must maintain a high level of stability in their expression throughout multiple stages of organism development, under different treatments and environmental conditions, and across diverse cell or tissue types [ 11 , 12 ]. In studies on internal insect reference gene screening, 18S ribosomal RNA ( 18S rRNA ), ribosomal protein S18 (PRS18 ), beta-tubulin ( TUB ), and glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) are commonly mentioned [ 13 , 14 ]. These genes are all engaged in the typical physiological and metabolic activities of cells and are frequently utilized as internal reference genes [ 7 , 15 ].…”

Section: Introductionmentioning

confidence: 99%

Identification and Validation of Reference Genes for Expression Analysis Using RT-qPCR in Leptocybe invasa Fisher and La Salle (Hymenoptera: Eulophidae)

Liu

Zhou

Qiu

et al. 2023

Insects

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Leptocybe invasa (Hymenoptera: Eulophidae) is a globally intrusive pest. Despite extensive research into the physiological responses of this pest, our understanding of the molecular mechanisms still needs to be improved. We want to accurately investigate the expression of L. invasa’s target genes, so it is imperative to select fitting reference genes. In this study, eight housekeeping genes’ stability (RPS30, ACTR, 18S rRNA, ACT, RPL18, GAPDH, 28S rRNA, and TUB) was tested under five different experimental conditions, including male or female adults, somites (head, thorax, and abdomen), temperatures (0 °C, 25 °C, and 40 °C), diets (starvation, clear water, 10% honey water, Eucalyptus sap), and pesticides (acetone was used as a control, imidacloprid, monosultap). Gene stability was calculated using RefFinder, which integrates four algorithms (the ∆Ct method, geNorm, NormFinder, and BestKeeper). The findings implied that ACT and ACTR were the most accurate when comparing sexes. For analyzing different somites, 28S rRNA and RPL18 were ideal; the 28S rRNA and RRS30 were perfect for analyzing at different temperatures. The combination of ACT and GAPDH helped to analyze gene expression in different diets, and GAPDH and 28S rRNA were suitable for various pesticide conditions. Overall, this research offers a complete list of reference genes from L. invasa for precise analysis of target gene expression, which can improve the trustworthiness of RT-qPCR and lay the foundation for further investigations into the gene function of this pest.

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“…However, to date, no such reference genes have been proven to provide stable expression profiles across all investigational conditions throughout the plant and animal kingdom. In recent reports, the most commonly used reference genes including Actin, tubulin, glyceraldehyde-3-phosphate dehydrogenase, and elongation factor 1α, etc., proved that their transcript levels vary in different plant species under different experimental conditions 36 – 38 . This might be due to differences in the cell basal metabolism and specific cellular functions and thus have the extensive molecular regulation changes of reference genes of the species under different environmental conditions 14 , 38 – 40 .…”

Section: Discussionmentioning

confidence: 99%

Selection and validation of reliable reference genes for quantitative real-time PCR in Barnyard millet (Echinochloa spp.) under varied abiotic stress conditions

Renganathan,

Renuka,

Vanniarajan

et al. 2023

Sci Rep

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Quantitative real-time polymerase chain reaction (RT-qPCR) using a stable reference gene is widely used for gene expression research. Barnyard millet (Echinochloa spp.) is an ancient crop in Asia and Africa that is widely cultivated for food and fodder. It thrives well under drought, salinity, cold, and heat environmental conditions, besides adapting to any soil type. To date, there are no gene expression studies performed to identify the potential candidate gene responsible for stress response in barnyard millet, due to lack of reference gene. Here, 10 candidate reference genes, Actin (ACT), α-tubulin (α-TUB), β-tubulin (β-TUB), RNA pol II (RP II), elongation factor-1 alpha (EF-1α), adenine phosphoribosyltransferase (APRT), TATA-binding protein-like factor (TLF), ubiquitin-conjugating enzyme 2 (UBC2), ubiquitin-conjugating enzyme E2L5 (UBC5) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were selected from mRNA sequences of E. crus-galli and E. colona var frumentacea. Five statistical algorithms (geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder) were applied to determine the expression stabilities of these genes in barnyard millet grown under four different abiotic stress (drought, salinity, cold and heat) exposed at different time points. The UBC5 and ɑ-TUB in drought, GAPDH in salinity, GAPDH and APRT in cold, and EF-1α and RP II in heat were the most stable reference genes, whereas ß-TUB was the least stable irrespective of stress conditions applied. Further Vn/Vn + 1 analysis revealed two reference genes were sufficient to normalize gene expression across all sample sets. The suitability of identified reference genes was validated with Cu-ZnSOD (SOD1) in the plants exposed to different abiotic stress conditions. The results revealed that the relative quantification of the SOD1 gene varied according to reference genes and the number of reference genes used, thus highlighting the importance of the choice of a reference gene in such experiments. This study provides a foundational framework for standardizing RT-qPCR analyses, enabling accurate gene expression profiling in barnyard millet.

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Selection of reference genes for quantitative real-time PCR analysis in Lathyrus sativus L. under different development stages and drought stress

Cited by 6 publications

References 34 publications

Genome-wide identification, characterization, and validation of the bHLH transcription factors in grass pea

Genome-wide identification, characterization, and validation of the bHLH transcription factors in grass pea

Identification and Validation of Reference Genes for Expression Analysis Using RT-qPCR in Leptocybe invasa Fisher and La Salle (Hymenoptera: Eulophidae)

Selection and validation of reliable reference genes for quantitative real-time PCR in Barnyard millet (Echinochloa spp.) under varied abiotic stress conditions

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