Selection and Evaluation of Candidate Reference Genes for Quantitative Real-Time PCR in Aboveground Tissues and Drought Conditions in Rhododendron Delavayi

Zhang, Lu; Cai, Yafei; Zhang, Mingchao; Du, Guocheng; Wang, Jihua

doi:10.3389/fgene.2022.876482

Cited by 7 publications

(5 citation statements)

References 56 publications

(104 reference statements)

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“…Many researchers have conducted relatively quantitative comparisons between the most stable and unstable reference genes, and they have often obtained significant differences. The use of unstable reference genes leads to inaccurate quantitative results [ 30 , 31 , 32 ]. Specifically, in the case of fruits at different developmental stages, the two most stable reference genes, Act1 and UBQ , should be employed, while Act1 and His3 are advisable choices for different tissues.…”

Section: Discussionmentioning

confidence: 99%

Selection of Reference Genes in Evodia rutaecarpa var. officinalis and Expression Patterns of Genes Involved in Its Limonin Biosynthesis

Zhou,

Zhang,

et al. 2023

Plants

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E. rutaecarpa var. officinalis is a traditional Chinese medicinal plant known for its therapeutic effects, which encompass the promotion of digestion, the dispelling of cold, the alleviation of pain, and the exhibition of anti-inflammatory and antibacterial properties. The principal active component of this plant, limonin, is a potent triterpene compound with notable pharmacological activities. Despite its significance, the complete biosynthesis pathway of limonin in E. rutaecarpa var. officinalis remains incompletely understood, and the underlying molecular mechanisms remain unexplored. The main purpose of this study was to screen the reference genes suitable for expression analysis in E. rutaecarpa var. officinalis, calculate the expression patterns of the genes in the limonin biosynthesis pathway, and identify the relevant enzyme genes related to limonin biosynthesis. The reference genes play a pivotal role in establishing reliable reference standards for normalizing the gene expression data, thereby ensuring precision and credibility in the biological research outcomes. In order to identify the optimal reference genes and gene expression patterns across the diverse tissues (e.g., roots, stems, leaves, and flower buds) and developmental stages (i.e., 17 July, 24 August, 1 September, and 24 October) of E. rutaecarpa var. officinalis, LC-MS was used to analyze the limonin contents in distinct tissue samples and developmental stages, and qRT-PCR technology was employed to investigate the expression patterns of the ten reference genes and eighteen genes involved in limonin biosynthesis. Utilizing a comprehensive analysis that integrated three software tools (GeNorm ver. 3.5, NormFinder ver. 0.953 and BestKeeper ver. 1.0) and Delta Ct method alongside the RefFinder website, the best reference genes were selected. Through the research, we determined that Act1 and UBQ served as the preferred reference genes for normalizing gene expression during various fruit developmental stages, while Act1 and His3 were optimal for different tissues. Using Act1 and UBQ as the reference genes, and based on the different fruit developmental stages, qRT-PCR analysis was performed on the pathway genes selected from the “full-length transcriptome + expression profile + metabolome” data in the limonin biosynthesis pathway of E. rutaecarpa var. officinalis. The findings indicated that there were consistent expression patterns of HMGCR, SQE, and CYP450 with fluctuations in the limonin contents, suggesting their potential involvement in the limonin biosynthesis of E. rutaecarpa var. officinalis. This study lays the foundation for further research on the metabolic pathway of limonin in E. rutaecarpa var. officinalis and provides reliable reference genes for other researchers to use for conducting expression analyses.

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Section: Discussionmentioning

confidence: 99%

Selection of Reference Genes in Evodia rutaecarpa var. officinalis and Expression Patterns of Genes Involved in Its Limonin Biosynthesis

Zhou,

Zhang,

et al. 2023

Plants

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“…Total RNA was extracted from the samples using the OmniPlant RNA Kit (CW2598S, Cwbiotech, Beijing, China) and reverse transcribed utilizing RevertAid Reverse Transcriptase (EP0441, Thermo Scientific, Beijing, China), following the manufacturer’s protocol. The primer sequences for qRT-PCR are listed in Table S10 , with EF1α as the internal control [ 62 ]. qRT-PCR was performed utilizing an ABI real-time PCR system (Q1, ABI, Los Angeles, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

Early and Late Transcriptomic and Metabolomic Responses of Rhododendron ‘Xiaotaohong’ Petals to Infection with Alternaria sp.

Zhang

Xia

et al. 2023

IJMS

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In recent years, petal blight disease caused by pathogens has become increasingly epidemic in Rhododendron. Breeding disease-resistant rhododendron is considered to be a more environmentally friendly strategy than is the use of chemical reagents. In this study, we aimed to investigate the response mechanisms of rhododendron varieties to petal blight, using transcriptomics and metabolomics analyses. Specifically, we monitored changes in gene expression and metabolite accumulation in Rhododendron ‘Xiaotaohong’ petals infected with the Alternaria sp. strain (MR-9). The infection of MR-9 led to the development of petal blight and induced significant changes in gene transcription. Differentially expressed genes (DEGs) were predominantly enriched in the plant–pathogen interaction pathway. These DEGs were involved in carrying out stress responses, with genes associated with H2O2 production being up-regulated during the early and late stages of infection. Correspondingly, H2O2 accumulation was detected in the vicinity of the blight lesions. In addition, defense-related genes, including PR and FRK, exhibited significant up-regulated expression during the infection by MR-9. In the late stage of the infection, we also observed significant changes in differentially abundant metabolites (DAMs), including flavonoids, alkaloids, phenols, and terpenes. Notably, the levels of euscaphic acid, ganoderol A, (−)-cinchonidine, and theophylline in infected petals were 21.8, 8.5, 4.5, and 4.3 times higher, respectively, compared to the control. Our results suggest that H2O2, defense-related genes, and DAM accumulation are involved in the complex response mechanisms of Rhododendron ‘Xiaotaohong’ petals to MR-9 infection. These insights provide a deeper understanding of the pathogenesis of petal blight disease and may have practical implications for developing disease-resistant rhododendron varieties.

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“…The reaction was carried out as follows: 30 s at 95 °C for denaturation, followed by 40 cycles of 10 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C. The glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) gene was used as an internal control [ 66 ] to normalize the measured gene expression levels based on the standard curves, and the Ct values were automatically calculated by LightCycler ® 480 II (Roche Diagnostics, Germany) software. Finally, the relative expression of the target gene was calculated according to the 2 −ΔΔCT method [ 67 ].…”

Section: Methodsmentioning

confidence: 99%

Comprehensive Genomic Survey, Structural Classification, and Expression Analysis of WRKY Transcription Factor Family in Rhododendron simsii

Wan

Cheng

Zhang

et al. 2022

Plants

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(1) Rhododendron is one of the top ten traditional flowers in China, with both high ornamental and economic values. However, with the change of the environment, Rhododendron suffers from various biological stresses. The WRKY transcription factor is a member of the most crucial transcription factor families, which plays an essential regulatory role in a variety of physiological processes and developmental stresses. (2) In this study, 57 RsWRKYs were identified using genome data and found to be randomly distributed on 13 chromosomes. Based on gene structure and phylogenetic relationships, 57 proteins were divided into three groups: I, II, and III. Multiple alignments of RsWRKYs with Arabidopsis thaliana homologous genes revealed that WRKY domains in different groups had different conserved sites. RsWRKYs have a highly conserved domain, WRKYGQK, with three variants, WRKYGKK, WRKYGEK, and WRKYGRK. Furthermore, cis-acting elements analysis revealed that all of the RsWRKYs had stress and plant hormone cis-elements, with figures varying by group. Finally, the expression patterns of nine WRKY genes treated with gibberellin acid (GA), methyl jasmonate (MeJA), heat, and drought in Rhododendron were also measured using quantitative real-time PCR (qRT-PCR). The results showed that the expression levels of the majority of RsWRKY genes changed in response to multiple phytohormones and abiotic stressors. (3) This current study establishes a theoretical basis for future studies on the response of RsWRKY transcription factors to various hormone and abiotic stresses as well as a significant foundation for the breeding of new stress-tolerant Rhododendron varieties.

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Selection and Evaluation of Candidate Reference Genes for Quantitative Real-Time PCR in Aboveground Tissues and Drought Conditions in Rhododendron Delavayi

Cited by 7 publications

References 56 publications

Selection of Reference Genes in Evodia rutaecarpa var. officinalis and Expression Patterns of Genes Involved in Its Limonin Biosynthesis

Selection of Reference Genes in Evodia rutaecarpa var. officinalis and Expression Patterns of Genes Involved in Its Limonin Biosynthesis

Early and Late Transcriptomic and Metabolomic Responses of Rhododendron ‘Xiaotaohong’ Petals to Infection with Alternaria sp.

Comprehensive Genomic Survey, Structural Classification, and Expression Analysis of WRKY Transcription Factor Family in Rhododendron simsii

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