Transcriptomic and physiological analyses reveal changes in secondary metabolite and endogenous hormone in ginger (Zingiber officinale Rosc.) in response to postharvest chilling stress.

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The domains of unknown function (DUF) superfamilies contain proteins with conserved amino acid sequences without known functions. Among them, DUF668 was indicated widely involving the stress response of plants. However, understanding ZoDUF668 is still lacking. Here, 12 ZoDUF668 genes were identified in ginger by the bioinformatics method and unevenly distributed on six chromosomes. Conserved domain analysis showed that members of the same subfamily had similar conserved motifs and gene structures. The promoter region of ZoDUF668s contained the light, plant hormone and stress-responsive elements. The prediction of miRNA targeting relationship showed that nine ginger miRNAs targeted four ZoDUF668 genes through cleavage. The expression patterns of 12 ZoDUF668 genes under biotic and abiotic stress were analyzed using RT-qPCR. The results showed that the expression of seven ZoDUF668 genes was significantly downregulated under Fusarium solani infection, six ZoDUF668 genes were upregulated under cold stress, and five ZoDUF668 genes were upregulated under waterlogging stress. These results indicate that the ZoDUF668 gene has different expression patterns under different stress conditions. This study provides excellent candidate genes and provides a reference for stress-resistance research in ginger.

Genome-Wide Identification of DUF668 Gene Family and Expression Analysis under F. solani, Chilling, and Waterlogging Stresses in Zingiber officinale

Uncaria rhynchophylla (Miq.) Miq. ex Havil, a traditional medicinal herb, is enriched with several pharmacologically active terpenoid indole alkaloids (TIAs). At present, no method has been reported that can comprehensively select and evaluate the appropriate reference genes for gene expression analysis, especially the transcription factors and key enzyme genes involved in the biosynthesis pathway of TIAs in U. rhynchophylla. Reverse transcription quantitative PCR (RT-qPCR) is currently the most common method for detecting gene expression levels due to its high sensitivity, specificity, reproducibility, and ease of use. However, this methodology is dependent on selecting an optimal reference gene to accurately normalize the RT-qPCR results. Ten candidate reference genes, which are homologues of genes used in other plant species and are common reference genes, were used to evaluate the expression stability under three stress-related experimental treatments (methyl jasmonate, ethylene, and low temperature) using multiple stability analysis methodologies. The results showed that, among the candidate reference genes, S-adenosylmethionine decarboxylase (SAM) exhibited a higher expression stability under the experimental conditions tested. Using SAM as a reference gene, the expression profiles of 14 genes for key TIA enzymes and a WRKY1 transcription factor were examined under three experimental stress treatments that affect the accumulation of TIAs in U. rhynchophylla. The expression pattern of WRKY1 was similar to that of tryptophan decarboxylase (TDC) under ETH treatment. This research is the first to report the stability of reference genes in U. rhynchophylla and provides an important foundation for future gene expression analyses in U. rhynchophylla. The RT-qPCR results indicate that the expression of WRKY1 is similar to that of TDC under ETH treatment. It may coordinate the expression of TDC, providing a possible method to enhance alkaloid production in the future through synthetic biology.

Evaluation of Reference Genes for Normalizing RT-qPCR and Analysis of the Expression Patterns of WRKY1 Transcription Factor and Rhynchophylline Biosynthesis-Related Genes in Uncaria rhynchophylla

Ardisia kteniophylla A. DC, widely known as folk medicinal herb and ornamental plant, has been extensively investigated due to its unique leaf color, anti-cancer and other pharmacological activities. The quantitative real-time PCR (qRT-PCR) was an excellent tool for the analysis of gene expression with its high sensitivity and quantitative properties. Normalizing gene expression with stable reference genes was essential for qRT-PCR accuracy. In addition, no studies have yet been performed on the selection, verification and stability of internal reference genes suitable for A. kteniophylla, which has greatly hindered the biomolecular researches of this species. In this study, 29 candidate genes were successfully screened according to stable expression patterns of large-scale RNA seq data that from a variety of tissues and the roots of different growth stages in A. kteniophylla. The candidates were then further determined via qRT-PCR in various experimental samples, including MeJA, ABA, SA, NaCl, CuSO4, AgNO3, MnSO4, CoCl2, drought, low temperature, heat, waterlogging, wounding and oxidative stress. To assess the stability of the candidates, five commonly used strategies were employed: delta-CT, geNorm, BestKeeper, NormFinder, and the comprehensive tool RefFinder. In summary, UBC2 and UBA1 were found to be effective in accurately normalizing target gene expression in A. kteniophella regardless of experimental conditions, while PP2A-2 had the lowest stability. Additionally, to verify the reliability of the recommended reference genes under different colored leaf samples, we examined the expression patterns of six genes associated with anthocyanin synthesis and regulation. Our findings suggested that PAP1 and ANS3 may be involved in leaf color change in A. kteniphella. This study successfully identified the ideal reference gene for qRT-PCR analysis in A. kteniphella, providing a foundation for future research on gene function, particularly in the biosynthesis of anthocyanins.

Systematic identification of reference genes for qRT-PCR of Ardisia kteniophylla A. DC under different experimental conditions and for anthocyanin-related genes studies

Botanical pesticides have gradually become accepted for use in the control of agricultural pests. In order to clarify the active compounds of the ginger (Zingiber officinale) shoot extract (GSE) and its inhibitory effect on the growth of sorghum aphids (Melanaphis sorghi). In this study, LC-MS/MS was used to determine the major active compounds of the GSE, and leaf disc method was used to explore the insecticidal effect of the active compounds of ginger on sorghum aphids and the response mechanism of sorghum aphids. The results showed that phenolic acids were identified as the main active compounds, followed by flavonoids. The aphidicidal activity test using the above compounds found that 6-gingerol, and quercetin-3-O-rutinoside exhibited aphidicidal activity (GSE > quercetin-3-O-rutinoside > 6-gingerol). The growth of sorghum aphid was evaluated by using different concentrations of the GSE. It was found that with the increase of concentration and treatment time, the litter size, longevity and molting of aphids significantly decreased, and the mortality of aphids increased. The enzyme activity of aphids treated with 15 mg·mL−1 GSE was determined, and it was found that the GSE could significantly inhibit the activities of pepsin, lipase and α-amylase of aphids, while the activity of superoxide dismutase (SOD) was significantly activated. The activities of peroxidase (POD) and catalase (CAT) increased at first and then decreased. In detoxification enzymes, the carboxylesterase (CarE) activity was significantly activated, the acetylcholinesterase (AChE) activity was significantly inhibited, and the glutathione S-transferase (GST) activity increased at first and then decreased. The above results indicated that the GSE may become a botanical pesticide for aphid control and provide new resources for the development of aphid biological agents.

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Evaluation of the Contact Toxicity and Physiological Mechanisms of Ginger (Zingiber officinale) Shoot Extract and Selected Major Constituent Compounds against Melanaphis sorghi Theobald

Gene expression analysis largely improves our understanding of the molecular basis underpinning various plant biological processes. Stable reference genes play a foundational role during the normalization of gene expression levels. However, until now, there have been few reference genes suitable for ginger reverse transcription-quantitative PCR (RT-qPCR) research. In this study, 29 candidate reference genes with stable expression patterns across multiple ginger tissues and 13 commonly used reference genes were selected to design RT-qPCR primers. After amplification specificity validation, 32 candidates were selected and further evaluated by RT-qPCR using samples from various organs subjected to NaCl, drought, heat, waterlogging, and chilling stress. Four strategies, including delta-CT, BestKeeper, geNorm, and NormFinder, were used to rank the stability of reference genes, and the ranks produced by these four strategies were comprehensively evaluated by RefFinder to determine the final rank. Overall, the top three stability reference genes indicated by RefFinder were RBP > ATPase > 40S_S3. Their expression pattern correlation analysis showed that the coefficients among each pair of RBP, ATPase, and 40S_S3 were larger than 0.96, revealing consistent and stable expression patterns under various treatments. Then, the expression of three pathogenesis-related (PR) genes and seven MYB genes in rhizomes during postharvest storage and subjected to pathogen infection was normalized by RBP, ATPase, 40S_S3, RBP and ATPase, ATPase and 40S-S3, and RBP and 40S-S3. The results showed that PR and MYB genes were induced by postharvest deterioration and pathogen infection. The correlation coefficients of RBP/ATPase, RBP/40S_S3, ATPase/40S_S3, RBP and ATPase/ATPase and 40S-S3, RBP and ATPase/RBP and 40S-S3, and ATPase and 40S-S3/RBP and 40S-S3 were 0.99, 0.96, 0.99, 0.99, 1.00, and 1.00, respectively, which confirmed the stability of these three reference genes in postharvest biology studies of ginger. In summary, this study identified appropriate reference genes for RT-qPCR in ginger and facilitated gene expression studies under biotic and abiotic stress conditions.

/pdf/identification-of-reference-genes-for-reverse-transcription-25jnbnrf.pdf

Identification of Reference Genes for Reverse Transcription-Quantitative PCR Analysis of Ginger Under Abiotic Stress and for Postharvest Biology Studies

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Transcriptomic and physiological analyses reveal changes in secondary metabolite and endogenous hormone in ginger (Zingiber officinale Rosc.) in response to postharvest chilling stress.

Evaluation of the Contact Toxicity and Physiological Mechanisms of Ginger (Zingiber officinale) Shoot Extract and Selected Major Constituent Compounds against Melanaphis sorghi Theobald

Identification of Reference Genes for Reverse Transcription-Quantitative PCR Analysis of Ginger Under Abiotic Stress and for Postharvest Biology Studies