Induction of Phlorotannins and Gene Expression in the Brown Macroalga Fucus vesiculosus in Response to the Herbivore Littorina littorea

Mechanisms related to the induction of phlorotannin biosynthesis in marine brown algae remain poorly known. Several studies undertaken on fucoid species have shown that phlorotannins accumulate in the algae for several days or weeks after being exposed to grazing, and this is measured by direct quantification of soluble phenolic compounds. In order to investigate earlier inducible responses involved in phlorotannin metabolism, Fucus vesiculosus was studied between 6 and 72 h of grazing by the sea snail Littorina littorea. In this study, the quantification of soluble phenolic compounds was complemented by a Quantitative real-time PCR (qRT-PCR) approach applied on genes that are potentially involved in either the phlorotannin metabolism or stress responses. Soluble phlorotannin levels remained stable during the kinetics and increased significantly only after 12 h in the presence of grazers, compared to the control, before decreasing to the initial steady state for the rest of the kinetics. Under grazing conditions, the expression of vbpo, cyp450 and ast6 genes was upregulated, respectively, at 6 h, 12 h and 24 h, and cyp450 gene was downregulated after 72 h. Interestingly, the pksIII gene involved in the synthesis of phloroglucinol was overexpressed under grazing conditions after 24 h and 72 h. This study supports the hypothesis that phlorotannins are able to provide an inducible chemical defense under grazing activity, which is regulated at different stages of the stress response.

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Comprehensive genomic analysis and expression profiling of the BTB and TAZ (BT) genes in cucumber (Cucumis sativus L.)

Czech Journal of Genetics and Plant Breeding ◽

10.17221/34/2019-cjgpb ◽

2019 ◽

Vol 56 (No. 1) ◽

pp. 15-23 ◽

Cited By ~ 1

Author(s):

Yong Zhou ◽

Guanghua Li ◽

Lin Zhang ◽

Jie Xu ◽

Lifang Hu ◽

...

Keyword(s):

Stress Responses ◽

Expression Patterns ◽

Genomic Analysis ◽

Biological Processes ◽

Quantitative Real Time Pcr ◽

Cucumis Sativus L ◽

Btb Domain ◽

Qrt Pcr ◽

Cucumber Genome ◽

Bt Genes

BTB-TAZ (BT) proteins are plant-specific transcription factors containing a BTB domain and a TAZ domain. They play vital roles in various biological processes and stress responses. In this study, a total of three BT genes (CsBT1–3) were identified from cucumber genome, and they were unevenly distributed in two of the seven chromosomes. Phylogenetic analysis of the BT proteins from cucumber, Arabidopsis, apple, tomato, and rice revealed that these proteins could be distinctly divided into two groups in accordance with their motif distributions. We also determined the structures of BT genes from cucumber, Arabidopsis, and rice to demonstrate their differences. The quantitative real-time PCR (qRT-PCR) results showed that the CsBT genes displayed differential expression patterns in cucumber tissues, and their expression was regulated by cold, salt, and drought stresses. These findings suggest that CsBT genes may participate in cucumber development and responses to various abiotic stresses.

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Determination the Gene Expression Levels of adhesins and Extracellular Enzymes Genes in Candida albicans biofilm producer by Quantitative Real Time PCR Technique (qRT-PCR)

Indian Journal of Forensic Medicine & Toxicology ◽

10.37506/ijfmt.v15i2.14553 ◽

2021 ◽

Keyword(s):

Gene Expression ◽

Candida Albicans ◽

Real Time ◽

Real Time Pcr ◽

Extracellular Enzymes ◽

Quantitative Real Time Pcr ◽

Expression Levels ◽

Qrt Pcr ◽

Gene Expression Levels

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Gene Expression Profiling in Multiple Myeloma Cells Treated with the Novel Anti-Myeloma Agents Zoledronate and Fluvastatin.

Blood ◽

10.1182/blood.v106.11.5078.5078 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5078-5078

Author(s):

Timothy J. Molloy ◽

Baulch-Brown Cindy ◽

Yi-Mo Deng ◽

Andrew Spencer ◽

David F. Ma

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Real Time ◽

Real Time Pcr ◽

Mevalonate Pathway ◽

Annexin V ◽

Quantitative Real Time Pcr ◽

Myeloma Cells ◽

Qrt Pcr

Abstract We have shown in vitro that multiple myeloma (MM) cells can be destroyed by treating them with the mevalonate pathway inhibitors zoledronate and fluvastatin. While the efficacy of these compounds singly and combination have been demonstrated, their exact modes of action remain largely unknown. The present study aimed to use microarray and quantitative real-time PCR (QRT-PCR) techniques to analyse gene expression in treated myeloma cells to identify novel genes and pathways involved in the anti-myeloma action of these compounds. The human MM cell line NCI-H929 was treated with zoledronate and fluvastatin singly and in combination, and RNA was extracted and used to interrogate oligonucleotide microarrays consisting of 19,000 features representing known and unknown genes. Quantitative real-time PCR was subsequently used to confirm the expression of several genes of interest. Flow cytometry with Annexin V FITC staining was used to detect apoptosis. It was observed that genes related to apoptosis (caspases and p53-related genes), cell cycle control (cyclins), GTPase signalling (Rabs), and growth and proliferation (growth factors) were particularly affected by zoledronate and fluvastatin, and some of these genetic effects were synergistic when a combination of zoledronate and fluvastatin was used. QRT-PCR confirmed the effects on the caspase- and p53-related apoptotic pathways, and these effects were correlated with increased apoptosis in the myeloma cells. The mevalonate pathway inhibitors fluvastatin and zoledronate are highly efficient at killing MM cells, and their effects appear to be synergistic. Our microarray and QT-PCR analyses demonstrated that the expression of specific groups of genes important to the survival and proliferation of myeloma cells are affected by these compounds. p53 and caspase-dependent pathways appear to be the key apoptotic cascades stimulated. Insights into the mechanisms of these novel therapeutics are important as they might help to define their roles in the treatment of multiple myeloma.

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Nutritional Homeostasis in Batch and Steady-State Culture of Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e04-04-0306 ◽

2004 ◽

Vol 15 (9) ◽

pp. 4089-4104 ◽

Cited By ~ 116

Author(s):

Alok J. Saldanha ◽

Matthew J. Brauer ◽

David Botstein

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Steady State ◽

Stress Response ◽

Stress Responses ◽

Global Pattern ◽

Batch Cultures ◽

Cycle Arrest ◽

Chemostat Cultures ◽

Limiting Nutrient

We studied the physiological response to limitation by diverse nutrients in batch and steady-state (chemostat) cultures of S. cerevisiae. We found that the global pattern of transcription in steady-state cultures in limiting phosphate or sulfate is essentially identical to that of batch cultures growing in the same medium just before the limiting nutrient is completely exhausted. The massive stress response and complete arrest of the cell cycle that occurs when nutrients are fully exhausted in batch cultures is not observed in the chemostat, indicating that the cells in the chemostat are “poor, not starving.” Similar comparisons using leucine or uracil auxotrophs limited on leucine or uracil again showed patterns of gene expression in steady-state closely resembling those of corresponding batch cultures just before they exhaust the nutrient. Although there is also a strong stress response in the auxotrophic batch cultures, cell cycle arrest, if it occurs at all, is much less uniform. Many of the differences among the patterns of gene expression between the four nutrient limitations are interpretable in light of known involvement of the genes in stress responses or in the regulation or execution of particular metabolic pathways appropriate to the limiting nutrient. We conclude that cells adjust their growth rate to nutrient availability and maintain homeostasis in the same way in batch and steady state conditions; cells in steady-state cultures are in a physiological condition normally encountered in batch cultures.

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Selection and Evaluation of Potential Reference Genes for Quantitative Real-Time PCR in Agaricus blazei Based on Transcriptome Sequencing Data

BioMed Research International ◽

10.1155/2021/6661842 ◽

2021 ◽

Vol 2021 ◽

pp. 1-13

Author(s):

Yuan-Ping Lu ◽

Jian-Hua Liao ◽

Zhong-Jie Guo ◽

Hui-Qing Zheng ◽

Ling-Fang Lu ◽

...

Keyword(s):

Gene Expression ◽

Cytochrome C ◽

Expression Pattern ◽

Real Time Pcr ◽

Reference Genes ◽

Fruit Body ◽

Pcr Analysis ◽

Agaricus Blazei ◽

Quantitative Real Time Pcr ◽

Qrt Pcr

Quantitative real-time PCR (qRT-PCR) is widely used to detect gene expression due to its high sensitivity, high throughput, and convenience. The accurate choice of reference genes is required for normalization of gene expression in qRT-PCR analysis. In order to identify the optimal candidates for gene expression analysis using qRT-PCR in Agaricus blazei, we studied the potential reference genes in this economically important edible fungus. In this study, transcriptome datasets were used as source for identification of candidate reference genes. And 27 potential reference genes including 21 newly stable genes, three classical housekeeping genes, and homologous genes of three ideal reference genes in Volvariella volvacea, were screened based on transcriptome datasets of A. blazei and previous studies. The expression stability of these genes was investigated by qRT-PCR analysis and further evaluated by four software packages, geNorm, NormFinder, BestKeeper, and RefFinder. Among these candidates, α-TUB (Tubulin alpha) and Cox5a (COX5A subunit VA of cytochrome c oxidase) were revealed as the most stable in fruit body, and suitable for 5 different developmental stages. α-TUB and ATP3 (ATP3 gamma subunit of the F1 sector of mitochondrial F1F0 ATP synthase) showed the most stable expression in stipe tissues and, Uqcrc (core subunit of the ubiquinol-cytochrome c reductase complex) and PUP3 (20S proteasome subunit beta 3) performed well in pileus tissues during the process of A. blazei development, while GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was among the least stable genes in all sample sets. Finally, the Ableln3 (homology of eln3 gene of Coprinus cinereus) was adopted to validate the reliability of these stable and unstable reference genes, indicating that the use of unsuitable reference genes as internal controls could change the target gene’s expression pattern. This study can provide guidance for choosing reference genes for analyzing the expression pattern of target genes and facilitate the functional genomic investigation on fruit body formation and development, as well as stipe elongation and pileus expansion in A. blazei.

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Selection of reference genes for quantitative real-time PCR analysis in cucumber (Cucumis sativus L.), pumpkin (Cucurbita moschata Duch.) and cucumber–pumpkin grafted plants

PeerJ ◽

10.7717/peerj.6536 ◽

2019 ◽

Vol 7 ◽

pp. e6536 ◽

Cited By ~ 5

Author(s):

Li Miao ◽

Xing Qin ◽

Lihong Gao ◽

Qing Li ◽

Shuzhen Li ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

Real Time Pcr ◽

Reference Genes ◽

Stable Expression ◽

Quantitative Real Time Pcr ◽

Graft Union ◽

Expression Levels ◽

Qrt Pcr

Background Quantitative real-time PCR (qRT-PCR) is a commonly used high-throughput technique to measure mRNA transcript levels. The accuracy of this evaluation of gene expression depends on the use of optimal reference genes. Cucumber–pumpkin grafted plants, made by grafting a cucumber scion onto pumpkin rootstock, are superior to either parent plant, as grafting conveys many advantages. However, although many reliable reference genes have been identified in both cucumber and pumpkin, none have been obtained for cucumber–pumpkin grafted plants. Methods In this work, 12 candidate reference genes, including eight traditional genes and four novel genes identified from our transcriptome data, were selected to assess their expression stability. Their expression levels in 25 samples, including three cucumber and three pumpkin samples from different organs, and 19 cucumber–pumpkin grafted samples from different organs, conditions, and varieties, were analyzed by qRT-PCR, and the stability of their expression was assessed by the comparative ΔCt method, geNorm, NormFinder, BestKeeper, and RefFinder. Results The results showed that the most suitable reference gene varied dependent on the organs, conditions, and varieties. CACS and 40SRPS8 were the most stable reference genes for all samples in our research. TIP41 and CACS showed the most stable expression in different cucumber organs, TIP41 and PP2A were the optimal reference genes in pumpkin organs, and CACS and 40SRPS8 were the most stable genes in all grafted cucumber samples. However, the optimal reference gene varied under different conditions. CACS and 40SRPS8 were the best combination of genes in different organs of cucumber–pumpkin grafted plants, TUA and RPL36Aa were the most stable in the graft union under cold stress, LEA26 and ARF showed the most stable expression in the graft union during the healing process, and TIP41 and PP2A were the most stable across different varieties of cucumber–pumpkin grafted plants. The use of LEA26, ARF and LEA26+ARF as reference genes were further verified by analyzing the expression levels of csaCYCD3;1, csaRUL, cmoRUL, and cmoPIN in the graft union at different time points after grafting. Discussion This work is the first report of appropriate reference genes in grafted cucumber plants and provides useful information for the study of gene expression and molecular mechanisms in cucumber–pumpkin grafted plants.

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Identification and evaluation of reference genes for quantitative real-time PCR analysis in Polygonum cuspidatum based on transcriptome data

BMC Plant Biology ◽

10.1186/s12870-019-2108-0 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 3

Author(s):

Xiaowei Wang ◽

Zhijun Wu ◽

Wenqi Bao ◽

Hongyan Hu ◽

Mo Chen ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Stress Responses ◽

Real Time Pcr ◽

Abiotic Stresses ◽

Reference Genes ◽

Expression Patterns ◽

Polygonum Cuspidatum ◽

Quantitative Real Time Pcr ◽

Different Tissues

Abstract Background Polygonum cuspidatum of the Polygonaceae family is a traditional medicinal plant with many bioactive compounds that play important roles in human health and stress responses. Research has attempted to identify biosynthesis genes and metabolic pathways in this species, and quantitative real-time PCR (RT-qPCR) has commonly been used to detect gene expression because of its speed, sensitivity, and specificity. However, no P. cuspidatum reference genes have been identified, which hinders gene expression studies. Here, we aimed to identify suitable reference genes for accurate and reliable normalization of P. cuspidatum RT-qPCR data. Results Twelve candidate reference genes, including nine common (ACT, TUA, TUB, GAPDH, EF-1γ, UBQ, UBC, 60SrRNA, and eIF6A) and three novel (SKD1, YLS8, and NDUFA13), were analyzed in different tissues (root, stem, and leaf) without treatment and in leaves under abiotic stresses (salt, ultraviolet [UV], cold, heat, and drought) and hormone stimuli (abscisic acid [ABA], ethylene [ETH], gibberellin [GA3], methyl jasmonate [MeJA], and salicylic acid [SA]). Expression stability in 65 samples was calculated using the △CT method, geNorm, NormFinder, BestKeeper, and RefFinder. Two reference genes (NDUFA13 and EF-1γ) were sufficient to normalize gene expression across all sample sets. They were also the two most stable genes for abiotic stresses and different tissues, whereas NDUFA13 and SKD1 were the top two choices for hormone stimuli. Considering individual experimental sets, GAPDH was the top-ranked gene under ABA, ETH, and GA3 treatments, while 60SrRNA showed good stability under MeJA and cold treatments. ACT, UBC, and TUB were suitable genes for drought, UV, and ABA treatments, respectively. TUA was not suitable because of its considerable variation in expression under different conditions. The expression patterns of PcPAL, PcSTS, and PcMYB4 under UV and SA treatments and in different tissues normalized by stable and unstable reference genes demonstrated the suitability of the optimal reference genes. Conclusions We propose NDUFA13 and EF-1γ as reference genes to normalize P. cuspidatum expression data. To our knowledge, this is the first systematic study of reference genes in P. cuspidatum which could help advance molecular biology research in P. cuspidatum and allied species.

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Relative and Absolute Quantitative Real-Time PCR-Based Quantifications ofhcnCandphlDGene Transcripts in Natural Soil Spiked withPseudomonassp. Strain LBUM300

Applied and Environmental Microbiology ◽

10.1128/aem.01387-10 ◽

2010 ◽

Vol 77 (1) ◽

pp. 41-47 ◽

Cited By ~ 21

Author(s):

Nadine J. DeCoste ◽

Vijay J. Gadkar ◽

Martin Filion

Keyword(s):

Gene Expression ◽

Real Time ◽

Real Time Pcr ◽

Transcriptional Analysis ◽

Relative Quantification ◽

Field Soil ◽

Quantitative Real Time Pcr ◽

Qrt Pcr ◽

The Absolute ◽

Bacterial Concentrations

ABSTRACTTranscriptional analysis of microbial gene expression using relative quantitative real-time PCR (qRT-PCR) has been hampered by various technical problems. One such problem is the unavailability of an exogenous standard robust enough for use in a complex matrix like soil. To circumvent this technical issue, we made use of a recently developed artificial RNA (myIC) as an exogenous “spike-in” control. Nonsterile field soil was inoculated with various concentrations of the test bacteriumPseudomonassp. strain LBUM300, ranging from 4.3- to 8.3-log bacterial cells per gram of soil. Total soil RNA was extracted at days 0, 7, and 14 postinoculation, and using two-step TaqMan assays,phlD(encoding the production of 2,4-diacetylphloroglucinol) andhcnC(encoding the production of hydrogen cyanide) gene expression was monitored. For relative quantification, a defined quantity ofin vitro-synthesized myIC RNA was spiked during the RNA extraction procedure. Absolute qRT-PCR was also performed in parallel. Both the absolute and relative quantifications showed similar transcriptional trends. Overall, the transcriptional activity ofphlDandhcnCchanged over time and with respect to the bacterial concentrations used. Transcripts of thephlDandhcnCgenes were detected for all five bacterial concentrations, but thephlDtranscript copy numbers detected were lower than those detected forhcnC, regardless of the initial bacterial concentration or sampling date. For quantifying a low number of transcripts, the relative method was more reliable than the absolute method. This study demonstrates for the first time the use of a relative quantification approach to quantifying microbial gene transcripts from field soil using an exogenous spike-in control.

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Evaluation and Validation of Reference Genes for Quantitative Real-Time PCR in Helopeltis theivora Waterhouse (Hemiptera: Miridae)

Scientific Reports ◽

10.1038/s41598-019-49479-1 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 5

Author(s):

Zheng Wang ◽

Qianqian Meng ◽

Xi Zhu ◽

Shiwei Sun ◽

Shengfeng Gao ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Real Time Pcr ◽

Reference Genes ◽

Developmental Stages ◽

Transcriptional Level ◽

Quantitative Real Time Pcr ◽

Systematic Analysis ◽

Helopeltis Theivora ◽

Qrt Pcr

Abstract Helopeltis theivora Waterhouse is a predominant sucking pest in many tropic economic crops, such as tea, cocoa and coffee. Quantitative real-time PCR (qRT-PCR) is one of the most powerful tools to analyze the gene expression level and investigate the mechanism of insect physiology at transcriptional level. Gene expression studies utilizing qRT-PCR have been applied to numerous insects so far. However, no universal reference genes could be used for H. theivora. To obtain accurate and reliable normalized data in H. theivora, twelve candidate reference genes were examined under different tissues, developmental stages and sexes by using geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder algorithms, respectively. The results revealed that the ideal reference genes differed across the treatments, and the consensus rankings generated from stability values provided by these programs suggested a combination of two genes for normalization. To be specific, RPS3A and Actin were the best suitable reference genes for tissues, RPL13A and GAPDH were suitable for developmental stages, EF1α and RPL13A were suitable for sexes, and RPL13A and RPS3A were suitable for all samples. This study represents the first systematic analysis of reference genes for qRT-PCR experiments in H. theivora, and the results can provide a credible normalization for qRT-PCR data, facilitating transcript profiling studies of functional genes in this insect.

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