Identification of reference genes for gene expression normalization in safflower (Carthamus tinctorius)

Liu, Fei; Guo, Dan; Tu, Yan; Xue, Ying Ru; Gao, Yue; Guo, Mei

doi:10.1016/j.bjp.2016.05.006

Cited by 13 publications

(7 citation statements)

References 50 publications

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“…The PCR procedure was as follows: holding for 3 min at 95 °C, followed by 40 cycles of denaturation for 10 s at 95 °C, annealing for 20 s at 58 °C, and elongation for 30 s at 72 °C. The specificity of the amplification was checked by analyzing the melting curves and its procedure was 60 °C for 1 min and 95 °C for 30 s. The relative expression levels of genes in at least two replicates of safflower samples were all normalized to Ct60s (KJ634810) as the reference gene and then compared with the expression levels of the control by using the 2 −∆∆Ct method [39].…”

Section: Methodsmentioning

confidence: 99%

CtACO1 Overexpression Resulted in the Alteration of the Flavonoids Profile of Safflower

Yanhua

Gao

et al. 2019

Molecules

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Background: Flavonoids with various structures play a vital role in plant acclimatization to varying environments as well as in plant growth, development, and reproduction. Exogenous applications of ethylene and 1-aminocyclopropane carboxylic acid (ACC), could affect the accumulation of flavonoids. Very few attempts have been made to investigate the effect of 1-aminocyclopropane carboxylic acid oxidase (ACO), a unique enzyme that catalyzes ACC to ethylene, on genes and metabolites in the flavonoid biosynthetic pathway. In this study, two ACOs in safflower (CtACOs) were cloned, and then transgenic safflower with overexpressed CtACO1 was generated through the Agrobacterium-mediated floral dipping method. Results: CtACO1 and CtACO2 were both characterized by the 2-oxoglutarate binding domain RxS and the ferrous iron binding site HxDxnH as ACOs from other plants. However, the transcript levels of CtACO1 in flowers at stages I, II, III, and IV were all higher than those of CtACO2. At the cellular level, by using electroporation transformation, CtACO1 was found to be localized at the cytomembrane in onion epidermal cells. CtACO1 overexpression had varying effects on genes involved in the ethylene and flavonoid biosynthetic pathways. The metabolites analysis showed that CtACO1 overexpression lines had a higher accumulation of quercetin and its glycosylated derivatives (quercetin 3-β-d-glucoside and rutin). In contrast, the accumulation of quinochalcones (hydroxysafflor yellow A and carthamin), kaempferol glycosylated derivatives (kaempferol-3-O-β-rutinoside and kaempferol-3-O-β-d-glucoside), apigenin, and luteolin in CtACO1 overexpression lines were decreased. Conclusion: This study confirmed the feasibility of applying the floral dipping method to safflower and showed a novel regulatory effect of CtACO1 in the flavonoid biosynthetic pathway. It provides hypothetical and practical groundwork for further research on regulating the overall metabolic flux of flavonoids in safflower, particularly hydroxysafflor yellow A and other quinochalcones, by using appropriate genetic engineering strategies.

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Section: Methodsmentioning

confidence: 99%

CtACO1 Overexpression Resulted in the Alteration of the Flavonoids Profile of Safflower

Yanhua

Gao

et al. 2019

Molecules

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“…Fold changes were represented by relative gene expression of target genes (icaA, smaI and esaL) that normalized to reference gene rpoB. The reference gene utilised to normalize the target gene expression was very important to estimate the fold change in gene expression and the accurate results, because is considered a highly conserved expressed RNA subunit and used as constant standard [52]. Calculations of the fold change gene expression of differences between the tested C T and the housekeeping gene are shown in Fig.…”

Section: G E N E E X P R E S S I O N O F B I O F I L M F O R M a T I mentioning

confidence: 99%

Boosting Antimicrobial Activity of Imipenem in Combination with Silver Nanoparticles towards S. fonticola and Pantoea sp.

Hasson¹,

Al-Awady²,

Al-Hamadani³

et al. 2019

Nano BioMed ENG

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Silver nanoparticles have been considered as powerful antimicrobial agents recently, especially with the increasing incidence of diseases associated with biofilm and multi-drug resistant pathogens. The aim of this study was to synthesize silver nanoparticles by biological and chemical methods and combination with imipenem to eradicate biofilm-forming bacteria at phenotypic and genotypic levels. The biosynthesis of silver nanoparticles was done by using Enterobacter cloacae (cell-free suspension) while chemosynthesis was conducted using sodium borohydride. Biological and chemical silver nanoparticles were characterized by ultraviolet-visible spectrophotometry which showed absorbance peak at 400 and 390nm respectively. Fourier transformer infrared analysis revealed that carboxylic and polyphenolic groups were coated on surface of both silver nanoparticles. Scanning electron microscope and size analyser showed that the sizes of biologically and chemically silver nanoparticles were 63 nm and 25 nm, respectively. In addition, it showed the formation of cubical nanoparticles. The antimicrobial effect of synthesized silver nanoparticles were evaluated by agar well diffusion and macrodilution method to determine minimum inhibitory concentration value. The results showed that biological silver nanoparticles were more effective on biofilm forming bacteria (Serratia fonticola and Pantoea sp.) than chemical synthesized ones. In addition, the combination effect between silver nanoparticles and imipenem displayed synergistic effect. Gene expression of biofilm encoding genes (smaI and esaL) were evaluated by real-time quantitative polymerase chain reaction (RT-qPCR) before and after treatment with silver nanoparticles in both types and imipenem and in combination between them. The results revealed that biological silver nanoparticles alone or in combination with antibiotics were more effective on biofilm gene expression by down regulation than other treatments.

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“…Quantitative real-time polymerase chain reaction (qRT-PCR), which is characterized by high sensitivity, strong specificity, a repetitive dynamic quantitative range and high throughput, is one of the most commonly used techniques for gene expression analysis. The accuracy of qRT-PCR results is largely dependent on the selected reference genes [ 1 – 6 ], the validity of which is a prerequisite for the correct application of qRT-PCR to analyze changes in target gene expression [ 7 – 9 ]. To obtain more accurate and reliable results, reference genes are required for standardized measurement of the expression levels of target genes [ 10 ].…”

Section: Introductionmentioning

confidence: 99%

Reference Gene Selection for Quantitative Real-Time PCR of Mycelia fromLentinula edodesunder High-Temperature Stress

Zhao

Yang

Chen

et al. 2018

BioMed Research International

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Housekeeping genes are important for measuring the transcription expression of functional genes; 10 traditional reference genes, TUB, TUA, GADPH, EF1, 18S, GTP, ACT, UBI, UBC, and H2A, were tested for their adequacy in Lentinula edodes (L. edodes). Using specific primers, mRNA levels of these candidate housekeeping genes were evaluated in mycelia of L. edodes, which were treated with high-temperature stress at 37°C for 0, 4, 8, 12, 18, and 24 hours. After treatment, expression stability of candidate genes was evaluated using three statistical software programs: geNorm, NormFinder, and BestKeeper. According to geNorm, TUB had the lowest M values in L. edodes strains 18 and 18N44. Using NormFinder, the best candidate reference gene in strain 18 was TUB (0.030), and the best candidate reference gene in strain 18N44 was UBI (0.047). In BestKeeper analysis, the standard deviation (SD) values of UBC, TUA, H2A, EF1, ACT, 18S, and GTP in strain 18 and those of GADPH and GTP in strain 18N44 were greater than 1; thus, these genes were disqualified as reference genes. Taken together, only UBI and TUB were found to be desirable reference genes by BestKeeper software. Based on the results of three software analyses, TUB was the most stable gene under all conditions and was verified as an appropriate reference gene for quantitative real-time polymerase chain reaction in L. edodes mycelia under high-temperature stress.

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Identification of reference genes for gene expression normalization in safflower (Carthamus tinctorius)

Cited by 13 publications

References 50 publications

CtACO1 Overexpression Resulted in the Alteration of the Flavonoids Profile of Safflower

CtACO1 Overexpression Resulted in the Alteration of the Flavonoids Profile of Safflower

Boosting Antimicrobial Activity of Imipenem in Combination with Silver Nanoparticles towards S. fonticola and Pantoea sp.

Reference Gene Selection for Quantitative Real-Time PCR of Mycelia fromLentinula edodesunder High-Temperature Stress

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