Identification and Validation of Reference Genes for the Normalization in Real-Time Rt-QPCR on Rice and Red Rice in Competition, Under Different Nitrogen Doses

Benemann, Daiane P.; Nohato, A.M.; Várgas, Leandro; Avila, Luis Antônio de; Agostinetto, Dirceu

doi:10.1590/s0100-83582017350100015

Cited by 4 publications

(2 citation statements)

References 30 publications

(34 reference statements)

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“…The reactions included 5 μl of EvaGreen 2X qPCR MasterMix (ABM, Canada), 0.3 μl (10 μM) of forward primer, 0.3 μl (10 μM) of reverse primer, 3.4 μl of nuclease-free water and 1 μl of cDNA, in a total volume of 10 μl. The qPCR conditions included 10 min at 95°C followed by 40 cycles of 30 s at 95°C, 45 s at 59°C, and 30 s at 77°C, interrupted by the dissociation curve with denaturation at 95°C (5 s), cooling at 70°C (1 min) and gradually heating at 0.11°C steps up to 95°C and cooling at 40°C (30 s), as described by Benemann et al (2017). The reactions were set-up in 96-well microtiter plates (Roche Life Science, Indianapolis, IN), using the cDNA dilution of 1:25, with three technical replicates and no-template controls.…”

Section: Methodsmentioning

confidence: 99%

The South American Fruit Fly: An Important Pest Insect With RNAi-Sensitive Larval Stages

et al. 2019

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RNA interference (RNAi) technology has been used in the development of approaches for pest control. The presence of some essential genes, the so-called “core genes,” in the RNAi machinery is crucial for its efficiency and robust response in gene silencing. Thus, our study was designed to examine whether the RNAi machinery is functional in the South American (SA) fruit fly Anastrepha fraterculus (Diptera: Tephritidae) and whether the sensitivity to the uptake of double-stranded RNA (dsRNA) could generate an RNAi response in this fruit fly species. To prepare a transcriptome database of the SA fruit fly, total RNA was extracted from all the life stages for later cDNA synthesis and Illumina sequencing. After the de novo transcriptome assembly and gene annotation, the transcriptome was screened for RNAi pathway genes, as well as the duplication or loss of genes and novel target genes to dsRNA delivery bioassays. The dsRNA delivery assay by soaking was performed in larvae to evaluate the gene-silencing of V-ATPase , and the upregulation of Dicer-2 and Argonaute-2 after dsRNA delivery was analyzed to verify the activation of siRNAi machinery. We tested the stability of dsRNA using dsGFP with an in vitro incubation of larvae body fluid (hemolymph). We identified 55 genes related to the RNAi machinery with duplication and loss for some genes and selected 143 different target genes related to biological processes involved in post-embryonic growth/development and reproduction of A. fraterculus . Larvae soaked in dsRNA (dsV-ATPase) solution showed a strong knockdown of V-ATPase after 48 h, and the expression of Dicer-2 and Argonaute-2 responded with an increase upon the exposure to dsRNA. Our data demonstrated the existence of a functional RNAi machinery in the SA fruit fly, and we present an easy and robust physiological bioassay with the larval stages that can further be used for screening of target genes at in vivo organisms’ level for RNAi-based control of fruit fly pests. This is the first study that provides evidence of a functional siRNA machinery in the SA fruit fly.

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Section: Methodsmentioning

confidence: 99%

The South American Fruit Fly: An Important Pest Insect With RNAi-Sensitive Larval Stages

et al. 2019

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“…The RT-qPCR used 10 μl of the cDNA sample (diluted at a ratio The relative abundances of PCR products were normalised using the average of two endogenous standard genes, 18S and Ubiquitin-10 rRNA, previously described in studies of relative expression in cultivated rice (Benemann et al, 2017). Means values of Ct (Cycle threshold), standard deviations and confidence intervals per treatment were calculated, and relative expression was defined using the ΔΔCt method of Dussault and Pouliot (2006), where ∆∆Ct is the relative expression of the gene, the application of the result in 2 −(∆∆Ct) gives the dimension of variation, and vertical bars indicate the confidence interval (α = 0.05).…”

Section: Real-time Rt-pcrmentioning

confidence: 99%

Genes related to flooding tolerance during germination and early growth of weedy rice

et al. 2020

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Evolution of flooding tolerance in weedy rice has occurred in several rice‐growing regions, but the genes related to this process and the environmental effects are unknown. The objective of this study was to analyse the expression of genes related to flooding tolerance in response to temperature and flooding during the initial establishment of weedy rice. The experiments were carried out with rice cultivars IRGA 417 and Nipponbare, which are sensitive to flooding, and weedy rice ITJ03 and AV04 genotypes that have high and intermediate tolerance to flooding, respectively. The expression of genes related to reserve mobilisation, anaerobic respiration, escape and quiescence strategies was analysed at periods up to 24 days after sowing. The flooding tolerance of weedy rice genotype ITJ03 was associated with the expression of RAmy3D and OsTPP7 , which are involved in the mobilisation of carbohydrate reserves, ADH1 and ADH2, which participate in anaerobic respiration, and SNRKL1 that triggers rapid elongation of the coleoptile and emergence. Although the genes PDC1, SUS3 and SUB1 are important for flooding tolerance in cultivated rice, their expression was not directly related to flooding tolerance in weedy rice. A temperature of 20°C reduced levels of expression of the RAmy3D, ADH2 and SNRKL1 genes and low temperature had a negative effect on the establishment of weedy rice. Breeding of rice genotypes with tolerance of low temperatures and anaerobic conditions may be a viable strategy to improve the control of weedy rice in paddy fields.

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Validation of suitable reference genes for normalization of quantitative reverse transcriptase- polymerase chain reaction in rice infected by Xanthomonas oryzae pv. oryzae

et al. 2020

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Identification and Validation of Reference Genes for the Normalization in Real-Time Rt-QPCR on Rice and Red Rice in Competition, Under Different Nitrogen Doses

Cited by 4 publications

References 30 publications

The South American Fruit Fly: An Important Pest Insect With RNAi-Sensitive Larval Stages

The South American Fruit Fly: An Important Pest Insect With RNAi-Sensitive Larval Stages

Genes related to flooding tolerance during germination and early growth of weedy rice

Validation of suitable reference genes for normalization of quantitative reverse transcriptase- polymerase chain reaction in rice infected by Xanthomonas oryzae pv. oryzae

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