Identification of reference genes for expression analysis by real-time quantitative PCR in drought-stressed soybean

Stolf-Moreira, Renata; Lemos, Eliana Gertrudes de Macedo; Abdelnoor, R. V.; Beneventi, Magda Aparecida; Rolla, Amanda Alves Paiva; Pereira, S. dos S.; Oliveira, M. C. N. de; Nepomuceno, Alexandre Lima; Marcelino-Guimarães, Francismar Corrêa

doi:10.1590/s0100-204x2011000100008

Cited by 28 publications

(24 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Here, ELF1B and ACTB, followed by TUA, were ranked as the most stable reference genes according to our GeNorm and NormFinder analyses, whereas the 18S gene was found to be the most unstable. Greater expression stability has already been demonstrated for the plant genes ELF1B (Jian et al, 2008;Hu et al, 2009), ACTB (Stolf-Moreira et al, 2011), and TUA (Jian et al, 2008;de Almeida et al, 2010). Previous plant gene expression studies have classified the 18S gene among the most stable (Stolf-Moreira et al, 2011;Xu et al, 2011), whereas in other studies, it is ranked as the least stable (de Almeida et al, 2010), which is consistent with our findings.…”

Section: Discussionsupporting

confidence: 92%

“…Compared with other studies of expression stability, such as adventitious rooting in Eucalyptus globulus (de Almeida et al, 2010) and Populus (Xu et al, 2011), in the different soybean tissues (Jian et al, 2008;Hu et al, 2009) and drought-stressed soybean roots and leaves (Stolf-Moreira et al, 2011), 18S transcripts were the most abundant (de Almeida et al, 2010;Stolf-Moreira et al, 2011). TUA2 [called TUB by Jian et al (2008)] and ELF1B exhibited the largest (Jian et al, 2008) and smallest (Hu et al, 2009) variations in Ct values, respectively.…”

Section: Discussionmentioning

confidence: 74%

“…The CRGs were chosen based on studies of gene expression under hypoxic conditions, such as the 18S gene analyzed in Arabidopsis (Liu et al, 2005) and soybeans (Komatsu et al, 2010); a number of genes validated as soybean reference genes, including ELF1B, TUA, and TUA2 in different tissues at different developmental stages (Jian et al, 2008); and ACTB in drought-stressed soybean roots and leaves (Stolf-Moreira et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Reference genes for quantitative real-time polymerase chain reaction studies in soybean plants under hypoxic conditions

et al. 2014

View full text Add to dashboard Cite

ABSTRACT. Quantitative real-time polymerase chain reaction (RTqPCR) is a powerful tool used to measure gene expression. However, because of its high sensitivity, the method is strongly influenced by the quality and concentration of the template cDNA and by the amplification efficiency. Relative quantification is an effective strategy for correcting random and systematic errors by using the expression level of reference gene(s) to normalize the expression level of the genes of interest. To identify soybean reference genes for use in studies of flooding stress, we compared 5 candidate reference genes (CRGs) with the NormFinder and GeNorm programs to select the best internal control. The expression Gene expression in flooding-stressed soybeans stability of the CRGs was evaluated in root tissues from soybean plants subjected to hypoxic conditions. Elongation factor 1-beta and actin-11 were identified as the most appropriate genes for RT-qPCR normalization by both the NormFinder and GeNorm analyses. The expression profiles of the genes for alcohol dehydrogenase 1, sucrose synthase 4, and ascorbate peroxidase 2 were analyzed by comparing different normalizing combinations (including no normalization) of the selected reference genes. Here, we have identified potential genes for use as references for RT-qPCR normalization in experiments with soybean roots growing in O 2 -depleted environments, such as flooding-stressed plants.

show abstract

Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 74%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Reference genes for quantitative real-time polymerase chain reaction studies in soybean plants under hypoxic conditions

et al. 2014

View full text Add to dashboard Cite

show abstract

“…The plates were assembled with cDNAs of the samples, AtAREB1∆QT primers (F: 5'-GGAGGTG GAGGGTTGACTAGA-3'; R: 5'-CACTGCTCTGAAACTCATCAAACG-3'), and the Gmβ-actin (accession No. GMU60500) primers for an endogenous control (F: 5'-GAGCTATGAATT GCCTGATGG-3'; R: 5'-CGTTTCATGAATTCCAGTAGC-3') according to Stolf-Moreira et al (2011). Reactions were calibrated with the GM sample A2639.34, which showed the lowest gene expression among all GM lines obtained.…”

Section: Relative Atareb1∆qt Gene Expression Levelmentioning

confidence: 99%

Overexpression of the activated form of the AtAREB1 gene (AtAREB1ΔQT) improves soybean responses to water deficit

Leite¹,

Barbosa²,

Marin³

et al. 2014

Genet. Mol. Res.

View full text Add to dashboard Cite

ABSTRACT. Abscisic acid-responsive element binding protein (AREB1) is a basic domain/leucine zipper transcription factor that binds to the abscisic acid (ABA)-responsive element motif in the promoter 6273 ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 13 (3): 6272-6286 (2014) Water deficit response in GM soybeans with AtAREB1∆QT region of ABA-inducible genes. Because AREB1 is not sufficient to direct the expression of downstream genes under non-stress conditions, an activated form of AREB1 (AREB1∆QT) was created. Several reports claim that plants overexpressing AREB1 or AREB1∆QT show improved drought tolerance. In our studies, soybean plants overexpressing AREB1∆QT were characterized molecularly, and the phenotype and drought response of three lines were accessed under greenhouse conditions. Under conditions of water deficit, the transformed plants presented a higher survival rate (100%) than those of their isoline, cultivar BR 16 (40%). Moreover, the transformed plants displayed better water use efficiency and had a higher number of leaves than their isoline. Because the transgenic plants had higher stomatal conductance than its isoline under well-watered conditions, it was suggested that the enhanced drought response of AREB1∆QT soybean plants might not be associated with altered transpiration rates mediated by ABA-dependent stomatal closure. However, it is possible that the smaller leaf area of the transgenic plants reduced their transpiration and water use, causing delayed stress onset. The difference in the degree of wilting and percentage of survival between the 35S-AREB1∆QT and wildtype plants may also be related to the regulation of genes that protect against dehydration because metabolic impairment of photosynthesis, deduced by an increasing internal CO 2 concentration, was not observed in the transgenic plants.

show abstract

“…The genes and their accession numbers were obtained from the NCBI similarity search. Gmβ-actin and rRNA18S were used as the reference for normalization (Stolf-Moreira et al, 2011b) For each period of time, total RNA was extracted using the Trizol reagent (Invitrogen, Carlsbad, USA) according to manufacturer instructions and synthesis of the complementary DNA (cDNA) was achieved using the Moloney murine leukemia virus (MMLV) (Invitrogen) reverse transcriptase, according to the method described by Schenck et al (2003). The Primer Express v.3.0 program (Applied Biosystems) was used to design the primers for qRT-PCR, being determined on the 3' regions.…”

Section: Qrt-pcr Analysismentioning

confidence: 99%

Transcription factors expressed in soybean roots under drought stress

Pereira¹,

Guimarães²,

Carvalho³

et al. 2011

Genet. Mol. Res.

View full text Add to dashboard Cite

ABSTRACT. To gain insight into stress-responsive gene regulation in soybean plants, we identified consensus sequences that could categorize the transcription factors MYBJ7, BZIP50, C2H2, and NAC2 as members of the gene families myb, bzip, c2h2, and nac, respectively. We also investigated the evolutionary relationship of these transcription factors and analyzed their expression levels under drought stress. The NCBI software was used to find the predicted amino acid sequences of the transcription factors, and the Clustal X software was used to align soybean and other plant species sequences. Phylogenetic trees were built using the Mega 4.1 software by neighbor joining and the degree of confidence test by Bootstrap. Expression level studies were carried out using hydroponic culture; the experiments were designed in completely randomized blocks with three repetitions. The blocks consisted of two genotypes, MG/ BR46 Conquista (drought-tolerant) and BR16 (drought-sensitive) and the treatments consisted of increasingly long dehydration periods (0, 25, 50, 75, and 100 min). The transcription factors presented domains and/or conserved regions that characterized them as belonging to the bzip, c2h2, myb, and nac families. Based on the phylogenetic trees, it was found that the myb, bzip and nac genes are closely related to myb78, bzip48 and nac2 of soybean and that c2h2 is closely related to c2h2 of Brassica napus. Expression of all genes was in general increased under drought stress in both genotypes.Major differences between genotypes were due to the lowering of the expression of the mybj7 and c2h2 genes in the drought-tolerant variety at some times. Over-expression or silencing of some of these genes has the potential to increase stress tolerance.

show abstract

Identification of reference genes for expression analysis by real-time quantitative PCR in drought-stressed soybean

Cited by 28 publications

References 30 publications

Reference genes for quantitative real-time polymerase chain reaction studies in soybean plants under hypoxic conditions

Reference genes for quantitative real-time polymerase chain reaction studies in soybean plants under hypoxic conditions

Overexpression of the activated form of the AtAREB1 gene (AtAREB1ΔQT) improves soybean responses to water deficit

Transcription factors expressed in soybean roots under drought stress

Contact Info

Product

Resources

About