Characterisation and validation of housekeeping genes for qRT-PCR expression analysis in Pterophyllum scalare

Author(s):  
Ngairangbam Sushila ◽  
Basanta Kumar Das ◽  
K. Pani Prasad ◽  
Gayatri Tripathi
2012 ◽  
Vol 40 (4) ◽  
pp. 3395-3407 ◽  
Author(s):  
M. Fernández-Aparicio ◽  
K. Huang ◽  
E. K. Wafula ◽  
L. A. Honaas ◽  
N. J. Wickett ◽  
...  

Genes ◽  
2019 ◽  
Vol 10 (9) ◽  
pp. 680 ◽  
Author(s):  
He ◽  
Liang ◽  
Lu ◽  
Wang ◽  
Liu ◽  
...  

Gibberellin (GAs) plays the important role in the regulation of grape developmental and growth processes. The bioinformatics analysis confirmed the differential expression of GA2, GA3, and GA20 gibberellin oxidase genes (VvGA2oxs, VvGA3oxs, and VvGA20oxs) in the grape genome, and laid a theoretical basis for exploring its role in grape. Based on the Arabidopsis GA2oxs, GA3oxs, and GA20oxs genes already reported, the VvGA2oxs, VvGA3oxs, and VvGA20oxs genes in the grape genome were identified using the BLAST software in the grape genome database. Bioinformatics analysis was performed using software such as DNAMAN v.5.0, Clustalx, MapGene2Chrom, MEME, GSDS v.2.0, ExPASy, DNAsp v.5.0, and MEGA v.7.0. Chip expression profiles were generated using grape Affymetrix GeneChip 16K and Grape eFP Browser gene chip data in PLEXdb. The expression of VvGA2oxs, VvGA3oxs, and VvGA20oxs gene families in stress was examined by qRT-PCR (Quantitative real-time-PCR). There are 24 GAoxs genes identified with the grape genome that can be classified into seven subgroups based on a phylogenetic tree, gene structures, and conserved Motifs in our research. The gene family has higher codon preference, while selectivity is negative selection of codon bias and selective stress was analyzed. The expression profiles indicated that the most of VvGAox genes were highly expressed under different time lengths of ABA (Abscisic Acid) treatment, NaCl, PEG and 5 °C. Tissue expression analysis showed that the expression levels of VvGA2oxs and VvGA20oxs in different tissues at different developmental stages of grapes were relatively higher than that of VvGA3oxs. Last but not least, qRT-PCR (Real-time fluorescent quantitative PCR) was used to determine the relative expression of the GAoxs gene family under the treatment of GA3 (gibberellin 3) and uniconazole, which can find that some VvGA2oxs was upregulated under GA3 treatment. Simultaneously, some VvGA3oxs and VvGA20oxs were upregulated under uniconazole treatment. In a nutshell, the GA2ox gene mainly functions to inactivate biologically active GAs, while GA20ox mainly degrades C20 gibberellins, and GA3ox is mainly composed of biologically active GAs. The comprehensive analysis of the three classes of VvGAoxs would provide a basis for understanding the evolution and function of the VvGAox gene family in a grape plant.


2017 ◽  
Vol 64 (4) ◽  
pp. 497-508
Author(s):  
M. Jiang ◽  
S.-Z. Xu ◽  
G.-S. Wen ◽  
C.-L. Zhao
Keyword(s):  

PLoS ONE ◽  
2020 ◽  
Vol 15 (2) ◽  
pp. e0226668 ◽  
Author(s):  
Xu Su ◽  
Liuyang Lu ◽  
Yashe Li ◽  
Congai Zhen ◽  
Guilei Hu ◽  
...  

2009 ◽  
Vol 10 (1) ◽  
Author(s):  
Valeria Valente ◽  
Silvia A Teixeira ◽  
Luciano Neder ◽  
Oswaldo K Okamoto ◽  
Sueli M Oba-Shinjo ◽  
...  

2019 ◽  
Vol 31 (1) ◽  
pp. 212
Author(s):  
Y. N. Cajas ◽  
K. Cañón-Beltrán ◽  
M. E. González ◽  
P. Ramos-Ibeas ◽  
A. Gutierrez-Adán ◽  
...  

One of the problems associated with in vitro production of embryos in bovine is the increase in reactive oxygen species (ROS), which leads to cell alterations and death. Nobiletin is a polymethoxyflavone isolated from citrus fruits with various beneficial effects on cell cycle regulation and inhibition of ROS production. In a preliminary study, we demonstrated that supplementation of 25 or 50 µM nobiletin to the in vitro maturation (IVM) medium reduces oxidative stress and improves oocyte nuclear and cytoplasmic maturation and embryo development. Thus, in this study, we aimed to evaluate the antioxidant activity of nobiletin during IVM on bovine matured oocytes, their cumulus cells (CC), and blastocysts by quantitative changes of gene expression. Immature cumulus oocytes complexes (COC) were aspirated from ovaries of slaughtered heifers. Selected COC underwent IVM in TCM-199+10% FCS and 10ng mL−1 epidermal growth factor (EGF; Control) supplemented with 25 µM (Nob25) or 50 µM (Nob50) nobiletin (MedChemExpress, Monmouth Junction, NJ, USA) or 0.001% dimethyl sulfoxide (DMSO control), a vehicle for nobiletin dilution, in 5% CO2 in air at 38.5°C. After 24h, 50 matured oocytes/group and their CC were snap-frozen in LN2 for gene expression analysis. The remaining oocytes were fertilized (Day 0) and cultured in vitro. Blastocysts (Day 7; n=50/group) were snap-frozen in LN2 for gene expression analysis (5 replicates). The mRNA abundance of candidate genes related with oxidative stress (SOD2, CYP51); apoptosis (BAX); quality (BMP15, BMP7, CLIC1, MAPK1, ABCB1); and cell junction (GJA1) was measured by quantitative PCR; H2AFZ and 18S rRNA were used as housekeeping genes. Statistical significance was assessed by one-way ANOVA. Supplementation of IVM medium with Nob25 or Nob50 produced changes in the expression levels of genes related to oxidative stress and apoptosis during IVM compared with controls. SOD2 and CYP51 were down-regulated in oocytes and CC (P<0.05) but not in blastocysts, whereas BAX was down-regulated only in CC (P<0.05). Nobiletin supplementation in IVM increased the expression of MAPK1 in oocytes and blastocysts (P<0.05); however, no differences were observed in CC. BMP15 for oocytes and their CC and GJA1 for CC were up-regulated in Nob25 and Nob50 groups compared with controls (P<0.05). The relative abundance of CLIC1 decreased in blastocysts from both nobiletin groups compared with controls (P<0.05). No significant differences in the expression in ABCB1 and BMP7 were detected. In conclusion, our results suggest that supplementation of 25 or 50 µM nobiletin to the IVM medium reduces oxidative stress in oocytes and CC, decreases CC apoptosis, and provokes positive changes in the expression of genes related to oocyte and embryo quality. This research was supported by Spanish MINECO (AGL2015-70140-R and AGL2015-66145-R). Y. N. Cajas was supported by a grant from SENESCYT-Ecuador.


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