Dataset Information

Identification and validation of superior housekeeping gene(s) for qRT-PCR data normalization in Agave sisalana (a CAM-plant) under abiotic stresses.

ABSTRACT: The adaptive mechanisms in Agave species enable them to survive and exhibit remarkable tolerance to abiotic stresses. Quantitative real-time PCR is a highly reliable approach for validation of targeted differential gene expression. However, stable housekeeping gene(s) is prerequisite for accurate normalization of expression data by qRT-PCR. Till date, no systematic validation study for candidate housekeeping gene identification or evaluation has been carried-out in Agave species. A total of 17 candidate housekeeping genes were identified from the de novo assembled transcriptomic data of A. sisalana and rigorously analyzed for expression stability assessment under drought, heat, cold and NaCl stress. Different statistical algorithms like geNorm, BestKeeper, NormFinder, and RefFinder on expression data determined the superior housekeeping gene(s) for accurate normalization of the gene of interest (GOI). The comprehensive evaluation revealed the ?-Tub 4, WIN-1 and CYC-A as the most stable, while EEF1?, GAPDH, and UBE2 were ranked as the least stable genes in pooled samples. Pairwise combination by geNorm showed that up to two housekeeping genes would be adequate to normalize the GOI expression data precisely. Validation of identified most and least stable housekeeping genes was carried-out by normalizing the expression data of AsHSP20 under abiotic stress conditions. Copy number of AsHSP20 gene supports the reliability of the genes used for normalization. This is the first report on the screening and validation of the housekeeping genes under abiotic stress condition in A. sisalana that would assist to understand the stress tolerance mechanisms by novel gene identification and accurate validation.

SUBMITTER: Sarwar MB

PROVIDER: S-EPMC7078421 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Publications

Identification and validation of superior housekeeping gene(s) for qRT-PCR data normalization in <i>Agave sisalana</i> (a CAM-plant) under abiotic stresses.

Sarwar Muhammad Bilal MB Ahmad Zarnab Z Anicet Batcho Agossa BA Sajid Moon M Rashid Bushra B Hassan Sameera S Ahmed Mukhtar M Husnain Tayyab T

Physiology and molecular biology of plants : an international journal of functional plant biology 20200204 3

The adaptive mechanisms in Agave species enable them to survive and exhibit remarkable tolerance to abiotic stresses. Quantitative real-time PCR is a highly reliable approach for validation of targeted differential gene expression. However, stable housekeeping gene(s) is prerequisite for accurate normalization of expression data by qRT-PCR. Till date, no systematic validation study for candidate housekeeping gene identification or evaluation has been carried-out in Agave species. A total of 17 c ...[more]

PMID: 32205931

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Project description:Best known as licorice, Glycyrrhiza Linn., a genus of herbaceous perennial legume, has been used as a traditional herbal medicine in Asia and a flavoring agent for tobacco and food industry in Europe and America. Abiotic stresses and hormonal treatments can significantly impact the development and metabolism of secondary metabolites in Glycyrrhiza. To better understand the biosynthesis of the trace-amount bioactive compounds, we first screened for the suitable reference genes for quantitative real-time reverse transcription PCR (qRT-PCR) analysis in Glycyrrhiza. The expression profiles of 14 candidate reference genes, including Actin1 (ACT), Clathrin complex AP1 (CAC), Cyclophilin (CYP), Heat-shock protein 40 (DNAJ), Dehydration responsive element binding gene (DREB), Translation elongation factor1 (EF1), Ras related protein (RAN), Translation initiation factor (TIF1), β-Tubulin (TUB), Ubiquitin-conjugating enzyme E2 (UBC2), ATP binding-box transpoter 2 (ABCC2), COP9 signal compex subunit 3 (COPS3), Citrate synthase (CS), and R3H domain protein 2 (R3HDM2) from two congeneric species, Glycyrrhiza uralensis F. and Glycyrrhiza inflata B., were examined under abiotic stresses (osmotic and salinity) and hormonal treatments (Abscisic acid (ABA) and methyl jasmonic acid (MeJA)) using a panel of software, including geNorm, NormFinder, BestKeeper, and Delta CT. The overall stability, however, was provided by RefFinder, a comprehensive ranking system integrating inputs from all four algorithms. In G. uralensis, the most stable reference genes under osmotic stress, salt stress, ABA treatment, and MeJA treatment were TIF1, DNAJ, CS, and ABCC2 for leaves and DNAJ, DREB, CAC, and CAC for roots, respectively. In comparison, the top ranked genes were TUB, CAC, UBC2, and RAN for leaves and TIF1, ABCC2, CAC, and UBC2 for roots, respectively, under stress and hormonal treatments in G. inflata. ACT and TIF1, on the other hand, were the least stable genes under the most experimental conditions in the two congeneric species. Finally, our survey of the reference genes in legume shows that EF, ACT, UBC2, and TUB were the top choices for the abiotic stresses while EF, UBC2, CAC, and ABCC2 were recommended for the hormonal treatments in Leguminosae. Our combined results provide reliable normalizers for accurate gene quantifications in Glycyrrhiza species, which will allow us to exploit its medicinal potential in general and antiviral activities in particular.

Dataset Information

Identification and validation of superior housekeeping gene(s) for qRT-PCR data normalization in Agave sisalana (a CAM-plant) under abiotic stresses.

Publications

Identification and validation of superior housekeeping gene(s) for qRT-PCR data normalization in <i>Agave sisalana</i> (a CAM-plant) under abiotic stresses.

Similar Datasets

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