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Identification of Stable Reference Genes for Quantitative Pcr in Jute Under Different Experimental Conditions: an Essential Assessment for Gene Expression Analysis

Ahlan Sabah Ferdous
Molecular Biology lab, Department of Biochemistry and Molecular Biology, University of Dhaka-1000, Bangladesh

Md. Tariqul Islam
Molecular Biology lab, Department of Biochemistry and Molecular Biology, University of Dhaka-1000, Bangladesh

Salma Sultana Alam
Molecular Biology lab, Department of Biochemistry and Molecular Biology, University of Dhaka-1000, Bangladesh

Haseena Khan
Molecular Biology lab, Department of Biochemistry and Molecular Biology, University of Dhaka-1000, Bangladesh

Abstract/ Executive Summary:

A reputation for accuracy and rapidity has made Quantitative reverse transcription PCR (qRT-PCR) a popular method for gene expression analysis. However, the precision of qRT-PCR critically relies on stable control genes for data normalization. To obtain consistent data, validation of internal controls is vital. We examined seven candidate genes of jute (ACT, ELF, TUB, UBC, U6, G3PDH, and 18S) under three abiotic (salinity, dehydration, and low temperature) and one biotic (fungal infection) stress conditions. Their stabilities were evaluated using four distinct algorithms (comparative deltaCT, geNorm, NormFinder, and BestKeeper) all integrated in a web-based tool, RefFinder. Our data suggest that ELF and UBC are most homogenously expressed under all three abiotic stress conditions, whereas TUB is most stable under fungal infestation. Contrasting expression was observed for G3PDH and ELF, the former being unsuitable for all abiotic conditions but was ranked second highest for biotic. The reverse was true for ELF. For validation of our data, expression of a cold responsive gene, CBF-1 was assessed under low temperature using the most and least stable reference genes. Striking differences in CBF-1 expression illustrate the accuracy of the reference genes. This set of genes will be useful for quantitative analyses of genes in jute.

Keywords: Reference gene, qRT-PCR, Jute, Biotic stress, Abiotic stress

Location: Molecular Biology lab, Department of Biochemistry and Molecular Biology, University of Dhaka

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Jute

Objectives:

To identify stable reference genes for quantitative PCR in jute under different experimental conditions.

Methodology:

Plant materials and experimental conditions - Seeds of farmer-popular O-9897 variety of tossa jute (Corchorus olitorius) were collected from Bangladesh Jute Research Institute (BJRI). Seeds were allowed to grow under controlled conditions (maintained in a growth chamber) for 3 days in petri dishes. Seedlings were then subjected to three different abiotic stressors (salinity, low temperature, and dehydration) and an abiotic stressor (fungal infestation) individually. Stressed seedlings were collected after 0, 6, 12, 24, and 48 hrs of stress application. These samples were immediately snap-frozen in liquid nitrogen and then kept at −80 °C for subsequent use. For salinity stress, seedlings were treated with a 300 mM sodium chloride solution on the 4thday of germination and collected at the different time intervals stated previously. For low temperature stress, seedlings were kept at 4 °C and collected at the desired intervals. Dehydration stress was simulated by treating the 4-day old seedlings with 150 mM mannitol solution and collected at specific time intervals of stress induction. For fungal stress, 1% solution of Macrophomina phaseolina was sprayed on the 4-day old seedlings and collected at different time intervals. Two independent biological replicates were collected for each of the samples. Candidate gene confirmation - A set of seven potential reference genes were selected based on literature reviews: ACT, ELF-α, G3PDH, TUB, UBC, U6-snRNA (U6), and 18S. Validation of stability of suitable reference genes was performed by expression profiling of transcription factor CBF-1 under low temperature stress. Because jute genome data are not publicly available, degenerate primers were at first designed from the sequences of gene homologs of other plant species: Gossipium, Vitis venifera, Ricinus, and Glycine max. Primers were designed using PRIMER 3.0 (http://frodo.wi.mit.edu/) and analyzed by the OligoAnalyzer (http://www.idtdna.com/analyzer/ Applications/OligoAnalyz er/) tool of IDT, with the aim of amplifying only the coding region. Primers were tested for expected product size by performing PCR using Mastercycler gradient (Eppendorf, Germany). The presence of a specific DNA band on an agarose gel following gel electrophoresis was the preliminary indication of the presence of these genes in jute. For further confirmation, the bands were extracted from the gel and sequenced. The sequences were then used against NCBI Blastn database with optimization to “somewhat similar sequences”.

Source of Information: AJCS 9(7):646-655 (2015); ISSN:1835-2707

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The set of experiments is the first attempt to validate candidate reference genes in jute for normalization of gene expression analysis using qRT-PCR. In this study, reference genes evaluated can be considered useful for future assessments of gene expression analysis when studying expression under stress conditions in jute. Moreover, this study provided useful guidelines for reference gene selection for researchers working on jute.

Knowledge Management: Journal