Gene expression analysis for selection and validation of suitable housekeeping gene(s) in cadmium exposed pigeonpea plants inoculated with arbuscular mycorrhizae

Abstract

The expression stability of six commonly used housekeeping genes (18S rRNA-18S ribosomal RNA, EF1α-elongation factor 1α, ACT1-Actin 1, GAPDH-Glyceraldehyde-3-phosphate dehydrogenase, TUB6-Tubulin/FtsZ family and UBC-Ubiquitin-conjugating enzyme) were scrutinized in leaves and roots of Cd stressed pigeonpea plants inoculated with arbuscular mycorrhizal (AM) species- Rhizoglomus intraradices (Ri), Funneliformis mosseae (Fm), Claroideoglomus etunicatum (Ce), C. claroideum (Cc). The stability profile of each gene was assessed using ΔCt, BestKeeper, NormFinder, RefFinder and geNorm algorithmic programs, which ranked different genes as most and least stable according to the tissues analysed. All the statistical algorithms ranked TUB6 as most stable and EF1α least stable housekeeping (HK) genes in both the plant tissues. The selected HK genes were verified using metallothionein (CcMT1) i.e. a stress responsive gene, whose expression altered under conditions of metal stress and AM inoculation. The expression pattern of CcMT1 varied highly when least stable reference gene was used for normalization as compared to most stable gene, under different treatments. Thus, there is a need of selecting suitable reference gene to achieve reliable results in gene expression studies using quantitative real time PCR (qRT-PCR). The study conducted will help future gene expression analysis in pigeonpea under specific stress.