Validation of a genus-specific gene; TPS, used as internal control inquantitative Real Time PCR of transgenic cotton

Identification of genes with invariant levels of gene expression is a prerequisite for validating transcriptomicchanges accompanying development. Ideally expression of these genes should be independent of themorphogenetic process or environmental condition.We report here the validation of internal control genei.e.TPS (trehalose 6-phosphate-synthase) in cotton (Gossypium spp), using TaqMan system in quantitativeReal Time PCR (qRT-PCR). The Gene expression was tested in five different G. hirsutum cultivars includingCoker 312, Acala SJ, ZETA 2, Taghva, Neishabour and a diploid wild type; G. barbadense. Identicalamplicons were obtained within these cultivars. No amplifications was achieved when DNA samples frombarley (Hordeum vulgare), maize (Zea mays), rice (Oryza sativa), Fig (Ficus carica), pistachio (Pistaciavera), yew (Taxus baccata), wheat (Tirticum aestivum), rose (Rosa hybrida) and soybean (Glycine max) wereused as template. Therefore, it was confirmed that the primers and probes designed in this study were specificfor the identification and quantification of internal control gene. These results reveal the possibility of usingthe TPS gene as an internal control in cotton. In another word, this gene would be a suitable candidate as areference gene in examination of gene expression, detection of transgenic cotton and determining e thezygosity as well as the copy number of the transgene.

Keywords:

Gossypium spp , trehalose 6-phosphate-synthase gene (TPS) , endogenous reference gene , RTPCR , transgene