Using bioinformatic data, we found that maize homologues of the Arabidopsis single subunit RING type ubiquitin ligase genes, COP1 (Constitutive Photomorphogenesis 1) and SINAT5 (Seven in Absentia in Arabidopsis thaliana 5) have been frequently predicted during large-scale cDNA sequencing project and released to databases since 2008. Despite this general information, no tissue specific expression profiles of these genes were published to our knowledge so far. In the present research, the expression and the relative levels of COP1 and SINAT5 transcripts were detected using reverse transcription and polymerase chain reaction (RT-PCR) with template materials collected separately from three different organs (root, leaf and seed) of maize plants grown under the normal growth conditions. The results not only confirmed the presence of COP1 transcript in all three test samples but also revealed its abundance in root tissues that may be consistent to its large number of targets for ubiquitination in darkness. Analysis of SINAT5 expression profile revealed a detectable band in leaf tissue sample that may be related to its specific roles in this organ.
Nowadays, identification of the novel physio-biological and therapeutic functions of plant cysteine proteinase inhibitors “plant cystatins / phytocystatins” are the great of interests for molecular biologists. Whether for biochemical, structural or functional studies, their successful expression along with an easy purification method is required. To date, fusion tags are the best available tools that meet all those requirements. We report here the cloning and simple functional expression and purification of a barley putative cystatin in Escherichia coli cells. For the first time, a part of barley coding sequence containing a predicted active cystatin was amplified by polymerase chain reaction and expressed as maltose binding fusion protein in TB1 strain of E. coli cells using pMALc2X over-expression vector system without affecting the bacterial growth. The expressed product was purified by single step affinity chromatography from the soluble fraction of induced culture at a yield of about 37 mg/ liter of bacterial cell culture. The purified fused protein could efficiently inhibit papain activity in vitro without the cleavage of the fusion partner.