Browsing by Author "Bandara, K.G.W.W."

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    Analysis of OsBBX13 Gene Responses to Salinity Stress Tolerance in Oryza sativa (Rice
    (Uva Wellassa University of Sri Lanka, 2020) Bandara, K.G.W.W.; Wijesundera, W.S.S.; Hettiarachchi, G.H.C.M.
    The B-box (BBX) proteins are a subgroup of zinc finger transcription factors that contain one or two B-box domains. The B-box domain is a conserved motif. Genes that encode for BBX proteins are highly conserved across all multicellular species. BBX proteins were first identified in Arabidopsis thaliana that play a significant role in light and abiotic stress signalling. In Oryza sativa (rice) only a few BBX genes have been identified which are involved in flowering. Our previous study on ortholog search identified OsBBX13 as a functional ortholog of the Arabidopsis AtBBX21 gene that is involved in light and abiotic stress regulation. This study aimed to conduct an in silico analysis of the OsBBX13 gene promoter to identify the presence of abiotic stress-responsive elements and to study the expression of the OsBBX13 gene under salinity stress. Promoter sequence (1.0 kb upstream of translation start site) of the OsBBX13 gene was retrieved from the Rice Annotation Project Database (RAP-DB). The tools of PlantCARE and New PLACE were used for scanning of abiotic stress-responsive cis-elements present on the promoter region of OsBBX13. The salinity stress-responsive elements, MYBCORE and GT1GMSCAM4, and Abscisic acid-responsive element (ABRE) which regulates the dehydration and salinity responses were identified in OsBBX13 promoter region. Quantitative Real Time PCR analysis of 7 days old rice seedlings exposed to 200 mM NaCl stress showed significant up-regulation of the OsBBX13 gene compared to the control. All these findings together suggested that the OsBBX13 gene is involved in salinity stress responses in rice. Keywords: BBX proteins, OsBBX13, Salinity stress, Abiotic stress
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    Expression of a Rabies Virus Specific Antigen by Cloning the Glycoprotein Gene into Escherichia con Expression System
    (Uva Wellassa University of Sri Lanka, 2016) Sewwandi, H.S.; Rodrigo, W.W.P.; Athapaththu, A.M.M.H.; Gunathilaka, P.A.D.H.N.; Bandara, K.G.W.W.; Wijesundara, R.R.M.K.K.; Bulumulla, P.B.A.I.K.
    Rabies is an infectious disease characterized by dysfunction of the central nervous system caused by Lyssavirus of family Rhabdoviridae. Detection of rabies antibodies are used to confirm if people have been successfully immunized. Currently, these detection methods require lots of expertise and are generally carried out in reference laboratories at a high cost. Therefore, it is vital to develop and standardize simple techniques such as Enzyme Linked Immunosorbent Assay (ELISA) for determining the level of antibodies against rabies virus at a lower cost. Hence, the aim of the present study was to clone rabies virus specific glycoprotein gene into bacterial expression vector for the production of recombinant protein. Initial attempts were made to isolate plasmid DNA of pET-28a (+) vector and pcDNA3. -RVG recombinant plasmid containing previously cloned Rabies Virus Glycoprotein gene (RVG). Both plasmids were successfully digested with BamHI and XhoI restriction enzymes. The purified Rabies Virus Glycoprotein gene was cloned into pET-28a (+) bacterial expression vector. The pET-28a (+)-RVG plasmids were successfully transformed into TOPIOP competent cells through electroporation. Transformants were screened by rapid screening method. Out of 20 colonies 8 were identified as recombinants. Further screening of recombinant colonies will be carried out by digesting with restriction enzymes. Putative correct recombinant construct will be transferred into bacterial expression system for the expression. Keywords: Rabies, Rabies virus glycoprotein gene, Cloning
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    Identification of Horse Meat and Beef using a Polymerase Chain Reaction Based Method with Cytochrome b Gene
    (Uva Wellassa University of Sri Lanka, 2013) Munasinghe, M. M. E.; Bulumulla, P. B. A. I. K.; Fernando, T. S. R.; Samaraweera, A. M.; Nanayakkara, A. K.; De Silva, D.P.D.C.; Bandara, K.G.W.W.; Senaratne, S.G.
    Adulteration of meat and meat products are taking place in many parts of the world and it is believed that numerous such incidents have even occurred in Sri Lanka as well. Demanding meat and meat products are being adulterated with cheaper or unconventional meat types (eg. dog, horse or rat meat) with phenotypic similarities. This situation created a scenario where the food analyst from many developing countries needs to pay special attention to identify meat. A recent disclosure in UK about an adulteration of beef with equine meat created a paranoid situation, which drastically affected genuine brands produced by large companies and international food chains (Thomson, 2013). Hence, the adulteration must be prevented to ensure traceability of meat from farm to form. In general, molecular methods facilitate accurate and more reliable analysis of meat adulteration. Compared to nuclear DNA, application of mitochondrial DNA (mtDNA) in meat identification experiments provides many advantages, such as maternal inheritance and ubiquitous natures (Kvist, 2000). Thus, mtDNA can be used when the evidentiary material supply is limited. Among the Mitochondrial genes mitochondrial cytochrome b (Cyt b) gene has often used to detect the source of meat (Farias, et al., 2001). Hence, the aim of this study is to establish qualitative polymerase chain reaction method to detect horse meat adulteration in beef using mitochondrial DNA Cyt b region. Methodology Beef and horse (Equus ferus caballus) meat samples were collected from slaughter house at Dematagoda and from Faculty of Veterinary Medicine and Animal Science, at University of Peradeniya, respectively. Genomic DNA was extracted following the protocol as described in Abdel-Rahman et al. (2009) with modifications. First, 600 mg of tissue was homogenized in 6000 μL STE buffer and 48 μL of 10% SDS and 24 μL of proteinase K (10 mg/mL) was added. Then, the mixture was incubated at 37 °C overnight. After that, DNA was purified by equal and chloroform–isoamylalcohol (24:1), successively. Then, DNA was precipitated by adding chilled ethanol in the presence of 3 M sodium acetate. The resulting pellet was washed with 70% ethanol, air-dried and subsequently dissolved in 80 μL of TE buffer. Species specific primers were designed (horse forward (HF) - ATC ATC ACA GCC CTG GTA GTC GTA CAT, horse reverse (HR) - ATG TGG AGG GTG GGG ATG AGT GCT A, cattle forward (CF) - CAT CGG CAC AAA TTT AGT CG and cattle reverse (CR) - GAG CTA GAA TTA GTA AGA GGG CC) to amplify mitochondrial Cyt b gene of cattle and horse. The PCR products were electrophoresed on 2% agarose gel containing 0.5 µg/mL Ethidium bromide and were visualized and imaged using a UV trans-illuminator (Gel Dox XP+ system, BioRad) and gel documentation system (Image Lab 3.0, BioRad) to distinguish the species origin. Furthermore, to investigate the detection limit of the PCR system, DNA was extracted from 600 mg of beef which was mixed separately with 1%, 5% and 20% of horse meat. Results and Discussion MtDNA evolve at a much faster rate than nuclear DNA. At the same time different regions of the mitochondrial genome would evolve at different rates. Therefore, mtDNA maintain variable regions but with a certain level of conservation. Similarly, mitochondrial Cyt b gene contains both slow and rapid evolving regions with conservative and variable regions. The evolution of the Cyt b gene is prevented due to the functional restrictions in the conservative regions (Farias, et al., 2001). Therefore, Cyt b gene is used to identify horse meat from beef as the sequence variability in Cyt b gene between different species is extremely high.