IRANIAN JOURNAL OF BIOTECHNOLOGY
Year:2015 | Volume:13 | Issue:3|Papers Count:9

Background: Selectable marker gene (SMG) systems are critical for generation of transgenic crops. Transgenic crop productionwithout using SMG is not economically feasible. However, SMGs are non-essential once an intact transgenic planthas been established. Elimination of SMGs from transgenic crops both increases public acceptance of GM crops and preparesgene stacking possibility for improvement of complex traits. Synthetic inducible promoters provide an efficient andflexible strategy to regulate transgene expression.Objectives: This study aimed to construct a transformation vector based on Cre/loxP recombination system to enhanceefficiency of SMG-free transgenic plant production followed by post-excision expression of gene of interest in transgenicplants by a pathogen inducible promoter.Materials and Methods: In pG-IPFFDD-creint-gusint construct, cre recombinase and selectable marker gene (nptII) cassetteswere placed between the two loxP recognition sites in direct orientation. Seed-specific Napin promoter was used forregulation of Cre expression in transgenic seeds. In the construct, loxP flanked sequence containing nptII and recombinasecassettes, located between a pathogen inducible promoter containing FFDD cis-acting elements and β-glucuronidase codingregion. The cunstuct was transformed into Nicotiana tabaccum via Agrobacterium-mediated transformation.Results: The results showed that both cre and nptII excision occurs in T1 progeny tobacco plants through seed-specificcre expression. The excisions were confirmed by methods activation of the gus gene, germination test on kanamycin-containingmedium and molecular analysis. Inducibility of gus expression by FFDD-containing promoter in T1 leaf tissueswas confirmed by histochemical Gus staining assay.Conclusions: The established system is not only an efficient tool for marker gene elimination but also provides possibilityfor inducible expression of the transgene.

Background: Xanthomonas citri subsp. citri (Xcc), the causative agent of bacterial citrus canker, has affected citricultureworldwide. Varieties of means have been used to minimize its devastating effects, but no attention has been given tobacteriocins.Objectives: Here and for the first time, we report the isolation and characterization of two novel bacteriocins.Materials and Methods: Secretome containing bacteriocins of isolated bacteria was separated via SDS-PAGE. Each isolatedprotein band was characterized and checked for its efficacy in controlling two pathogenic isolates of Xcc via disk diffusionassay. The effects of varieties of carbon, nitrogen and phosphate sources were evaluated on both bacterial growthand bacteriocin production via Taguchi orthogonal method.Results: The two bacteriocins showed an activity up to 55ºC that were sensitive to proteases suggesting being protein in nature.Analysis of SDS-PAGE purified protein bands of bacterial secretomes with demonstrated potency against Xcc revealed thepresence of peptides with relative molecular masses of 16.9 and 17 kDa for Cronobacter and Enterobacter, respectively.Sequence analysis of peptides revealed an HCP1 family VI secretion system homologue for Cronobacter (YP_001439956) andpilin FimA homologue for Enterobacter (CBK85798.1). A Taguchi orthogonal array was also implemented to determine theeffect of temperature and eight other chemical factors on bacteriocin production for each bacterium.Conclusions: Two peptides with novel antibacterial activities effective against Xcc were isolated, characterized and conditionswere optimized for their higher production.

Background: Watermelon silver mottle virus (WSMoV), which belongs to the genus Tospovirus, causes significant lossin Cucurbitaceae plants.Objectives: Development of a highly sensitive and reliable detection method for WSMoV.Materials and Methods: Recombinant plasmids for targeting the sequence of nucleocapsid protein gene of WSMoV wereconstructed. SYBR Green I real-time PCR was established and evaluated with standard recombinant plasmids and 27watermelon samples showing WSMoV infection symptoms.Results: The recombinant plasmid was used as template for SYBR Green I real-time PCR to generate standard and meltingcurves. Melting curve analysis indicated no primer-dimers and non-specific products in the assay. No cross-reactionwas observed with Capsicum chlorosis virus (genus Tospovirus) and Cucumber mosaic virus (genus Cucumovirus).Repeatability tests indicated that inter-assay variability of the Ct values was 1.6%.Conclusions: A highly sensitive, reliable and rapid detection method of SYBR Green I real-time PCR for timely detectionof WSMoV plants and vector thrips was established, which will facilitate disease forecast and control.

Background: Recently, some new nanobiosensors using different nanoparticles or microarray systems for detection ofmycotoxins have been designed. However, rapid, sensitive and early detection of aflatoxicosis would be very helpful todistinguish high-risk persons.Objectives: We report a highly sensitive competitive immunoassay using magnetic/silica core shell as a signal intensifierfor the determination of aflatoxin B1 using fluorescence resonance energy transfer (FRET) from Cd/Te quantum dots (antiaflatoxinB1 antibody immobilized on the surface of Cd/Te quantum dots) to Rhodamine 123 (Rho 123-labeled aflatoxinB1 bound to albumin). The specific immune-reaction between the anti-aflatoxin B1 antibody on the QDs and the labeledaflatoxinB1 brings the Rho 123 fluorophore (acting as the acceptor) and the QDs (acting as the donor) in close spatialproximity and causes FRET to occur upon photo-excitation of the QDs. Using magnetic/silica core shell to intensify theobtained signal is the novelty of this study.Materials and Methods: Cd/Te QDs were synthesized by the simultaneous reduction of cadmium chloride and telluriumin the presence of sodium borohydride under nitrogen atmosphere. Magnetic nanoparticles were synthesized using FeSO4 and FeCl3 (1: 2 molar ratio) and ammonia as an oxidizing agent under nitrogen atmosphere. The prepared magneticnanoparticles shelled by silica using tetraethoxysilane in the presence of ammonia. Nanoparticles synthesis and monodispersity confirmed by TEM. Immobilization of Cd/Te QDs to antibodies and labeling of aflatoxin B1-albumin by Rho 123 were performed by EDC/NHS reaction in reaction mixture buffer, pH 6, at room temperature.Results: By using the magnetic/silica core shell sensitivity of the system changed from 2×10-11 in our previous study to2×10-12 in this work. The feasibility of the method established by the detection of aflatoxin B1 in spiked human serum.There is a linear relationship between the decreased fluorescence intensity of Rho 123 with increasing concentration ofaflatoxin B1 in spiked samples, over the range of 0.01-0.06 μmol.mL-1.Conclusions: This homogeneous competitive detection scheme is simple, rapid and efficient, and does not require multipleseparation steps and excessive washing.

Background: Collagen, the most abundant protein in the human body, and as an extracellular matrix protein, has an importantrole in the fiber formation. This feature of the collagen renders establishment of the structural skeleton in tissues.Regarding specific features associated with the collagen, such as, formation of the porous structure, permeability andhydrophilicity, it can also be used as a biocompatible matrix in the enzyme engineering.Objectives: The aim of the present study was to investigate the application of the type I collagen as a matrix for alkalinephosphatase immobilization using cross-linking method.Material and Methods: The Alkaline phosphatase was covalently immobilized on collagen matrix by using 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC). The source of the alkaline phosphatase was from the bovineintestinal mucous. After that, the activity of the immobilized enzyme was assayed under different experimental conditions.Results: The optimum pH was similar to that of the free enzyme, whereas the optimum temperature and thermal stabilitywere shown some increments. The surface topography of the collagen matrix containing immobilized enzyme and ALP(Alkaline phosphatase) deficient was investigated by Atomic-force microscopy (AFM). Images that have been obtainedapplying AFM show significant differences between uncovered and immobilized enzyme- matrix surface topography.Conclusions: Our findings suggest that type I collagen can be utilized as a matrix for alkaline phosphatase immobilizationvia cross-linking method.

Background: Lipase is an enzyme with immense application potential. Ester synthesis by lipase catalysis in organic mediais an area of key industrial relevance. Enzymatic preparations with traits that cater to the needs of this function are hencebeing intensely researched.Objective: The objectives of the study were to immobilize the lipase from Bacillus sp. PS35 by cross-linking and adsorptiononto styrene-divinyl benzene (Sty-Dvb) hydrophobic resin and to comparatively characterize the free and immobilizedlipase preparations. The work also aimed to apply the immobilized lipase for catalysing the fatty acid methyl ester (FAME)synthesis from palm oil and optimize the process parameters for maximizing the yield.Materials and Methods: In this study, the purified lipase from Bacillus sp. PS35 was immobilized by adsorption ontostyrene-divinyl benzene hydrophobic resin with gluteraldehyde cross-linking.Results: The immobilized enzyme showed better pH and temperature stabilities than the free lipase. Organic solvent stabilitywas also enhanced, with the relative activity in the presence of methanol being shifted from 53% to 81%, therebyfacilitating the enzyme’s application in fatty acid methyl ester synthesis. It exhibited remarkable storage stability over a30-day period and after 20 repetitive uses. Cross-linking also reduced enzyme leakage by 49%. The immobilized lipasewas then applied for biodiesel production from palm oil. Methanol and oil molar ratio of 5: 1, three step methanol additions, and an incubation temperature of 50oC were established to be the ideal conditions favoring the transesterificationreaction, resulting in 97% methyl ester yield.Conclusions: These promising results offer scope for further investigation and process scale up, permitting the enzyme’scommercial application in a practically feasible and economically agreeable manner.

Background: Gossypium thurberi is a wild diploid species that has been used to improve cultivated allotetraploid cotton.G. thurberi belongs to D genome, which is an important wild bio-source for the cotton breeding and genetic research. Toa certain degree, chloroplast DNA sequence information are a versatile tool for species identification and phylogeneticimplications in plants. Different chloroplast loci have been utilized for evaluating phylogenetic relationships at each classificationlevel among plant species, including at the interspecies and intraspecies levels. Present study was conducted inorder to analyse the sequence of its chloroplast.Objectives: Present study was conducted to study and compare the complete chloroplast sequence of G. thurberi, analysesof its genome structure, gene content and organization, repeat sequence and codon usage and comparison with two cultivatedallotetraploid sequenced cotton species.Materials and Methods: The available sequence was assembled by DNAman (Version 8.1.2.378). Gene annotation wasmainly performed by DOGMA. The map of genome structure and gene distribution were carried out using OGDRAWV1.1. Relative synonymous codon usage (RSCU) of different codons in each gene sample was calculated by codonW inMobyle. To determine the repeat sequence and location, an online version of REPuter was used.Results: The G. thurberi chloroplast (cp) genome is 160264 bp in length with conserved quadripartite structure. Singlecopy region of cp genome is separated by the two inverted regions. The large single copy region is 88, 737 bp, and the smallsingle copy region is 20, 271 bp whereas the inverted repeat is 25, 628 bp each. The plastidic genome has 113 single genesand 20 duplicated genes. The singletones encode 79 proteins, 4 ribosomal RNA genes and 30 transfer RNA genes.Conclusions: Amongst all plastidic genes only 18 genes appeared to have 1-2 introns and when compared with cpDNAof two cultivated allotetraploid, rps18 was the only duplicated gene in G.thurberi. Despite the high level of conservationin cp genome SSRs, these are useful in analysis of genetic diversity due to their greater efficiency as opposed to genomicSSRs. Low GC content is a significant feature of plastidic genomes, which is possibly formed after endosymbiosis byDNA replication and repair.

Background: Hepatitis C virus (HCV) is a main public health problem causing chronic liver infection and subsequentlyliver cirrhosis and lethal hepatocellular carcinoma (HCC). Vaccination based on HCV capsid proteins has attracted a specialinterest for prevention of viral infections. The core protein is a basic and evolutionary most conserved protein, whichregulates the cellular processes related to viral replication and pathogenesis. The envelope E1 and E2 proteins involve ingeneration of the infectious particles, viral entry by binding to a host cell receptor, and modulation of the immune responses.Objectives: In current study, the efficient generation of recombinant core and core-E1E2 proteins was developed in bacterialexpression systems.Materials and Methods: The expression of HCV core and core-E1E2 proteins was performed using prokaryotic pET-28aand pQE-30 expression systems in BL21/ Rosetta, and M15 strains, respectively. The recombinant proteins were purifiedusing affinity chromatography under native conditions and also reverse staining method. Finally, the levels of recombinantproteins were assessed by BCA kit and spectrophotometer.Results: The data showed a clear band of ~573 bp for HCV core and ~2238 bp for core-E1E2 genes in agarose gel.Moreover, a ~21 kDa band of core protein and a ~83 kDa band of core-E1E2 protein were revealed in SDS-PAGE. Theaffinity chromatography could not purify the core and core-E1E2 proteins completely, because of low affinity to Ni-NTAbead in comparison with reverse staining method.Conclusions: This study is the first report for purification of HCV core and core-E1E2 proteins using the reverse stainingprocedure with no need of any chromatography columns. The BL21 strain was more potent than Rosetta strain for HCVcore protein in pET 28a expression system. Furthermore, M15 strain was suitable for expression of coreE1E2 in pQE-30bacterial system.

Background: Weissellicin 110 is the only bacteriocin reported in Weissella cibaria up to now. This bacteriocin representsseveral unique features. This is the first report on the gene sequence that encodes for the bacteriocin.Objectives: Providing a rapid detection method to isolate the weissellicin 110 encoding gene and determination of the bacteriocindistribution were the objectives.Materials and Methods: Bacteriocin from W. cibaria 860106 was purified and analyzed using mass spectrometry for proteinssequencing. The draft genome sequence of W. cibaria 860106 was generated using next generation sequencing. PCRwas applied to detect the weissellicin 110 encoding gene.Results: The molecular weight and partial protein sequence were obtained for the bacteriocin from W. cibaria 860106. Anopen reading frame (ORF) was identified as weissellicin 110 from the draft genome sequence. PCR primers were designedto amplify the weissellicin 110 encoding gene and these primers detected sequences from other 27 BLIS-producing W.cibaria strains previously isolated from either various Taiwanese fermented foods or the respective raw materials.Conclusions: The genetic information of weissellicin 110 was obtained, enabling rapid detection of the weissellicin 110encoding gene. Results suggest that weissellicin 110 producing W. cibaria strains are widely distributed inTaiwanese fermentedfoods.