[Cloning and expression of the binding subunit of shiga-like toxin type 2 gene and immunization study in an animal model]

Objective: Enterohemorrhagic Escherichia coli [EHEC] which produces shiga-like toxin type 2 [Stx2] is a major cause of bloody diarrhea. This pathogen can lead to hemolytic uremic syndrome [HUS] and renal failure with a high mortality rate. Stx2 is the major virulence factor of EHEC. Neutralization of toxin by specific antibodies is known to be the best way to prevent and cure HUS. In this study, we describe the cloning, expression, purification, and immunization of the Stx2B subunit which is responsible for toxin binding to the target cell surface

Methods: The Stx2B gene was amplified by PCR and subcloned into a pET28a expression vector and transformed into E. coli BL21-DE3. We evaluated recombinant protein expression and rSTX2B was purified by the Ni-NTA column. The purified rSTX2B was administered subcutaneously to BALB/c mice in three separate doses as an immunogenic candidate. The raising of anti-rSTX2B antibodies in immunized mice sera was evaluated by Elisa assay. The neutralizing immune response was verified by an in vitro assay on HeLa cells and an in vivo assay on mice by challenging them with a lethal dose of Stx2

Results: The IgG titration verified the induction of a humeral response in immunized mice. The HeLa cell assay indicated that the Stx2 toxin was neutralized by immune mice sera. In the challenge assay, 70% of immunized mice survived

Conclusion: Recombinant rSTX2B can induce a neutralizing immune response in mice. It can be used as a major component in development of EHEC vaccines

Enhancement of shikalkin production in arnebia euchroma callus by a fungal elicitor, rhizoctonia solani

Background: There is a growing demand for mass production of shikalkin [a natural pigment consisted of shikonin and alkannin] due to its increasing applications in cosmetics, pharmaceutical and nutrition industries. The root of Iranian Arnebia euchroma produces shikalkin. The promising capability of this plant for shikalkin production has already been demonstrated in cell culture studies

Objectives: Elicitation effect of Rhizoctonia solani [R. solani] in comparison with the effects of Cu[2+], methyl jasmonate [MJ], and salicylic acid [SA] on the shikalkin production was investigated in A. euchroma callus

Materials and Methods: The calli from different origins [leaf, collar and root] were proliferated on a modified Linsmaier-Skoog [mLS] medium and were subsequently transferred onto the pigment production medium containing various amounts of the desirable elicitor. Observations were quantified and the pigment production was precisely measured spectrophotometrically

Results: Pigment biosynthesis was induced on White medium containing IAA [1 micro M] and kinetin [10 micro M] in dark at 25[degree]C. Use of R. solani increased the pigment production by 7 fold greater than normal White medium. Cu[2+] only doubled the shikalkin production. MJ and SA showed enhancing effects comparable to that of Cu[2+]

Discussions: It is assumed that upon binding of the polysaccharides of the fungal cells to the plant cell surface, a cascade of signaling is initiated that led to expression of genes involving in the biosynthesis of shikalkin

Sugarcane [nco310] transient transformation using uida reporter gene

Sugarcane is a monocotyledonous crop that is cultivated in the tropical and subtropical regions of the world. One of the most important criteria, influencing the efficiency of the sugarcane transformation is known to be related to physical and biological factors during the transformation procedure. The objective of this research was to study the response of callus induction and embryogenic callus production and to identify the major parameters controlling DNA delivery by particle bombardment into sugarcane [Saccharum officinarum L.] cv. NCo310. For callus induction and embryogenic callus production, leaf base segments were subjected to in vitro culture medium supplemented with two plant growth regulators [2,4-D and Dicamba]. Results showed that 1 mg.L -1 2,4-D was significantly influential in callus induction and embryogenic callus production. Considering both physical and biological factors, the efficiency of DNA [uidA gene] delivery was assessed by scoring transient GUS [gene [beta-glucuronidase]] expression in bombarded tissues. The highest transient GUS expression was obtained when callus was bombarded with the construct harboring rice Act1 promoter, and having 9 cm target distance, 25 inHg vacuum pressure, 1 microm gold particles, 12.5 micro g DNA per bombardment and one day pre-culture prior to the bombardment. A bombardment procedure suitable for elite sugarcane varieties was developed, which allowed high-efficiency DNA delivery combined with reduced damage to target tissues.

Expression of genes encoding protein kinases during flower opening in two cut rose cultivars with different longevity

Ethylene plays an important role in wide-ranging aspects of plant growth and development, including fruit ripening, leaf and flower senescence. In this study, the expression patterns of two genes involved in the ethylene signal transduction pathway [RhCTR1 and RhCTR2] were investigated during the flower opening stages in two Rosa hybrida cultivars, 'Black magic' and 'Maroussia', which are characterized by short and long vase lives, respectively. RhCTR1 expression increased significantly during flower opening in both cultivars, but its expression level in cv. Maroussia was significantly higher than that in cv. Black magic. No variation in gene expression was detected for RhCTR2 in both cultivars. Therefore, this study showed that the vase life of the two cultivars correlated with the expression of RhCTR1, but not with that of RhCTR2, the behavior of which is typical of a constitutive gene

Regeneration of glyphosate-tolerant Nicotiana tabacum after plastid transformation with a mutated variant of bacterial aroA gene

Presence of antibiotic resistance markers has always been considered as one of the main safety concerns in transgenic plants and their derived products. Elimination of antibiotic selectable markers from transgenics is a major hurdle for finding efficient and safe candidates. Herbicide tolerance genes might be attractive alternatives. In this study, a variant form of the 5-enoylpyruvyl shikimate-3-phosphate synthase [EPSPS] gene that harbors glycine at position 96 to alanine and alanine 183 to threonine substitutions and confers higher resistance to the broad-spectrum herbicide, glyphosate, was substituted against the spectinomycin resistant gene as a sole selectable marker for plastid transformation of Nicotiana tabacum. Plastid transformation was carried out using the biolistic delivery procedure while delivery parameters such as rupture disk pressure, bombardment distance, etc had been optimized first. A previous study showed that the glyphosate herbicide imposes lethal effects on the structure and integrity of the plastid membrane, even at low concentrations. In order to overcome this problem, a modified procedure for selection of transplastomic cells was used. A long preculture incubation period followed by a gradual increased in glyphosate concentration led to sufficient expression of the transgene. Tolerant calli were thus regenerated through direct selection of transformed plastids in the presence of the glyphosate

Analysis of induced mutants of salinity resistant banana [Musa acuminata cv. Dwarf Cavendish] using morphological and molecular markers

Genetic diversity amongst 21 induced mutant clones tolerant to salinity along with one non-irradiated sensitive clone of banana [Musa acuminata cv. Dwarf Cavendish [AAA]] were studied using morphological and random amplified polymorphic DNA [RAPD] markers. Out of the 30 phenotypic indices screened, 23 were polymorph and two traits, leaf habit and blotches color, were differentiated by non-irradiated clone. RAPDs established 106 major amplified products using 14 primers. Out of 106 markers, eight were monomorph, and the remaining [98] were polymorph. The extent of polymorphism indicated the existence of considerable variation DNA level within induced mutant clones. Primer OPA-02 revealed banding patterns specific to salinity resistant clones. Both morphological and RAPD analyses successfully detected genetic variation within induced mutant clones, RAPD also detected variation between the irradiated and non-irradiated clones, which were morphologically indistinguishable. Results were indicative that induced mutations bear a great potential in improving banana for salinity resistant

Site-directed mutagenesis, expression and biological activity of E. coli 5-enolpyruvylshikimate 3-phosphate synthase gene

Site-directed mutagenesis [SDM] as a powerful technique was used to change two important and conserved amino acids in 5-enolpyruvylshikimate 3- phosphate synthase [EPSPS] gene of E. coli. The mutations changed glycine 96 to alanine and alanine 183 to threonine. These two amino acids are very important for intraction of the wide spectrum herbicide, glyphosate, to EPSP synthase enzymes. By designing mutagen primers and overlapping extension method, three kinds of altered bacterial EPSPS enzymes with first, second and both mutations were produced. These modified enzymes are expected to show decreased affinity for herbicide, with least alteration in their enzymatic activity. These altered genes were cloned under the control of chemically inducible T7 promoter and over expressed in E. coli. Biological activity analyses in the presence of glyphosate show that the bacteria containing the mutated enzymes, especially the enzyme with two mutations, were more tolerant to glyphosate

Transformation of rapeseed [Brassica napus L.] plants with sense and antisense constructs of the fatty acid elongase gene

The biosynthetic pathways of saturated and unsaturated fatty acids consist of many steps controlled by various enzymes. One of the methods for improving oil quality is to change the fatty acid profile through genetic manipulation which requires isolation and characterization of the genes and other cis-acting elements, such as the promoter, involved in fatty acid biosynthesis. pketoacylCoA synthase [KCS] that is the key enzyme in erucic acid biosynthesis. This enzyme is involved in producing eicosanoeic [C20:1] and erucic acids [C22:1] from C18 fatty acids, and is encoded by the fatty acid elongase [FAE] gene. Specific primers were used to amplify the FAE gene and its promoter from genomic DNA by using PCR technique. The putative gene and its promoter were cloned in sense and antisense orientation into the plant expression vector [pBI121]. The sense and antisense constructs of the FAE gene were transformed via Agrobacterium-mediated transformation into low erucic acid rapeseed [LEAR such as PF] and high erucic acid cul-tivars [HEAR such as Maplus]. The transformed plants were screened on kanamycin-containing media and then analysed by PCR and Southern blotting techniques. Moreover, erucic acid content of the first generation of transgenic [T[0]] plants analysed with gas chromatography, showed significant changes in fatty acid composition of transgenic rapeseed plants containing sense and antisense constructs of the FAE gene