Screening of new isolates of Bacillus thuringiensis (Bt) and cloning of the cry genes

Nine new indigenous isolates of Bacillus thuringiensis (Bt) were characterized for their colony type, crystal inclusion and toxicity analysis with Helicoverpa armigera Hubner and Spodoptera litura Linn. Genomic deoxyribonucleic acid (DNA) isolated from all the new isolates were subjected to screening for cry1, cry2, cry4, cry10 and cry11 genes and predicted possible potential DNA amplicons were cloned and sequenced. Partial cry1 gene fragment (~1.5 kb) amplified by degenerate primers and about 450 bp DNA fragment amplified by cry10 gene specific primers from two isolates T109 and T136 were cloned in to T/A cloning vector. DNA sequencing of about 1.5 kb amplicon showed 99% homology to the holotype sequence of cry1Ac1. Nucleotide sequence of about 450 bp fragments of isolate T109 and T136 showed homology to a hypothetical protein and serine/threonine phosphatase respectively.Keywords: Bacillus thuringiensis (Bt), cloning, cry genes, polymerase chain reaction (PCR), toxicity analysi

Cloning of partial cry1Ac gene from an indigenous isolate of Bacillus thuringiensis

The discoveries of novel cry genes of Bacillus thuringiensis (Bt) with higher toxicity are important for the development of new products. The cry1 family genes are more toxic to the lepidopteran insects ­­­­­­according to the previous reports. In the present study, nine indigenous isolates of Bt were used for screening of cry1 genes by PCR using degenerate primers specific to cry1 family genes. Two of the nine new isolates of Bt showed amplification with cry1 family primers. Partial cry1gene(s) was cloned from one of the cry1 positive isolate of Bt, T133. Nucleotide sequence data generated for partial cry1 gene of T133 showed 98% homology with 1420 bp of the partial cry1Ac1 gene. Deduced amino acid sequence of the partial cry1Ac of Bt strain, T133 showed two per cent variation in comparison to Cry1Ac1 by differing at 5 positions; one deletion at 442th position and five substitutions at the following positions, 296, 367, 440 and 563.Keywords: Bacillus thuringiensis, cry1 genes, degenerate primer, cloningAfrican Journal of Biotechnology, Vol 13(18), 1845-184

Employees demographic factors and its relationship with work motivation and performance of employees in sugar industry

The Securities and Exchange Board of India (SEBI) laid down several regulatory measures to protect the investors of capital market. Effective investor protection is not simply a function of �law on the books�, but it depends heavily on �law in action� which depends on issues like innovative supervisory strategies, product design initiatives, capital market research, investors redress and investors education. This study is considered to be interesting for two reasons, firstly, it covers the recent trends in grievance redressal rate and secondly, it examines the fluctuations in pending grievance redressal rate. The present research is based on purely secondary data available at SEBI web sites and used analytical tools like simple percentage analysis and hypothesis testing. The paper pleads for implementation of effective investor grievance redressal mechanism

Identification of acid-stress-tolerant proteins from promising cyanobacterial isolates

Cyanobacterial cultures were isolated from acidic (pH 4.9-6.2) rice grown soils in Tamil Nadu, India. The predominant genera were Anabaena (50%), Westiellopsis (17.5%), Nostoc (15%), Oscillatoria (5%) and others that were unicellulars (12.5%) viz., Microcystis, Calothrix and Phormidium. The levels of tolerance to acidity varied among these strains, which were tested and authenticated for their acid tolerance capacity under both in vitro and pot culture conditions. Westiellopsis sp. was found to predominate from pH 4.9 to pH 6.2, indicating its adaptability. Cultures tolerant to acidic conditions were characterized for growth, biomass production and biochemical constituents. Under acidic conditions, Westiellopsis sp. showed pronounced chlorophyll a content, phycobilin pigment content, ammonia excretion and nitrogenase activity compared to normal conditions. Molecular characterization, particularly isozyme and random amplified polymorphic DNA (RAPD) analysis, were also carried out. Three strains of Westielliopsis sp. strains were selected, of which two were able to grow at an acidity level of pH 4.0, while one strain was able to sustain growth at an acidity level of 5.0. These three cultures, along with acid susceptible strains of Westielliopsis sp. and Anabaena sp. PCC 7120 (standard check) were subjected to acid shock for different time intervals. Protein profiling of both the acid-tolerant and acid-susceptible strains was carried out with samples collected at different time intervals. Based on the presence/absence of protein bands in the tolerant/susceptible strains, some low- and medium-molecular weight proteins can be linked to conferring acid tolerance

Characterization of parasporin gene harboring Indian isolates of Bacillus thuringiensis

Bacillus thuringiensis (Bt) is popularly known as insecticidal bacterium. However, non-insecticidal Bt strains are more extensively available in natural environment than the insecticidal ones. Parasporin (PS) is a collection of genealogically heterogeneous Cry proteins synthesized in non-insecticidal isolates of Bt. An important character generally related with PS proteins is their strong cytocidal activity preferentially on human cancer cells of various origins. Identification and characterization of novel parasporin protein which are non-hemolytic and noninsecticidal but having selective anticancer activity raise the possibility of a novel application of Bt in medical field. In the present study, seven new indigenous isolates (T6, T37, T68, T98, T165, T186, and T461) of Bt showed variation in colony morphology, crystal characters and protein profiles with each other. Out of the seven new isolates screened for parasporin (ps) and cry genes, two of the new indigenous isolates (T98 and T186) of Bt showed the presence of ps4 gene. Partial ps4 gene was cloned from the two new isolates and the sequence of partial ps4 gene showed high homology with its holotype ps4Aa1. These two isolates were characterized based on the proteolytic processing of the inclusion proteins and the proteolytic products were found to be comparable to the PS4 reference strain A1470. The two isolates of Bt did not show toxicity toward Spodoptera litura and Helicoverpa armigera. Based on the results of this study, it can be concluded that the isolates T98 and T186 are parasporin producers

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Not AvailableTomato (Solanum lycopersicum L.,) is an important vegetable crop extensively damaged by lepidopteran insect pest, Helicoverpa armigera Hubner (tomato fruit borer). In order to mitigate this problem, an attempt was made to generate transgenic tomato plants resistant to fruit borer. In this study, a regeneration protocol was standardized for a local high yielding genotype, cv. PKM1. Out of nine different combinations of media tested, modified MS medium containing B5 vitamins along with zeatin exhibited higher regeneration efficiency. Cotyledonary explants of cv. PKM1 were transformed with an indigenous synthetic cry2AX1 gene encoding insecticidal crystal protein of Bacillus thuringiensis through Agrobacterium mediated transformation. Screening by PCR revealed presence of cry2AX1 gene in five out of nine putative transformants; two of the five PCR positive transformants showed expression of Cry2AX1 protein in qualitative ELISA. The two ELISA positive plants showed low level of mortality in H. armigera and significant reduction in feeding of leaf area, in detached leaf bioassay. Selection of cry2AX1 transgenic tomato plants by quantitative ELISA for high level of gene expression will be useful for getting plants with desirable level of insect protection.Not Availabl

Expression and inheritance of chimeric cry2AX1 gene in transgenic cotton plants generated through somatic embryogenesis

Transgenic cotton plants expressing a novel cry2AX1 gene consisting of sequences from the cry2Aa and cry2Ac genes, driven by the CaMV35S or the EnCaMV35S promoters, were generated through Agrobacterium-mediated transformation. Among 40 independent PCR-positive events, 14 produced Cry2AX1 protein at detectable levels. Cry2AX1 levels in T-0 plants ranged from 0.012 to 1.068 mu g/g fresh leaf tissue. Insect bioassays on Cry2AX1-expressing T-0 transgenic lines showed 27.0 to 88.8% mortality of Helicoverpa armigera. Southern hybridization analysis revealed single locus integration of the cry2AX1 gene in two T-0 plants which caused 87.5 and 88.8% mortality in H. armigera. The presence and expression of the cry2AX1 gene were confirmed in T-1 progeny using PCR and quantitative ELISA. The highest expression level of Cry2AX1 protein recorded in the T-1 generation was 0.766 mu g/g fresh leaf tissue, far less than the expression of Cry2Ab protein (23 mu g/g leaf tissue) reported in the commercialized Bt cotton, Bollgard II. In spite of the low Cry2AX1 level, the mortality against H. armigera ranged between 33.3 and 85.7% in the T-1 generation. These results indicate that this novel cry2AX1 gene produced a potent insecticidal protein that was effective against H. armigera in transgenic cotton plants at very low concentrations