Search results for: transgenic%20tobacco.

Authors: Sadegh Lotfieblisofla, Arash Khodabakhshi

Abstract:

Tissue plasminogen activator (tPA) as a serine protease plays an important role in the fibrinolytic system and the dissolution of fibrin clots in human body. The production of this drug in plants such as tobacco could reduce its production costs. In this study, expression of tPA gene and protein targeting to different plant cell compartments, using various signal peptides has been investigated. For high level of expression, Kozak sequence was used after CaMV35S in the beginning of the gene. In order to design the final construction, Extensin, KDEL (amino acid sequence including Lys-Asp-Glu-Leu) and SP (γ-zein signal peptide coding sequence) were used as leader signals to conduct this protein into apoplast, endoplasmic reticulum and cytosol spaces, respectively. Cloned human tPA gene under the CaMV (Cauliflower mosaic virus) 35S promoter and NOS (Nopaline Synthase) terminator into pBI121 plasmid was transferred into tobacco explants by Agrobacterium tumefaciens strain LBA₄₄₀₄. The presence and copy number of genes in transgenic tobacco was proved by Southern blotting. Enzymatic activity of the rt-PA protein in transgenic plants compared to non-transgenic plants was confirmed by Zymography assay. The presence and amount of rt-PA recombinant protein in plants was estimated by ELISA analysis on crude protein extract of transgenic tobacco using a specific antibody. The yield of recombinant tPA in transgenic tobacco for SP, KDEL, Extensin signals were counted 0.50, 0.68, 0.69 microgram per milligram of total soluble proteins.

Keywords: Recombinant tissue plasminogen activator, plant cell comportment, leader signals, transgenic tobacco.

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Authors: Ahmad Ali Shahid, Muhammad Shakil Shaukat, Kamran Shehzad Bajwa, Abdul Qayyum Rao, Tayyab Husnain

Abstract:

Agriculture is the backbone of economy of Pakistan and cotton is the major agricultural export and supreme source of raw fiber for our textile industry. To combat severe problems of insect and weed, combination of three genes namely Cry1Ac, Cry2A and EPSPS genes was transferred in locally cultivated cotton variety MNH-786 with the use of Agrobacterium mediated genetic transformation. The present study focused on the molecular screening of transgenic cotton plants at T3 generation in order to confirm integration and expression of all three genes (Cry1Ac, Cry2A and EPSP synthase) into the cotton genome. Initially, glyphosate spray assay was used for screening of transgenic cotton plants containing EPSP synthase gene at T3 generation. Transgenic cotton plants which were healthy and showed no damage on leaves were selected after 07 days of spray. For molecular analysis of transgenic cotton plants in the laboratory, the genomic DNA of these transgenic cotton plants were isolated and subjected to amplification of the three genes. Thus, seventeen out of twenty (Cry1Ac gene), ten out of twenty (Cry2A gene) and all twenty (EPSP synthase gene) were produced positive amplification. On the base of PCR amplification, ten transgenic plant samples were subjected to protein expression analysis through ELISA. The results showed that eight out of ten plants were actively expressing the three transgenes. Real-time PCR was also done to quantify the mRNA expression levels of Cry1Ac and EPSP synthase gene. Finally, eight plants were confirmed for the presence and active expression of all three genes at T3 generation.

Keywords: Agriculture, Cotton, Transformation, Cry Genes, ELISA and PCR.

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Authors: Zilya R. Vershinina, Andrei K. Baymiev, Aleksei K. Baymiev, Aleksei V. Chemeris

Abstract:

This paper reports optimization of characteristics of bioballistic transformation of spring soft wheat (Triticum aestivum L. cultivar Raduga) and getting of transgenic plants, carrying pea lectin gene. This gene will let to create new associative wheat symbiosis with nodule bacteria of field pea, which has growth encouraging, fungistatic and other useful characteristics.

Keywords: transgenic wheat, pea lectin, rhizobia root colonization, symbiosis

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Authors: R. Weinzierl, R. Estes, N. Tinsley, M. Keshlaf

Abstract:

The corn earworm, Helicoverpa zea Boddie, is a serious pest of corn. Larval feeding in ear tips destroys kernels and allows growth of fungi and production of mycotoxins. Infested sweet corn is not marketable. Development of improved transgenic hybrids expressing insecticidal toxins from Bacillus thuringiensis (Bt) may limit or prevent crop losses. The effectiveness of Attribute^® II Bt resistance and applications of Voliam Xpress insecticide were evaluated for effectiveness in controlling corn earworm in plots near Urbana, IL, USA, in 2013. Where no insecticides were applied, ear infestations and kernel damage in Attribute^® II ‘Protector’ plots were consistently lower (near zero) than in plots of the non-Bt isoline ‘Garrison.’ Multiple applications of Voliam Xpress significantly reduced the number of corn earworm larvae and kernel damage in the Garrison plots, but infestations and damage in these plots were greater than in Protectorplots that did not receive insecticide applications. Our results indicate that Attribute^® II Bt resistance is more effective than multiple applications of an insecticide for preventing losses caused by corn earworm in sweet corn.

Keywords: Bacillus thuringiensis, Helicoverpa zea, insect pest management, transgenic sweet corn.

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Authors: M. Kaomek, J. R. Ketudat-Cairns

Abstract:

The cDNA encoding the 326 amino acids of a Class I basic chitinase gene from Leucaena leucocephala de Wit (KB3, Genbank accession: AAM49597) was cloned under the control of CaMV35S promoter in pCAMBIA 1300 and transferred to Koshihikari. Calli of Koshihikari rice was transformed with agrobacterium with this construct expressing the chitinase and β- glucouronidase (GUS). The frequencies of calli 90 % has been obtained from rice seedlings cultured on NB medium. The high regeneration frequencies, 74% was obtained from calli cultured on regeneration medium containing 4 mg/l BAP, and 7 g/l phytagel at 25°C. Various factors were studied in order to establish a procedure for the transformation of Koshihikari Agrobacterium tumefaciens. Supplementation of 50 mM acetosyringone to the medium during coculivation was important to enhance the frequency to transient transformation. The 4 week-old scutellum-derived calli were excellent starting materials. Selection medium based on NB medium supplement with 40 mg/l hygromycin and 400 mg/l cefotaxime were an optimized medium for selection of transformed rice calli. The percentage of transformation 70 was obtained. Recombinant calli and regenerated rice plants were checked the expression of chitinase and gus by PCR, northern blot gel, southern blot gel, and gus assay. Chitinase and gus were expressed in all parts of recombinant rice. The rice line expressing the KB3 chiitnase was more resistant to the blast fungus Fusarium monoliforme than control line.

Keywords: chitinase, Leucaena leucocephala de Wit, Koshihikari, transgenic rice.

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Authors: Kobra Nalbandi, Bahram Baghban Kohnehrouz, Khalil Alami Saeed

Abstract:

The low level of foreign genes expression in transgenic plants is a key factor that limits plant genetic engineering. Because of the critical regulatory activity of the promoters on gene transcription, they are studied extensively to improve the efficiency of the plant transgenic system. The strong constitutive promoters, such as CaMV 35S promoter and Ubiqutin 1 maize are usually used in plant biotechnology research. However the expression level of the foreign genes in all tissues is often undesirable. But using a strong seed-specific promoter to limit gene expression in the seed solves such problems. The purpose of this study is to isolate one of the seed specific promoters of Hordeum vulgare. So one of the common varieties of Hordeum vulgare in Iran was selected and their genomes extracted then the D-Hordein promoter amplified using the specific designed primers. Then the amplified fragment of the insert cloned in an appropriate vector and then transformed to E. coli. At last for the final admission of accuracy the cloned fragments sent for sequencing. Sequencing analysis showed that the cloned fragment DHPcontained motifs; like TATA box, CAAT-box, CCGTCC-box, AMYBOX1 and E-box etc., which constituted the seed-specific promoter activity. The results were compared with sequences existing in data banks. D-Hordein promoters of Alger has 99% similarity at 100 % coverage. The results also showed that D-Hordein promoter of barley and HMW promoter of wheat are too similar.

Keywords: Barley, Seed specific promoter, Hordein.

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Authors: M. Keshlaf, R. Mensah, O. Nicetic, R. Spooner-Hart

Abstract:

The effectiveness of a commercial bee attractant, synthetic honey bee queen mandibular pheromone (Fruit Boost^®) for enhancing pollination of Gossypium hirsutum was evaluated in a transgenic (Bt) cotton crop. The study assessed the number of bee visitations to blossoms of plants treated with Fruit Boost^® as well, as effects on fruit set, yield, and lint quality. Bee activity on plots sprayed with pheromone concentrations of 50 and 500 queen equivalents (QEQ) /ha did not differ significantly from water-only control, on the day of application or the subsequent day. Application of the pheromone did not increase fruit set, yield, or lint quality. Two consecutive pheromone applications, applied two days apart, were not significantly different from a single application for any parameter.

Keywords: Apis mellifera, cotton, pollination, QMP pheromone

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Authors: Mouse, cardiac myocytes, computer simulation, action potential.

Abstract:

Heterogeneous repolarization causes dispersion of the T-wave and has been linked to arrhythmogenesis. Such heterogeneities appear due to differential expression of ionic currents in different regions of the heart, both in healthy and diseased animals and humans. Mice are important animals for the study of heart diseases because of the ability to create transgenic animals. We used our previously reported model of mouse ventricular myocytes to develop 2D mouse ventricular tissue model consisting of 14,000 cells (apical or septal ventricular myocytes) and to study the stability of action potential propagation and Ca2+ dynamics. The 2D tissue model was implemented as a FORTRAN program code for highperformance multiprocessor computers that runs on 36 processors. Our tissue model is able to simulate heterogeneities not only in action potential repolarization, but also heterogeneities in intracellular Ca2+ transients. The multicellular model reproduced experimentally observed velocities of action potential propagation and demonstrated the importance of incorporation of realistic Ca2+ dynamics for action potential propagation. The simulations show that relatively sharp gradients of repolarization are predicted to exist in 2D mouse tissue models, and they are primarily determined by the cellular properties of ventricular myocytes. Abrupt local gradients of channel expression can cause alternans at longer pacing basic cycle lengths than gradual changes, and development of alternans depends on the site of stimulation.

Keywords: Mouse, cardiac myocytes, computer simulation, action potential

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