Codon optimization of <Emphasis Type="Italic">cry1Ab</Emphasis> gene for hyper expression in plant organelles |
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Authors: | Rasheda Jabeen Muhammad Sarwar Khan Yusuf Zafar Tehmina Anjum |
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Institution: | (1) Institute of Mycology and Plant Pathology, University of the Punjab, Lahore, 54590, Pakistan;(2) National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan;(3) National Institute for Genomics and Advanced Biotechnology, Park road, Islamabad, 45500, Pakistan; |
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Abstract: | With the advent of genetic manipulation techniques, it has become possible to clone and insert gene into the genome of crop
plants to confer resistance to insects and pests. Resistance to insects has been demonstrating in transgenic plants either
by triggering defense system of plants or by expressing heterologous cry genes for delta-endotoxins from Bacillus thuringiensis. In the present study, synthetic cry1Ab gene was developed with optimized chloroplast preferred codons and is expressed in tobacco plastid genome called plastome,
following chloroplast transformation strategy, which is environment friendly technique to minimize out-crossing of transgenes
to related weeds and crops. In addition, due to high polyploidy of plastid genome transformation of chloroplast permits the
introduction of thousands of copies of foreign genes per plant cell, leading to extraordinarily high levels of foreign protein
expression. The chloroplast transformation technology aims to insert stably into the plastome through homologous recombination
into pre-decided position. To characterize the synthetic cry1Ab gene, chloroplast transformation vectors were developed and bombarded to the leaf cells of tobacco plants maintained under
aseptic conditions. After bombardment, the drug resistant shoots were selected and regenerated on drug containing regeneration
medium. Homoplasmic shoots were recovered after successive rounds of selection and regeneration. Proliferated plants were
subjected to genomic DNA analysis by using polymerase chain reaction (PCR) technique where cry1Ab gene-specific primers were used. PCR positive plants were subjected to protein analysis, and functionally expressed proteins
were detected using Immuno-Strips specific for cry1Ab/Ac gene products. Transgenic plants carrying cry1Ab gene were found expressing Bt toxins confirming that engineered gene could be expressed in other plants as well. |
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