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Title: Evaluation of Synthetic EPSPS Gene in Genetically Transformed Plants
Authors: Imran, Muhammad
Keywords: Biotechnology
Issue Date: 2018
Publisher: Pakistan Institute of Engineering & Applied Sciences, Islamabad.
Abstract: Codon optimized synthetic epsps gene was used to develop transgenic tobacco and sugarcane lines using Agrobacterium and biolistic approach, respectively. Putative transgenic tobacco lines analyzed by qRT-PCR showed variable mRNA transcript levels. Southern hybridization performed for determining gene integration patterns showed multiple copy inserts (1-4) of transgene in various tested lines. Immunoblot strips confirmed the translation of cp4-epsps mRNA transcripts in T1 line. Malachite green assay showed variable EPSPS activity levels in various lines in response to glyphosate application. Transgenic tobacco lines exposed to 1% (v/v) glyphosate at 4 to 5 leaf stage showed variable resistance levels ranging from moderate level tolerance to high tolerance. The cp4-epsps mRNA transcript levels varied from genotype to genotype in tested transgenic sugarcane lines regardless of the integration of multiple copy number (3-6) of transgene. Genetic transformation with pyramided constructs results in the integration of multiple genes at a single locus, which is desirable in terms of expression efficiency of transgenes. The cp4-epsps gene was pyramided with insect resistant cry1Ac and cry2Ab genes and cloned in pSb187 vector was used to genetically transform tobacco using Agrobacterium mediated approach. Transgenic lines were confirmed through PCR using gene specific primers of respective genes. The mRNA transcript level of cp4-epsps gene detected through qRT-PCR using GAPDH gene as an internal control was found to be variable among different transgenic lines. Transgenic lines were tested for checking the translational efficiency of all three genes using immunoblot strips. Transgenic tobacco lines found expressing all three proteins were subjected to detached leaf insect bioassays to evaluate the effectiveness of Bt genes in putative transgenic tobacco against insect attack using first instar larvae from Spodoptera littura and Helicoverpa armigera. Transgenic tobacco found to be resistant against these insects showed that cry1Ac and cry2Ab genes were efficient enough in pyramided form. When these transgenic tobacco lines were exposed to glyphosate at 10 to 12 leaf stage, chlorotic symptoms were observed which hinted that herbicide tolerance potential of transgene declined with increasing age of the plants. In order to overcome the spatio temporal gene expression issues, a dual copy epsps expression cassette synthesized under different regulatory elements, cloned in pGreen0029 binary vector was used to generate transgenic tobacco. These tobacco lines exhibited excellent resistance against 19% (v/v) glyphosate at 10-12 leaf stage. This construct has shown tremendous potential regarding transgene expression levels which is required in the field crops. This dual cassette cp4-epsps gene has been recommended for generation of glyphosate resistant cotton and other commercially important cultivars.
Gov't Doc #: 17116
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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