Construction of plant expression vectors harboring synthetic cry2AX1 gene and its expression in tobacco (Nicotiana tabacum)

Abstract

The cry2AX1 is a potential synthetic Bt gene for developing transgenic crops resistant to lepidopteran insect pests. By using this gene, two new plant expression vectors were constructed in pCAMBIA2300 backbone. In the construct p2300:CaMV:2AX1, the cry2AX1 gene was driven by CaMV35S promoter and in p2300:CaMV:ctp-2AX1, the chloroplast transit peptide (ctp) sequence of cotton rbcS1b was N-terminally fused in frame with synthetic cry2AX1 coding sequence driven by CaMV35S promoter. These constructs were confirmed by PCR, restriction analysis and nucleotide sequencing. Further the plant transformation vectors were mobilized to Agrobacterium tumefaciens LBA4404. Leaf discs of tobacco (Nicotiana tabacum L. cv Petite Havana) were used as explants for transformation with the two constructs through Agrobacterium mediated transformation. Plantlets were generated in vitro on medium containing kanamycin as selection agent. Screening by PCR revealed presence of cry2AX1 gene in all twelve putative transformants generated from p2300:CaMV:2AX1 and eighteen out of nineteen putative transformants generated from p2300:CaMV:ctp-2AX1 construct. The expression of Cry2AX1 protein in PCR positive T0 tobacco plants ranged from 3 to 42 ng/g and 1.6 to 28 ng/g of fresh leaf tissue in p2300: CaMV:2AX1 and p2300:CaMV:ctp- 2AX1 constructs, respectively. The two new plant transformation vectors were validated for expression of synthetic cry2AX1 gene in tobacco as model plant.