Reconstruction and validation of three different binary vectors suitable for generation of genetically engineered Helicoverpa protected crops

Abstract

Availability of a suitable plant transformation binary vector is necessary for the generation of transgenic crops with an adequate expression of transgenic proteins. Therefore, three binary vectors were constructed viz., pBK204, pBK205, and pBK206 harboring either a truncated or a full-length version of a Cry1Ac gene for the generation of Helicoverpa protected crops. Two different promoters viz., Arabidopsis Rubisco small subunit (AtSSU) gene promoter or CaMV35S promoters were used to regulate the various versions of Cry1Ac gene. The binary vectors were reconstructed either by the Gibson assembly method and others by ligating the restriction enzyme digested fragments. The reconstructed binary vectors were mobilized into Agrobacterium strain AGL1 and validated by Agrobacterium infiltration assays of Nicotiana benthamiana. The amount of Cry1Ac protein accumulated in the Agroinfiltrated tobacco leaves was quantified using the quantitative ELISA assay. The expression of the Cry1Ac protein in the tobacco leaves ranged from 0.25 to 0.26 µg /g fresh weight (FW) when transformed with these three constructs. Thus, the vectors constructed in this study appeared to be suitable for generation of Helicoverpa resistant transgenic crops by Agrobacterium-mediated genetic transformation method.