Simultaneous Expression of AhBTF3, AhNF-YA7 and EcZF Modulates Acclimation Responses to Abiotic Stresses in Rice (Oryza sativa L)

AbstractIntroductionAbiotic stress responses are under the strict control of transcriptional regulation. Gene stacking approach using two to three transcription factors (TFs), which aid in stable transcription would be promising. An attempt was made to co express a basal transcription factor (AhBTF3); Nuclear factor (AhNFYA7), an activator protein and zinc finger protein (EcZF), a stress inducible TF. It is hypothesised that a combination of different classes of TFs enhances transcriptional stability that can regulate stress acclimation.MethodsThe gene cassette harbouring NFYA7, BTF3 and ZF expressed under rbcS, Ubi and rd29A promoters respectively, was cloned in the destination vector pi12GW (PsAKR1 as selectable marker), by modified gateway cloning strategy. Agrobacterium mediated in planta transformation technique was used to generate rice (AC-39020) transgenic plants. The putative transgenics were screened for glyphosate tolerance and confirmed by PCR and RT-PCR. The select lines were subjected for physiological characterisation to assess their stress response.ResultsThe transformants screened for glyphosate tolerance exhibited varied response (Fig. 1). The select lines maintained higher chlorophyll stability when subjected for excised leaf disc stress tolerance assay using PEG and NaCl (Fig. 2A and B). The transgenics exhibited better growth as evidenced by plant height and seed yield (Fig. 2C and D).The promising transgenic lines were challenged with different abiotic stresses using NaCl, PEG and cadmium chloride which revealed their superior performance at seedling level (Fig. 3A). The integration of marker gene and expression of each transgene was confirmed by PCR (Fig. 3B) and RT-PCR respectively (Fig. 3C).DiscussionCo expression of stress relevant TFs can enhance the levels of tolerance tomultiple abiotic stresses as demonstrated in this study. The roles of individual TF in modulating cellular stress responses need to be examined