Molecular and physiological analysis of transgenic rice harbouring chimeric PDH47 gene against abiotic stress tolerance

Abstract

Abiotic stress especially drought can severely affect the physiological status of any plants; thereby impart a significant negative impact on growth, development, and metabolism. The major emphasis of most of the recent studies was the identification of stress-regulated genes and transcription factors which play an important role in governing tolerance/resistance against abiotic stresses. DEAD-box helicases (Asp-Glu-Ala-Asp amino acid) are one of the important genes which confer tolerance to various abiotic stresses. They are involved in unwinding of nucleic acids by utilizing the energy from ATP hydrolysis. PDH47 (Pea DNA Helicase 47kDa) gene, one of the DEAD-box helicases is known to impart various abiotic stress tolerance. In the present study rice transgenic line cv. IR64 was developed through Agrobacterium-mediated genetic transformation using immature embryos as explants. The putative transgenic lines showed the presence of transgene when subjected to PCR analysis using gene-specific primers. Three previously developed transgenic rice lines in our laboratory namely ASD16-46/1, ASD16-66/1 and ASD16-68/1 expressing PDH47 gene were selected for drought stress tolerance study. Quantitative Real-Time PCR analysis showed a varied level of expression of PDH47 gene both in the root and leaf tissues of the transgenic line before and after drought stress. The expression of PDH47 gene induced during drought stress in the transgenic lines showed the varied level of drought tolerance in the vegetative stage without any negative effects on the morphological and agronomical traits. The physiological and biochemical analyses confirmed that the expression of PDH47 gene in the transgenic lines was associated with increased leaf relative water content, water retention capacity, maintenance of chlorophyll, stomatal conductance, net photosynthetic rate, transpiration rate and water use efficiency. These transgenic lines also showed an increased accumulation of the osmolytes like proline, glycine betaine and decreased electrolyte leakage, lipid peroxidation, less accumulation of H2O2 during drought stress. These transgenic lines showed better root architecture system such as root length, root number, root thickness, root biomass, shoot biomass and root to shoot ratio as compared to control untransformed plants during drought stress. The western blot analysis confirmed that the expression of RUBISCO large subunit (55kDa) protein was more in the transgenic rice lines as compared to untransformed control plants during drought stress treatment.