Cloning, characterization and analysis of the Arabidopsis RD29A promoter for its inducible expression in rice under salinity and drought stress

Abstract

Traditional breeding as well as genetic engineering have been utilized to improve drought and salinity tolerance of rice with the goal of increasing its productivity. Use of modern molecular biology tools for engineering stress tolerant crops is based on the expression of specific stress-related genes. So, the choice of promoter can play an important role in abiotic stress tolerance as it controls gene expression. Thus inducible expression of gene is crucial for transgenic rice research which is usually implemented by making use of inducible promoters. In this study, therefore, a stress inducible promoter RD29A from Arabidopsis was characterized in Binnatoa rice variety. For this characterization, Agrobacterium mediated transformation of Binnatoa rice was carried out using the RD29A-GUS construct. The transgene was confirmed up to T₂ generation by RD29A specific primer. Southern analysis indicated single-copy integration of the promoter in the rice genome. Histochemical assay was performed in rice leaves, roots and seeds to test the inducibility of RD29A under both drought and salt stress (100 mM and 200 mM). Salt stress was applied at both seedling and reproductive stages whereas drought stress was applied only at seedling stage. Under drought stress, no expression was visible in root samples but a very prominent expression was found in leaves at day 7, 10, 13 and 16. At 100 mM salt stress, color intensity indicated gradual GUS expression increase up to 24h but a decline at 48h in both leaves and roots. At 200 mM salt stress however, expression of GUS in leaf sample was found remarkably higher at 5, 10, 24 and 48 hours. In the seedling root however, highest color was found after 5 hours of 200 mM stress. At reproductive stage stress, at both 100 and 200 mM salt exposure 6 days after flowering, GUS expression was maximal in leaves and seeds at 24 hours, but no expression was detected in roots. The time required for the toxicity of the salt to take effect, particularly in photosynthesizing leaves is about 24 hours. Therefore, maximal expression of the GUS gene at 24 hours indicates the suitability of the RD29A promoter for driving transgenes suitable in conferring salt tolerance. This promoter was also found suitable for driving transgenes in leaves under drought stress.