Development of multiple gene construct with regulatory genes and their functional validation

Abstract

Rice is widely cultivated under irrigated condition and its water requirement is very newlinehigh. Due to dwindling water resources, to save irrigation water growing rice under newlinesemi-irrigated aerobic condition has phenomenal relevance. In this ecosystem decreased water availability, high VPD affects crop growth and productivity. From this context it is important to improve adaptation of rice under aerobic condition by improving water relations and cellular level tolerance mechanisms. Stress adaptation at cellular level involves activation of stress responsive genes which are regulated by transcription factors. Hence, under stress, co-ordinated expression of multiple transcription factors is crucial to increase cellular level tolerance. As a first step a multigene construct coexpressing GUS, AtbHLH17 and AtWRKY28 was developed using modified multisite gateway technology and developed transgenics in Arabidopsis. These transgenics showed improved tolerance to diverse stresses like drought, salinity and oxidative stress by upregulation of large number of downstream genes. Further, three stress responsive TFs from different families i.e EcNAC1, EcMYC57, EcbZIP60 were cloned from fingermillet and validated their relevance in stress tolerance in tobacco. Tobacco transgenics co-expressing all the three TFs showed superior phenotype under stress compared to single TFs expressing transgenics signifying the importance of co newlineexpression of relevant stress specific TFs. To improve adaptation of rice under semiirrigated aerobic conditions rice transgenics were developed co-expressing EcNAC1, EcMYC57 and EcbZIP60 in the background of genotype having superior water relations. Desirable transformants were identified based on dessication response. Molecular characterization of transformants showed the integration and expression of all three genes. The transgenics showed improved tolerance to salinity and oxidative stress.