Molecular Physiological Analyses of the T1-T2 Lines of OsmiR399–Overexpressing Transgenic Rice

Abstract

Abscisic acid (ABA) plays a pivotal role in plant response to abiotic stresses such as drought, cold and salinity. Among abiotic stresses, drought stress causes many problems during plant growth and development. As a result, it considerably reduced the productivity of rice (Oryza sativa L.) cultivation. The Arabidopsis miR399 has been demonstrated to be induced by ABA under drought conditions and enhance drought tolerance by down-regulating PHO2 that encodes UBIQUITIN-CONJUGATING E2 ENZYME 24 (UBC24) (MS thesis by Jae Young Jang). The miR399s in rice (OsmiR399s) are highly homologous to Arabidopsis miR399s, and the T0 transgenic rice plants that overexpress OsmiR399, such as PGD1::OsmiR399f and Rab21::OsmiR399c lines, were previously produced by using either the constitutive PGD1 or the stress-inducible Rab21 promoter (MS thesis by Min Kyung Lee). In this study, the subsequent T1 and T2 generations of the OsmiR399-overexpressing transgenic rice were obtained, and the transgenic plants homozygotic for the single copy transgene were selected by TaqMan PCR and Southern hybridization analyses. Intact insertions of the transgenes without DNA rearrangements and sequence changes were confirmed based on DNA sequencing as well as PCR analyses of the entire transgenes that included T-DNA border sequences. In addition, it was also confirmed that the levels of the OsmiR399 transcripts were increased with the decreases in the accumulation of OsPHO2 transcripts in the transgenic plants compared with wild type (WT). Furthermore, OsmiR399-overexpressing transgenic plants were demonstrated to have higher survival rates and photosynthetic capabilities compared with WT under drought conditions. Taken all together, these results suggested that the OsmiR399 down-regulate OsPHO2 in order to obtain the drought tolerance in rice.