Phenotypic characterization of the transgenic rice plants over-expressing
. (A) Schematic diagram of the OsPUB15 overexpression vector. The ORF fragment of OsPUB15 is placed between the maize Ubiquitin promoter (Pro-Ubq) and nos terminator (Tnos). The Hyg gene driven by the CaMV 35S promoter (Pro-35S) is included for hygromycin resistance. (B) Spontaneous cell death phenotype of transgenic rice plants over-expressing OsPUB15 (referred to simply as OsPUB15ox) under sterile conditions. L3, L8, L11 and L16 indicate the corresponding OsPUB15ox lines while Con represents the control plants expressing the empty vectors. (C) qRT-PCR analysis of OsPUB15 expression in two-week-old OsPUB15ox plants and the control plants. The expression level of OsPUB15 in the control plants was set as 1.0. The expression level of the rice ACTIN1 gene was used as an internal control for normalization of the data. Data represent means ± SDs of three replicates. (D) Histochemical assays of the OsPUB15ox plants and the control plants. Leaves of two-week-old transgenic plants were stained with trypan blue (left panel), DAB (middle panel) or NBT (right panel) to indicate cell death, superoxide or hydrogen peroxide accumulation, respectively. L3 and Con represent OsPUB15ox line 3 and the control plants, respectively. (E) Transcript expression analysis of PR genes in transgenic rice plants. Total RNA was extracted from leaves of two-week-old transgenic plants. RT-PCR was performed with specific primers for PR1a, PR1b, PR10 and PBZ1, respectively. Control RT-PCR reactions were conducted with rice ACTIN1. Con, L-, L+, L5 and L6 represent leaves of the control plants, later-emerged young leaves of OsPUB15ox before lesion formation, early-emerged old leaves of OsPUB15ox with lesions, total leaves of OsPUB15ox-L5 and OsPUB15ox-L6, respectively. (F) Disease resistance determination of OsPUB15ox seedlings to M. oryzae isolates. The control rice seedlings and the OsPUB15ox seedlings were treated with indicated blast strains, and the responses were analyzed two days later. The above experiments were repeated three times with similar results obtained.