Transformation and characterization of transgenic rice and Cleome spinosa plants carrying the maize phosphoenolpyruvate carboxylase genomic DNA

Abstract

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme in the C4 photosynthetic pathway for the initial fixation of atmospheric CO2 into the four-carbon (C4) acid, oxaloacetate. Here, we report that a vector carrying the intact maize C4pepc gene was used to transform C3 monocot japonica rice cultivar Tainung 67 (TNG67) which is an important cultivar in Taiwan, and C3 dicot Cleome spinosa which is closely related to the C4 dicot Cleome gynandra, via an Agrobacterium-mediated method. In total, 29 transgenic rice and 4 transgenic C. spinosa plants were obtained. Genomic PCR and southern blot analyses revealed that the maize pepc and selective antibiotic resistant genes were present in almost all randomly-selected transgenic plants but not in wild-type plants. Reverse transcription-PCR showed that maize pepc mRNA was detected in transgenic plants. Western blot analysis confirmed the presence of 110 kDa maize PEPC protein in transgenic plants but not in wild-type plants. Furthermore, higher PEPC enzyme activities were detected in transgenic rice plants than in transgenic C. spinosa plants. Taken together, we clearly demonstrated monocot maize C4pepc gene is functional in both C3 monocot rice and C3 dicot C. spinosa plants. Two T5 homozygous rice lines, each harboring a single insertion of maize pepc gene, showed 7- to 9.4-fold and 40–54% PEPC enzyme activity as compared to the untransformed rice and maize, respectively. These stable transgenic rice lines will be valuable material for studying the effect of maize PEPC on rice photosynthesis under different conditions.