Cloning Of Riboflavin Receptor Gene From Turtle And Expression Of The Gene In Transgenic Plants

Riboflavin (vitamin B2) participates in many physiological processes in organisms. The vitamin often is phosphorated and combined with nucleotides to form flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), important components of the respiratory electron-transmission chain, which affects antioxidation through the role of vitamin C. FMN and FAD often serve as coenzyme of many enzymes (flavoproteins), which control many physiological reactions, and particularly production of reactive oxygen intermediates (ROIs). ROIs are essential for the hypersensitive cell death and growth, defense against pathogens and insects, and many aspects of responses to environmental stress. Levels of riboflavin in plants are believed to be important to these processes. As such, if riboflavin levels could be manipulated, the important processes could be controlled as a consequence. This would result in great improvement of plant defense and productivity. This also could provide us with a particular insight into studying coordination of plant defense with growth regulation, which may be affected by levels of riboflavin. The author cloned a riboflavin receptor (riboflavin binding protein, RfBP) gene from Trionyx sinensis japonicus and generated transgenic Arabidopsis and tobacco plants that express the gene. Studies with transgenic plants could reveal whether the modulation of free riboflavin affects plant defense and growth signal transduction.The RfBP gene was cloned by PCR from the soft-shelled turtle, using primers made based on the region conserved in the riboflavin receptor proteins of chicken and turtle. Computer analyses by several programs revealed that the sequence we cloned is 99% identical to the gene reported previously. The cloned gene was inserted into the plant transformation vector pBI121. After confirming the correct orientation of RfBP in the vector, the recombinant unit was introduced into Arabidopsis and tobacco plants. Transgenic lines in Tl to T3 generations were obtained based on screening seeds on medium and molecular analyses of the plants. Southern blot hybridization and PCRanalysis suggested that the transgene was integrated into chromosomes of both plants. RT-PCR and northern blot RNA gel analyses indicated that the gene was expressed in transgenic plants. Fluorescent spectrophoresis showed that the transgenic expression of the RfBP gene caused changes of riboflavin levels. The vitamin levels decreased in transgenic Arabidopsis plants tested, compared with untransformed and the pBIl21-only-transformed plants. However, levels of riboflavin increased 24hours after treatment with riboflavin, compared with the controls. These results suggest that expression of the RfBP gene in transgenic plants modulates contents of free riboflavin in plant tissues. Finally, transgenic plants were different from control plants in several morphological properties, including plant height, leaf colors and blossom numbers. These results provide an insight into the role of modulating riboflavin contents in plant growth and development regulation.