Molecular Cloning, Bioinformatics Analysis and Overexpression of the Tyrosine Aminotransferase Gene in Rehmannia Glutinosa

Rehmannia glutinosa is an important medicinal plant producing many bioactive compounds such as catalpol, acteoside and so on. Tyrosine aminotransferase (TAT) is the first key enzyme that catalyzes the reversible interconversion of tyrosine and 4-hydroxyphenylpyruvate in the tyrosine-derived branch pathway of acteoside biosynthesis. To confirm its role for acteoside accumulation, we isolated a full-length cDNA from Rehmannia glutinosa Libosch. Sequence analysis indicated that it contained a 1266 bp open reading frame, encoding a TAT of 421 amino acid residues. Multiple sequence alignment revealed that the homology of RgTAT amino acid sequence to that of Sesamum indicum (XP_011100354.1) was the highest (89.94%). Evolutionary tree showed that Sesamum indicum TAT and RgTAT were grouped together. Quantitative real-time PCR analysis indicated that the expression of RgTAT in leaves was much higher than in roots and stems,and that the expression levels of RgTAT in the tuberous roots, stems and leaves of high-acteoside cultivar BJ-3 were higher than in that of low-acteoside cultivar Wen85-5. A plant expression vector was constructed containing the RgTAT and hygromycin resistance gene ( Hyg ). Transgenic Rehmannia glutinosa Libosch overexpressing RgTAT was obtained via an Agrobacterium tumefaciens-mediated transformation system, in which Hyg expression was confirmed by PCR. RgTAT expression in transgenic plantlets measured by real-time quantitative PCR was 7.72 ± 0.17 times greater than its expression in the untransformed plantlets. Moreover, HPLC analysis indicated that enhanced RgTAT expression corresponded to significantly increased acteoside for transgenic plantlets. Our results elucidate the role of RgTAT in the acteoside biosynthesis in Rehmannia glutinosa .