Abstract
Flavonoid compounds accumulating in the epidermal cell layers of plant tissues are considered to be among the most effective protectants against the ultraviolet-B (UV-B) radiation. To identify a transcription factor involved in the activation of the genes, such as chalcone synthase genes (CHS), in the pathway of flavonoid biosynthesis, a pair of degenerate primers was designed to amplify the most conservative region of MYB from soybean by polymerase chain reaction (PCR) or reverse transcriptase (RT)-PCR. Both of these amplification products were found to contain molecules of dozens of independent MYB-like sequences. The bacterial clones harboring a partial library of the RT-PCR products were differentially hybridized with the amplified cDNA fragments derived from total RNA of UV-B treat seedling (rMYB/UV-B+) and those from control RNA (rMYB/UV-B-). One clone designated as MYB29 showed a significantly stronger signal with rMYB/UV-B+ hybridization than with rMYB/UV-B-. Starting from the sequence information of MYB29 fragment, an entire sequence containing the complete gene designated as GmMYB29A1 was obtained by nested PCR of the flanking regions. In the course of this PCR cloning, we identified several independent products closely related to GmMYB29A1. In order to amplify the entire protein coding region of the closely related genes, two sets of primers were designed, two upstream primers containing the ATG start codon and the other two downstream primers containing the TGA stop codon. By sequencing those cDNAs amplified with RTPCR, a total of at least four members were found to comprise the subfamily, designated as GmMYB29. UV-B- responsive expression of the members of GmMYB29 was found to reach its peak within 2 hours after the onset of light exposure while in those of soybean (Gm)CHS it continued to rise for 6 hours. This finding suggested that these MYB transcription factors may activate genes for flavonoid biosynthesis including GmCHS in response to UV-B exposure.
