Poster: Transcription Regulation

Abs # 1036: Functional properties of UV-B responsive GmMYB29B1 as a transcription factor.

Presenter:	Akada, Shinji , akada@cc.hirosaki-u.ac.jp
Authors	Akada, Shinji  (A)   Kitajima, Hiroshi  (B)   Senda, Mineo  (A)   Ishikawa, Ryuji  (B)   Takeo, Harada  (B)   Minoru, Niizeki  (B)
Affiliations:	(A): Gene Research Center, Hirosaki University (B): Faculty of Agriculture and Life Sciense, Hirosaki University

GmMYB29 subfamily was previously isolated as a group of four genes encoding MYB transcription factors and was designated as GmMYB29A1, A2, B1, and B2. The family members showed more than 90 % amino acid sequence similarity in their N-terminal DNA binding domains (N-BD) and substantial diversity in their C-terminal halves. Expression analysis of the four genes indicated that each member was strongly up-regulated in response to UV-B exposure, with B1 the most prominently expressed. We show that B1 expressed on the yeast plasmid vector pBD-GAL4 functioned as a transcription factor in the yeast system. A series of deletion and replacement mutants were constructed to find that the third domain rich in acidic amino acids and its downstream C-terminal sequences, which in total consist of 26 amino acid residues, were important as the activator domain. Based on the amino acid sequence we speculated that B1 protein might be phosphorylated for an activation signal. In order to detect any phosphorylation event on B1, the protein expressed in E. coli was used as a substrate for the kinase activity of the crude extracts from hypocotyl tissues of soybean seedlings. The intact B1 protein was found strongly phosphorylated, but its N-BD was not, suggesting that the C-terminal half of B1 could serve as a target for a protein kinase. N-BD was also used for the gel-shift analysis to detect interaction of the protein with the promoter sequences of GmCHS gene family. The protein was found to interact specifically with H-box sequence (ACCTACC) on the promoter. H-box is very well conserved among the GmCHS gene family as a promoter core element and was conceived to serve as a cis-element for the UV-B responsive expression of GmCHS.