Structural characterization and promoter activity analysis of the gamma-kafirin gene from sorghum.

Abstract

A genomic clone encoding the gamma-kafirin gene from sorghum was isolated and sequenced. A 2938 bp sequenced fragment includes an intronless open reading frame of 636 nucleotides encoding a putative polypeptide of 212 amino acids. Comparison of the deduced amino acid sequence of gamma-kafirin with the published sequences of gamma-prolamins of maize, and Coix revealed highly conserved domains. The N-terminal region of these proteins contains the conserved hexapeptide PPPVHL, which is repeated eight times in gamma-zein, four times in gamma-kafirin and three times in gamma-coixin. The number of PPPVHL repeats accounts predominantly for the differences in the molecular weights of gamma-prolamins. Several putative regulatory sequences common to the gamma-kafirin and gamma-zein genes were identified in both the 5' and the 3' flanking regions. Putative GCN4-like regulatory sequences were found at positions -192 and -476 in the 5' flanking region of gamma-kafirin. In the 3' noncoding region, three putative polyadenylation signals, two AATAAT and one AATGAA, were found at positions +658, +716, and +785, respectively. In order to investigate the role of the putative GCN4-like motifs and other possible cis-acting element(s) of the gamma-kafirin promoter, a series of deleted and chimeric promoter constructs were introduced into maize, Coix and sorghum tissues by particle bombardment. Histochemical analysis of beta-glucuronidase (GUS) activity in different tissues indicated that the element(s) responsible for tissue specificity is probably located in the 285-bp proximal region of the promoter, while the remaining promoter sequence seems to carry the element(s) responsible for the quantitative response.