The relationship between the codon usage bias, gene expression level and the AUG context(from -20 to +6 positions relative to the initiator AUG codon) was examined in 541unigene sequences of rice. A significant correlation for CAI values (codon adaptationindex) was observed at five nucleotide positions (-19, -18, -9, -4, +5), eight (-19, -18,-14, -9, -6, -4, -1, +5) for CPP (codon preference parameter), and seven (-18, -16, -15,-9, -7, -1, +6) for mRNA abundance in the flanking sequence of the initiator AUG codonrespectively, but a significantly positive correlation for both CAI and CPP at twopositions (-4 and +5), indicating that both those positions are evolutionally under thenatural selection constraint at the translational level. By site-directed mutagenesis atseven specific positions (-18, -16, -15, -9, -7, -1 and +6) for allergenic protein thathad the highest mRNA abundance in this study, its expression level decreased dramatically63.3 and 72.5% respectively, indicating the importance of those 7 positions for geneexpression. A highly positive correlation (r=0.625, P<0.01) between AUGCAI and GCcontent in the flanking sequence of the initiator AUG codon showed a more effectivehigher GC content on translation initiation efficiency. The strong preference for G orC at those 8 positions (-6, -5, -3, -2, -1, +4, +5 and +6) in the AUG context suggestedthat an important factor in modulation of the translation efficiency, as well assynonymous codon usage bias, particularly in highly expressed genes.
