P34 has long been known as one of major allergenic proteins in soybean. Recently, two low-P34 soybean accessions, PI603570A and PI567476, were identified. In order to understand the molecular mechanism of a significant reduction of P34 protein level in PI567476, we performed comparative analysis of the P34 cDNAs and genomic sequences from low-P34 and normal soybean accessions. While no sequence variation was detected in P34 coding regions of the soybean accessions tested, the P34 gene from PI567476 contains ‘ATGT’ 4-bp insertion in front of the start codon. We also found the length polymorphism of ‘TA’ repeats in the P34 promoter regions among accessions. Northern and western blot analysis revealed that, even though mRNA levels of P34 are similar in low-P34 and normal soybean accessions, lower accumulation of P34 protein is only detected in the low-P34 accession, suggesting that the genetic polymorphisms in P34 promoters have a more significant effect on translation efficiency than transcription of the P34 gene. Transient expression analysis showed that the P34 promoter of PI567476 has significantly reduced activity compared to that of normal accession, cv. Clark. In addition, the chimeric promoter generated by introducing the 4-bp insertion in front of the start codon of the P34 gene from normal Clark accession showed markedly reduced activity. The results suggest that lower accumulation of P34 protein in low-P34 accession is largely due to the reduced translation efficiency caused by the 4-bp insertion in the P34 promoter, thus providing direct evidence for the molecular mechanism to explain the regulation of P34 gene expression in various soybean accessions.
