Functional analysis of the Lupinus luteus cyclophilin gene promoter region in Lotus japonicus

Abstract

Functional analysis of promoter sequences is important to understand the regulation of gene expression. This study aimed to investigate the promoter region of the Lupinus luteus cytoplasmic cyclophilin gene (LlCyP; AF178458). After bioinformatic analysis, four promoter deletion fragments were fused to the β-glucuronidase reporter gene. We used Lotus japonicus as a model plant. After Agrobacterium rhizogenes transformation of L. japonicus, only the longest promoter region (−1055 bp to ATG) supported the β-glucuronidase expression in root nodule parenchyma. Putative cis-elements located between −1055 and −846 bp were subjected to site-directed mutagenesis. Mutations incorporated in the TGATT and AGATT motifs (cytokinin response) abolished GUS expression in nodules, but the mutated AAAGAT motif (OSE, organ-specific element) still activated the GUS expression in root nodules, mainly in cells surrounding the vascular bundle. Promoter deletion and mutation experiments suggest that cis-elements responsible for gene expression in the nodule are located in the region spanning from −1055 to −846 bp. We constructed a deletion fragment, in which the DNA sequence located between −822 and −198 bp was removed (pCYPMG). The promoter region arranged in the pCYPMG supports the expression in the parenchyma of L. japonicus nodules, but it is lower than the whole promoter region. The obtained results may be useful for transgene expression in determinate and indeterminate root nodules.