Characterization of expression of the OsPUL gene encoding a pullulanase-type debranching enzyme during seed development and germination in rice.

ABSTRACT

Starch-debranching enzymes (DBEs) are key enzymes involved in starch metabolism in cereals, having a dual function, in both starch synthesis and degradation. However, their precise roles in this pathway, particularly their expression profiles, remain unclear. In the present study, we performed a quantitative real-time PCR (Q-PCR) analysis of the expression pattern of the OsPUL gene encoding a pullulanase-type DBE in different tissues as well as in seeds at different developmental stages. The results showed that this gene was expressed only in seeds. In addition, the 1177-bp OsPUL promoter sequence was cloned, and some endosperm-specific motifs such as the GCN4 and AACA motifs were observed to exist in this region. The promoter was then fused with the GUS reporter gene and its expression was carefully investigated in transgenic rice. The data from both histochemical and fluorometric analyses showed that the OsPUL promoter was capable of driving the target gene to have a high level of endosperm-specific expression. The OsPUL gene maintained a relatively high expression level during the entire period of seed development, and peaked in the middle and late stages. This observation was very consistent with that of the endogenous transcription analysis by Q-PCR. Furthermore, the seed germination experiment showed that the OsPUL promoter actively functions in the late stage of seed germination. The expression of the OsPUL gene was maintained at a significant level during the entire grain filling period and in the late stage of seed germination, which coincided with its involvement in starch anabolism and catabolism.