The objective of this study was to identify rice gene promoters that are specifically induced by feeding of the striped stemborer (Chilo suppressalis). Two PCR-selected cDNA subtractive libraries were constructed from the rice variety Minghui 63. Up- and down-regulated cDNAs induced by C. suppressalis feeding were arrayed on nylon membranes. After array hybridization and Northern blot analysis, a cDNA (B1-A04) encoding a putative subtilisin/chymotrypsin inhibitor was found to be rapidly and highly induced by C. suppressalis feeding, compared with mechanical wounding. The putative promoter region, spanning from −1,569 to +446 relative to the transcriptional initiation site was isolated, fused to the GUS gene (β-glucuronidase reporter gene) and introduced by Agrobacterium-mediated transformation to rice. In non-infested plants, the GUS activity driven by this promoter fragment was detected in culms and panicles, but not in leaves and sheaths. At 6 h after insect feeding, GUS activity was significantly induced in sheaths and culms, but not in leaves. GUS activity and native B1-A04 gene were not induced by JA and ABA treatment. A serial deletion analysis revealed two regions (−1,569 to −1,166 and −1,166 to −582) that negatively regulate the gene expression in sheaths of non-infested plants but not in insect-infested plants. An electrophoretic mobility shift assay (EMSA) identified 7 DNA fragments with various binding activities with nuclear proteins from mechanically wounded, insect-infested and untreated plants, and their possible roles in gene regulation were speculated. This promoter fragment should have utility in development of insect resistant transgenic crops.
Plant Molecular Biology – Springer Journals
Published: May 24, 2007
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