Characterization of the 5' Flanking Region of the Citrus d-Limonene Synthase Gene, Which Shows a Quantitatively Preferential Expression in Peel

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To obtain a peel-specific promoter useful for citrus transgenic research, the 5' flanking region of a citrus d-limonene synthase gene (CitMTSE2), which showed a high degree of expression in the peel of mature fruits, was isolated from the satsuma mandarin (Citrus unshiu Marc.) Bacterial Artificial Chromosome (BAC) library. Promoter activity was characterized by particle bombardment, agroinjection and Arabidopsis transformation analyses. Approximately 5kbps in the 5' upstream region (PCuMTSE2) contained several cis-motifs relating to guard cell-specific and stress-related genes. PCuMTSE2 fused to the uidA gene was directly incorporated into tissues from citrus and its relatives using particle bombardment. The results showed that PCuMTSE2 conferred β-glucronidase (GUS) activity in fruits rather than in seed and leaf tissues. Agroinjection analysis using citrus fruits showed PCuMTSE2 has promoter activity in peel but not in juice sacs. In a transgenic Arabidopsis-incorporated PCuMTSE2::uidA construct, GUS activity was detected in the joint between the stalk and silique. These results suggest that PCuMTSE2 could be utilized as a promoter regulating the quantitatively preferential expression in peel, and is useful for studies of manipulation by genetic engineering in citrus.
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