Identification of Stigma Specific Expression Fragment in the Promoter of a Soybean Chitinase Class I Gene

Abstract

The expression level of heterologous genes in transgenic plants serves as an important indicator of gene efficiency. The small number of currently known effective promoters, limits the possibilities in fine-tuning the expression of transgenes. We cloned and characterized a tissue-specific promoter fragment of the soybean chitinase class I gene (GmChi1). The GmChi1 promoter (GmChi1P) was cloned from Jungery soybean. The promoter sequence contains a number of putative cis-acting elements, including tissue-specific and stress-regulated motifs. By histochemical analysis, the GmChi1P-controlled β-glucuronidase (GUS) reporter enzyme activity was shown to be highest in the roots of transgenic Nicotiana tabacum cv. NC89 at the four-leaf sprout formation stage. Interestingly, the high GUS activity in transgenic tobacco roots was effectively suppressed by salicylic acid (SA) treatment. Deletion analysis of GmChi1P revealed that the sequences located between positions -719 and -382 contain key cis-elements responsible for the reporter uidA gene expression (encoding GUS) in leaves, roots, and wounds of Nicotiana tabacum. In addition, fluorometric analysis showed that the activity of the shortened ChiP(-1292) to ChiP(-719) promoters in the roots of transgenic tobacco was significantly suppressed by abscisic acid and completely suppressed by SA. The ChiP(-382) promoter was also found to be expressed exclusively in the stigma of transgenic tobacco flowers. Using the GUS reporter enzyme, no staining was detected in other flower organs in transgenic Nicotiana tabacum, including sepals, petals, anthers, filaments, and ovaries, or in any vegetative tissues. The results indicate that the promoter fragment ChiP(-382) can be used in tissue-specific regulation of gene expression and plant genetic engineering.