A NAPIN PROMOTER ACTIVATES GENE EXPRESSION IN DEVELOPING SEEDS OF LESQUERELLA FENDLERI

Abstract

Lesquerella fendleri produces industrial useful Hydroxy Fatty Acids (HFA) in seed oil. To improve oil and HFA of L. fendleri, it is desirable to use of seed-specific promoters to control the expression of target genes by genetic engineering. A seed-specific promoter fragment, -397 to -1 of a napin gene (PnapA) from Brassic napus was isolated by PCR and constructed to a small promoter-testing vector named pGPro4. A nopaline synthase (nos) promoter was used to control the expression of the selectable marker of pGPro4. pGPro4 also contains a bifunctional β-glucuronidase-enhanced Green Fluorescent Protein (gusA-eGFP) reporter gene that provides visual detection of reporter gene expression using either fluorescence in live cells or histochemical detection of β-glucuronidase activity. To demonstrate the usefulness of PnapA, L. fendleri was transformed with the pGPro4-PnapA vector. Primary transgenic shoots were generated from explants at an expected frequency of 13 to 23%, indicating that the nos promoter drove sufficient hptII expression to generate hygromycin resistant plants. Five independent transgenic L. fendleri lines were grown to maturity and generated T₁ seeds. Segregation analysis of T₁ seeds indicated that the transgenic L. fendleri lines contain one, two or more integration sites. The gusA-eGFP reporter gene activity was examined in various organs of all these transgenic lines by standard GUS assay. Only seeds showed positive GUS stain, confirming that PnapA confers seed-specific expression in transgenic L. fendleri.