A NAPIN PROMOTER ACTIVATES GENE EXPRESSION IN DEVELOPING SEEDS OF &lt;i&gt;LESQUERELLA FENDLERI&lt;/i&gt;

Chen Chen

doi:10.3844/ojbsci.2012.113.117

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 ?-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 ?-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.

Highlights

To improve oil and Hydroxy Fatty Acids (HFA) of L. fendleri, it is desirable to use of seed-specific promoters to control the expression of target genes by genetic engineering
A nopaline synthase promoter was used to control the expression of the selectable marker of pGPro4. pGPro4 contains a bifunctional β-glucuronidase-enhanced Green Fluorescent Protein reporter gene that provides visual detection of reporter gene expression using either fluorescence in live cells or histochemical detection of β-glucuronidase activity
L. fendleri is amenable to Agrobacterium-mediated transformation (Chen, 2011; Skarjinskaia et al, 2003; Wang et al, 2008), genetic transformation provides an alternative means to improve this crop

Summary

INTRODUCTION

We have cloned and sequenced the upstream regulatory region of napA gene to test the tissue specific expression pattern of this promoter in L. fendleri. We constructed the promoter sequence from -397 to -1 (corresponding to Genbank accession J02798, 708 to 1145) in pGPro vector (Chen et al, 2011) (GenBank accessions JN593324) This vector contains a promoterless gusA-eGFP (β-glurcuronidase-enhanced Green Fluorescent Protein) reporter gene, a small size plasmid backbone and many unique restriction sites. When an organ-specific promoter-target gene cassette is placed within a such vector, the enhancer can bidirectionally interfere with the transcription of the target gene as well as nearby genes, affecting the fidelity of the organ-specific gene expression and/or causing unintended misexpression of nearby genes (Yoo et al, 2005; Xie et al, 2001) To avoid this problem, a nopaline sythase promoter (nosP) is used to drive a selectable marker gene, hptII (hygromycin phosphotransferase II), in pGPro. We report here the construction of the napA promoter to upstream of gusA gene and analysis the tissue specific pattern of this promoter in transgenic L. fendleri carrying this construct

MATERIALS AND METHODS

Detection of β-Glucuronidase Gene Activity

RESULTS AND DISCUSSION

Tissue Specificity of PnapA

CONCLUSION