Transformation of <i>Lesquerella Fendleri</i> with the New Binary Vector pGPro4-35S

Abstract

Problem statement:Crop genetic engineering requires the use of various promoters to control the expression of introduced transgenes. Some of the binary vectors currently available for promoter characterization in dicotyledonous plants have pitfalls due to their construction, such as containing a selectable marker cassette with enhancer sequences that can potentially interfere with the expression specificity of nearby promoters. Also, many binary vectors are quite large in size and contain few useful restriction sites making their in vitro manipulation technically challenging. Approach: A small (7698 bp) and flexible binary vector named pGPro4 was constructed to possess unique features favorable for promoter analysis in dicot plants. A nopaline synthase (nos) promoter was used to control the expression of the selectable marker of pGPro4 to prevent the problem of interference with the neighboring promoter-reporter fusion. In pGPro4, the nos promoter and hygromycin phosphotransferase II (hptII) sequences are flanked by loxP sites, which allow for Cre recombinase-mediated removal when hygromycin resistance is no longer desired. pGPro4 also contains a bifunctional ß-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. Results and Conclusion: To demonstrate the usefulness of the pGPro4 vector, a CaMV35S promoter was fused to gusA-eGFP and the resulting plasmid, pGPro4-35S, was used to transform Lesquerella fendleri. Primary shoots were generated from explants at an expected frequency of 10-27.5%, indicating that the nos promoter drove sufficient hptII expression to generate hygromycin resistant plants. Six independent transgenic L. fendleri lines were grown to maturity and generated T 1 seeds. The bifunctionality of the gusA-eGFP reporter gene was verified by detecting both green fluorescence and β-glucuronidase activity in multiple T 1 L. fendleri seedlings from 5 of the 6 the independent transgenic lines.

A NAPIN PROMOTER ACTIVATES GENE EXPRESSION IN DEVELOPING SEEDS OF <i>LESQUERELLA FENDLERI</i>

“…The vector map of the pGPro4 vector was described (Chen et al, 2011) and the annotated sequence is available from Genbank (accession JN593324). The promoter sequence of napA from -397 to -1 was synthesized by using PCR and the genomic DNA of Brassic napus, cultivar svaloefs karab (USDA-ARS germplams accession number PI 535869) as template.…”

“…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 pGPro4 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.…”

Comparative Genomics of Domesticated and Wild Sunflower: Complete Chloroplast and Mitochondrial Genomes

“…In addition, WGS data make possible creation of DNA markers (SSR, CAPS etc). As well as complete genome sequences can be used for developing specific transformation vectors (Chen et al, 2011) or other genetic engineering applications.…”