Submitted to: Online Journal of Biological Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/5/2012
Publication Date: 1/19/2012
Citation: Chen, G.Q., Thilmony, R.L., Lin, J.T. 2012. Transformation of Lesquerella fendleri with the new binary vector pGPro4-35S. Online Journal of Biological Sciences. 11(3)90-95. DOI: 10.3844/ojbsci.2011.90.95.
Interpretive Summary: Lesquerella fendleri, under development as a new industrial oilseed crop in the southwestern region of the U.S., is valued for its unusual hydroxy fatty acid (HFA). HFAs and derivatives are used as raw materials for numerous industrial products, such as lubricants, plasticizers and surfactants. L. fendleri is amenable to Agrobacterium-mediated transformation, thus genetic transformation provides an alternative means to improve this crop. One of the essential components of Agrobacterium-mediated transformation system is a binary vector. One sometimes overlooked problem in many popular vectors is the use of the CaMV35S promoter with one or more enhancer sequences to drive selectable marker gene expression. When an organ-specific promoter-target gene casette 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. To avoid this problem, we describe the construction of two new binary vectors, pGPro4 and its derivative pGPro4-35S. The pGPro4-35S vector carries the double enhanced version of the CaMV 35S promoter fused to the bifunctional ß-glucuronidase-enhanced Green Fluorescent Protein (gusA-eGFP) reporter gene. We examine the utility of this novel vector by genetic transformation of L. fendleri and investigate its functionality by visualizing reporter gene activity in the transgenic plants produced.
Technical Abstract: 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 ß-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% to 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 T1 seeds. The bifunctionality of the gusA-eGFP reporter gene was verified by detecting both green fluorescence and ß–glucuronidase activity in multiple T1 L. fendleri seedlings from 5 of the 6 the independent transgenic lines.