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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Bioproducts Research » Research » Publications at this Location » Publication #287581
Title: A Napin promoter activates gene expression in developing seeds of Lesquerella fendleri

Author
item Chen, Grace
item Lin, Jiann

Submitted to: Online Journal of Biological Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/25/2012
Publication Date: 9/17/2012
Citation: Chen, G.Q., Lin, J.T. 2012. A Napin promoter activates gene expression in developing seeds of Lesquerella fendleri. Online Journal of Biological Sciences. 12(3):113-117. DOI: 10.3844/ojbssp.2012.113.117.

Interpretive Summary: Lesquerella fendleri (A. Gray) S. Wats. (Brassicaceae), 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. To engineer oils or fatty acid contents in L. fendleri seed, it is preferable to utilize seed specific promoters, to limit the changes of oils and fatty acids to the seed, and to avoid the changes in membrane lipids in other parts of the plant. It is known that napins, a group of storage proteins, are tightly regulated by seed developmental process. The physiological role of napins is to provide the growing seedling with essential nutrients prior to the establishment of the photosynthetic capacity. 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 in pGPro4 vector. This vector contains a promoterless gusA-eGFP (ß-glurcuronidase-enhanced Green Fluorescent Protein) reporter gene, a small size plasmid backbone and many unique restriction sites. More importantly, pGPro4 was designed for precise testing an organ-specific promoter. Because in many popular vectors such as pCAMBIA and pGreen, one overlooked problem 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 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. To avoid the problem, a nopaline sythase promoter (nosP) is used to drive a selectable marker gene, hptII (hygromycin phosphotransferase II), in pGPro4. 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. The results indicate that the napA promoter only drive gene expression in seeds of L. fendleri.

Technical Abstract: Problem statement: 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. Approach: 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 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 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. 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 in pGPro4 vector. This vector contains a promoterless gusA-eGFP (ß-glurcuronidase-enhanced Green Fluorescent Protein) reporter gene, a small size plasmid backbone and many unique restriction sites. More importantly, pGPro4 was designed for precise testing an organ-specific promoter. Because in many popular vectors such as pCAMBIA and pGreen, one overlooked problem 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 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. To avoid the problem, a nopaline sythase promoter (nosP) is used to drive a selectable marker gene, hptII (hygromycin phosphotransferase II), in pGPro4. 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. The results indicate that the napA promoter only drive gene expression in seeds of L. fendleri.