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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Crop Improvement and Genetics Research » Research » Publications at this Location » Publication #261160
Title: Pollen-specific transgene excision in tobacco using the codon optimized serine resolvase CinHo-RS2 site-specific recombination system

Author
item MOON, HONG - University Of Tennessee
item ABERCROMBIE, LAURA - University Of Tennessee
item EDA, SHIGETOSHI - University Of Tennessee
item BLANVILLAIN, ROBERT - University Of California
item Thomson, James - Jim
item OW, DAVID - Chinese Academy Of Sciences
item STEWART, NEAL - University Of Tennessee

Submitted to: Plant Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/1/2011
Publication Date: 4/10/2011
Citation: Moon, H.S., Abercrombie, L.L., Eda, S., Blanvillain, R., Thomson, J.G., Ow, D.W., Stewart, N.C. 2011. Pollen-specific transgene excision in tobacco using the codon optimized serine resolvase CinHo-RS2 site-specific recombination system. Plant Molecular Biology. 75(6):621-631.

Interpretive Summary: This research investigates the utility of the recombinase CinH for use in tobacco. A system was designed to study CinH for auto-excision (deletion) of unwanted transgenic DNA from the genome of tobacco pollen to limit gene flow to surrounding fields. Results indicate that the CinH recombinase can be up to 99% effective at transgene excision from the host genome.

Technical Abstract: Transgene escape, a major environmental and regulatory concern in transgenic crop cultivation, could be alleviated by removing transgene from pollen, the most frequent carrier of transgene. A vector containing a codon optimized serine resolvase CinH recombinase (CinHo) and its recognition sites RS2 was constructed and transformed into tobacco (Nicotiana tobacum cv. Xanthi). Codon usage optimized CinHo recombinase recognized 119 bp of nucleic acid sequences, RS2, in pollen and excised the transgene flanked by the RS2 sites. In this system, the pollen-specific LAT52 promoter from tomato was employed to control the expression of CinHo recombinase. Loss of expression of green fluorescent protein (GFP) gene under the control of the LAT59 promoter from tomato was used as an indicator of transgene excision. Efficiency of transgene excision from pollen was determined by flow cytometry (FCM)-based transgenic pollen screening. While a transgenic event in the absence of CinHo recombinase contained about 70% of GFP-synthesizing pollen, three single-copy transgene events contained less than 1% of transgenic GFP-synthesizing pollen based on 30,000 pollen grains analyzed per event. This suggests that CinHo-RS2 recombination system could be effectively utilized for transgene biocontainment strategy after further optimization. This research represents the first instance of using the CinHo-RS2 recombination system in transgenic plants.