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Genetically Engineering Plants With A Gene For Mercuricion Reduction And Resistance

We are engineering a bacterial gene encoding mercuric reductase for the production of plants with the ability to electrochemically reduce toxic, ionic mercury. Bacterial mercury resistance operons have been described in detail (Summers, 1986). One gene contained in the polycistron is merA, which codes for mercuric ion reductase. This enzyme catalyzes the reduction of toxic Hg++ to far less toxic Hgo . The coding sequence of the bacterial gene is very GC-rich, possibly preventing observable expression in transgenic plants. We designed a merA sequence that contained codons more typical of highly expressed plant genes. Overlap Extension-PCR (OE-PCR) was used to modify the codons in a 9% block of coding sequence and to alter the non-coding regulatory sequences. Agrobacterium-mediated transformation was used to produce Arabidopsis having the modified gene, merA9. These plants are capable of germinating and growing to seed set on medium containing up to 100 mercuric chloride, whereas control seeds fail to grow on 25 µ.M Hg++. Full length mRNA was detected in mercury resistant plants using northern blot hybridization. Mercury-resistant transgenic Arabidopsis seedlings were observed to evolve Hg o vapor from buffered HgC1 2 at up to three times the rate of control seedlings. We are continuing to investigate the effect of further sequence modification for the optimization of merA gene expression in transgenic plants. Ultimately, we intend to develop transgenic tree species transformed with modified merA constructs. We have developed reliable and efficient protocols for tissue culture propagation, plant regeneration and gene transfer for yellow-poplar (Liriodendron tulipifera). Our progress towards the production of merA transgenic yellow-poplar is discussed.


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Author(s): Clayton L. Rugh, H. Dayton Wilde, Scott A. Merkle, Richard B. Meagher

Publication: Tree Improvement and Genetics - Southern Forest Tree Improvement Conference - 1995