The expressed sequence tags, derived from the developing tilapia brain, were established in our laboratory. An unique gene, high mobility group proteins 2 (HMG2), were investigated. HMG2 is a non-histone chromatin protein. HMG2 cloned from tilapia, Oreochromis mossambicus, is a gene with open reading frame 642bps, and deduced as 213 amino acid protein sequence. NCBI Conserved Domain search, the Gene Ontology, NEBcutter restrict enzyme analysis, NCBI blastx, and neighbor phylogenetic tree were used for the bioinformatic analysis. In the present study, the protein expression of HMG2 in the BL21 E.coli system, a prokaryotic system, and purified with 6X His Tag NI-NTA affinity chromatography.

The cyst nematode, Globodera tabacum tabacum Behrens, and the parasitic angiosperm, Egyptian broomrape, Orobanche aegyptiaca Pers., are obligate root parasites that cause severe yield and quality loss of many important crop hosts. Although these represent two diverse classes of parasites, they have significant similarities in the modes of parasitism and complex interactions with their hosts. Conventional control methods have had limited success in controlling these parasites. The overall objective of this research was to engineer resistance to the cyst nematode and Egyptian broomrape by expressing genes encoding parasite specific toxins under the control of parasite-responsive promoters using tobacco (Nicotiana tabacum L. cv. Xanthi). For nematode resistance, an anti-feeding strategy was employed utilizing the tomato proteinase inhibitor I (PI-I) gene as a nematode specific toxin. Transgenic tobacco plants were generated that expressed genes encoding an intracellarly retained or secreted form of tomato PI-I under the control of the nematode-inducible promoter, derived from tomato (Lycopersicon esculentum L.) Hmg2 gene. Our goals were to determine the effectiveness of local PI-I expression on nematode resistance and to determine if intracellular or extracellular PI-I deposition enhances resistance. Two constructs were generated that contained either the coding region of the tomato PI-I gene, lacking the signal sequence (EM1), or the coding region of PI-I including the signal sequence (EM2), fused to the nematode-responsive Hmg2 promoter. Transgenic PI-I plants were inoculated with G. t. tabacum cysts and evaluated for nematode interactions. Our results suggest that local expression of intercellular of PI-I significantly reduced cyst production when compared to the nontransformed controls. For broomrape resistance, a well characterized R/avr gene pair, the tobacco N resistance gene and the tobacco mosaic virus replicase (TMV) gene, was utilized to create novel gene-for-gene resistance via a N gene-mediated hypersensitive response (HR) to limit broomrape parasitism. The bean (Phaselous vulgaris L.) chalcone synthase 8 (CHS8) promoter has been characterized as a broomrape–responsive promoter. We introduced the CHS8:TMV replicase gene construct into tobacco plants that contains an endogenous N gene. Transgenic tobacco plants were inoculated with O. aegyptiaca seeds and monitored for parasite attachment and development. The expression of the TMV replicase leads to a significant reduction in broomrape parasitism. These genetic engineering strategies show promise in enhancing resistance to these destructive parasites. / Ph. D.