Yemets A. Key role of tubulin in molecular and cellular mechanisms of plant resistance to herbicides with antimitotic mode of action

The thesis is devoted to investigation a tubulin structural, molecular and biological peculiarities permitting a key role in realization molecular and cell mechanisms of plant resistance to herbicides with anti-mitotic mode of action (dinitroanilines and phosphorothioamidates); in elaboration a new approaches for goal-seeking search of new compounds with anti-microtubular activity, effective methods for polyploidisation and micronucleation of plant cells, and usage of a mutant tubulin genes in the capacity of selective marker genes in genetic engineering for production transgenic plants resistant to these herbicides. Particularly, for the first time the mechanism of resistance to isopropyl-N-phenyl carbamate have been investigated in obtained mutants Nicotiana sylvestris and have been shown that they are the mutants on proteins of microtubule organizing centres. It was established that obtained lines Nicotiana plumbaginifolia resistant to dinitroaniline trifluralin (with cross-resistance to another dinitroanilines and phosphorothioamidates) possess a mutant beta-tubulin subunit carrying substitution of Phen317 to Ser, and lines N. plumbaginifolia resistant to phosphorothioamidate amiprophosmethyl - of Ser248 to Pro. Using cell cultures of goosegrass Eleusine indica as a model to study the tubulin role in the mechanisms of resistance to dinitroanilines and phosphorothioamidates it has been proved that missens mutation in alpha-tubulin molecule (substitution The239 to Ile) provides the resistance to these herbicides in E.indica cells. It was shown also that beta- and gamma-tubulin molecules do not possess the same site of herbicide interaction in homological fragments of their molecules. Based on analysis of structural peculariaties of dinitroaniline and phosphorothioamidate binding sites with alpha-tubulin the chemical design of new dinitroaniline derivatives and the screening their biological activities were carried out. High-performance methods of plant cell polyploidisation and micronucleation with use dinitroanilines, phosphorothioamidates and phenylcarbamates have been worked out. For the first time it has been demonstrated the application of mutant tubulin gene carrying dinitroaniline-resistance as as a safe selective marker plant gene in cellular and genetic engineering of plants. For the first time transgenic lines of monocotyledonous (finger millet) and dicotyledonous (tobacco, soybean, flax) plants with resistance to dinitroanilines and phosphorothioamidates have been obtained using mutant alpha-tubulin gene.