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Faculty of Biology

University of Warmia & Mazury Olsztyn. Faculty of Biology. Dariusz J. Michalczyk. Department of Biochemistry. The effects of chemical modifications on biologically active substances Physico-chemical characteristics of potentially bioactive proteins, peptides and lipophyllic compounds

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Faculty of Biology

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  1. University of Warmia & Mazury Olsztyn Faculty of Biology Dariusz J. Michalczyk

  2. Department of Biochemistry • The effects of chemical modifications on biologically active substances • Physico-chemical characteristics of potentially bioactive proteins, peptides and lipophyllic compounds • Studies on the mechanisms of stress and dormancy in seeds • Biochemical background of seed germination

  3. Department of Animal Physiology • Expression of genes encoding precursors of peptide hormones and receptors • Mechanism of action of prolactin, oxytocin, opioids and xenobiotics in different animal cells

  4. Department of Genetics • Biotechnological methods in plant improvement – DH barley lines, mutagenesis in barley and Avena strigosa; exploitation of somaclonal and gametoclonal variation of in vitro cultures • Molecular evolution of plants

  5. Department of Plant Physiology and Biotechnology • Molecular aspects of plant development (floral induction in Pharbitis nil; genetic regulation of aquaporins; the role of RFOs and galactosyl cyclitols in seed development) • Biotechnological methods in studies of plant secondary metabolism and plant responses to stresses (regulation of PAL, effects of STS transgene expression)

  6. Biosynthesis of resveratrol in transgenic tomato and tobacco

  7. Resveratrol • 3,5,4’- trihydoxystilbene • phytoalexin of grapes and peanuts • health-promoting agent of wine? (antioxidant, cholesterol reducing- and anticancer agent)

  8. Other stilbenes resveratrol glucopyranoside

  9. STS&CHSreactions

  10. STS gene isolation

  11. Transformation method • Mc Cormick S., 1991. • cotyledonary explants from 7 – 10 d. seedlings (cv. Zorza); • 48 h. preculture; transformation with GV3101; • regeneration on MS with 2% sucrose, IAA (0,02 mg/l), zeatine (2 mg/l) + kanamycine + cefotaxime

  12. PCR & RT-PCR transgene plant extracts no transcript callus extracts no transcript transgene

  13. Thin layer chromatography of tomato leaf extracts (under UV) separation start resveratrol standard leaf extracts

  14. TLC of tobacco callus and leaf extracts calluses leaves Rs K_ W C 26 ? reswer. III W1034 IIINTViC26

  15. HPLC st.r. resveratrol – 8,6mg/250ml T NT sts+ K-

  16. p.CaMV+sts gttcctactgaatcaaaggccatggagtcaaaaattcagatcgaggatctaacagaactcgccgtgaagacctggcgaacagttcatacagagtcttttacgactcaatgacaagaagaaaatcttcgtcaacatggtggagcacgacactctcgtctactccaagaatatcaaagatacagtctcagaagaccaaagggctattgagacttttcaacaaagggtaatatcgggaaacctcctcggattccattgcccagctatctgtcacttcatcaaaaggacagtagaaaaggaaggtggcacctacaaatgccatcattgcgataaaggaaaggctatcgttcaagatgcctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgatatctccactgacgtaagggatgacgcacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttggagaggacacgctgaaatcaccagggatccttcctcaacttaatcttaggctttaatttgagtacgtagctgggatcaatggcttcagtcgaggaaattagaaacgctcaacgtgccaagggtccggccaccatcctagccattggcacagctacccccgaccactgtgtctaccagtctgattatgctgattactatttcagggtcactaagagcgagcacatgactgcgttgaagaagaagttcaatcgcatatgtgacaaatccatgatcaagaagcgttacattcatttgaccgaagaaatgcttgaggagcacccaaacattggtgcttatatggctccatctcttaacatacgccaagagattatcactgctgaggtacccaagctcggtaaggaagcagcattgaaggctcttaaagagtggggtcagcctaaatcgaagatcacccaccttgtattttgtaccacctcaggtgtagaaatgcctggtgcagattataaactcgctaatctcttaggcctcgaaccatctgtcagaagagtgatgttggaccagcaagggtgctatgcaggtggaactgtccttcgaaccgctaaggatcttgcagagaataatgcaggagcacgagttcttgtggtgtgctctgagagcacagttgttacatttcgtggcccttccgaagatgctttggactctttagttggccaagccctttttggtgatgggtctgcagctgtaatcgtaggatcagatccggatatctcaattgaacgaccactcttccagcttgtctcagcagcccaaacatttattcctaattctgcaggtgccattgcaggaaacttacgtgaggtgggactcacctttcatttgtggcccaatgtgcccactttaatttctgagaacatagagaaatgtttgactcaggcttttgacccacttggtattagcgattggaactccttattttggattgctcacccaggtggccctgcaattcttgatgcagttgaagcaaaactcaatttagataaaaagaaactcgaagcaacgaggcatgtgctaagtgagtatggaaacatgtcaagtgcatgtgtgttgtttattttggatgagatgagaaagaaatcccttaagggggagagggccaccacgggtgaaggattggattggggagtattatttggttttggaccaggcttgactattgaaactgttgtgttgcatagcattcctatggtgacaaattaagtgaaggaaaagagaatggtcccttcaatgtcctattatgttgaataggagtaaggtattgagctcgaatttccccgatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcg KSKITHLVFCTTSGVEMPGADYKLANLLGLEPSVRRVMLDQQGCYAGGTVLRTAKDLAEN ....................................... !! ................... KSKITHLVFCTTSGVEMPGADYKLANLLGLEPSVRRVMLYHQGCYAGGTVLRTAKDLAEN NAGARVLVVCSESTVVTFRGPSEDALDSLVGQALFGDGSAAVIVGSDPDISIERPLFQLV ............ !............................................... NAGARVLVVCSEITVVTFRGPSEDALDSLVGQALFGDGSAAVIVGSDPDISIERPLFQLV stilbene synthase 3 [Vitis sp. cv. 'Norton'] stilbene synthase [Vitis riparia] stilbene synthase [Vitis labrusca] stilbene synthase [Parthenocissus henryana] stilbene synthase [Cissus rhombifolia] stilbene synthase [Parthenocissus quinquefolia] Gudrun i Joachim Schrőder (1992):The exchange of just one hisitidine to glutamine changes the substrate specificity of stilbene synthase

  17. Basic physiological parameters of transgenic and nontransformed (control) tomato plants control plants transformants

  18. Conclusions • transfer of STS gene to plants lacking it is a simple form of metabolic engineering • strong expression of sts in tobacco plants does not necessarily lead to pollen sterility • tobacco cells can modify substantial part of resveratrol pool e.g. by glucosylation • more detailed analyses are needed and planned for

  19. dr Jacek Olszewski • dr Agnieszka Piotrowicz-Cieślak • dr Wioletta Pluskota • dr Grzegorz Panasiewicz • Michał Kaszuba, Jolanta Nej, Piotr Pupel

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