Zbornik - Društvo genetičara Srbije

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68 ZBORNIK ABSTRAKATA III KONGRESA GENETIÈARA SRBIJE III-Usm-1 Subotica, 30. novembar - 4. decembar 2004. PRIMENA GENETIÈKOG ININJERINGA KOD OTPORNOSTI BILJAKA PREMA PATOGENIMA Jelena Boškoviæ 1 , eljana Prijiæ 1 i M. Boškoviæ 2 1 Megatrend univerzitet primenjenih nauka, Beograd 2 Poljoprivredni fakultet, Novi Sad Genetièki ininjering najavljuje novu eru za nauènike, koji se bave istraivanjem odravanja zdravih biljaka, sa optimalnim prinosima useva i minimalnom primenom pesticida. Nauènici iz celog sveta istrauju biohemijsku prirodu i vanost biljnih reakcija prema patogenima i njihovom razvoju. Geni otpornosti kod biljaka i oni koji su odgovorni za reakcije otpornosti, identifikuju se i ininjeringom prenose u biljne vrste u cilju njihove zaštite prema patogenima. Odreðivanje ovih gena stvara moguænosti za postepeno vrednovanje njihovih specifiènih uloga i vanosti u reakciji patogeneze prema parazitima upotrebom transgenih biljaka, koje su dobijene tehnikom genetièkog ininjeringa. Poslednje primenjene tehnike molekularne biologije kod biljaka i genetièki ininjering za studije interakcija domaæina i patogena su rezultirale za identifikacije kloniranja brojnih gena ukljuèenih u reakciju odbrane biljaka posle infekcije sa patogenima. To obuhvata gene sa ekspresijom proteina, peptida ili antimikrobnih jedinjenja koji su direktno toksièni prema patogenima. Genetièki produkti neposredno spreèavaju proizvode virulentnosti patogena ili podstièu strukturalne gene odbrane, koji direktno ili indirektno aktiviraju opšte reakcije odbrane biljke, i gene otpornosti kod hipersenzitivnih reakcija u interakcijama sa avirulentnim faktorima. U ovom radu se pregledno iznose ostvareni rezultati kod korišæenja širokog spektra kloniranih gena u poveæanju otpornosti prema gljiviènim patogenima i virusima u transgenim biljkama i upuæuju na buduæa osporovanja i perspektive. Koncept programiranog izumiranja æelija (PCD) i fitoaleksini imaju posebnu ulogu kod razjašnjenja molekularne osnove induciranih reakcija odbrane biljke od patogena. APPLICATIONS OF GENETIC ENGINEERING IN RESISTANCE TO PLANT PATHOGENS Genetic engineering ushers in new era for plant scientists working tomaintainhealthy plants, optimize crop yields, and minimize pesticide usage. Scientists from all over the world are investigating the biochemical nature of, and the signals involved in, a plants reactions to pathogens invasion and pathogens development. Plant resistance genes and genes involved in resistance reactions are being identified an engineered into crop plants to protect them against plant pathogens. The identifications of these genes have made it possible to subsequently evaluate their specific roles and importance in disease response pathways using transgenic plants developed with genetic engineering techniques. Recent applications of technique in plant molecular biology and genetic engineering to study of host pathogen interactions have resulted in the identifications on cloning of numerous genes involved in the defense responses of plants following pathogen infection. These include genes that expresses proteins, peptides or antimicrobial compounds that are directly toxic to pathogens. Genes products directly inhibit pathogen virulence products or enhance plant structural defense genes, that directly or indirectly activate general plant defense response, and resistance genes in the hypersensitive response and in the interactions with avirulence factors. In this paper is reviewed advances made in utilizing a broad range of cloned genes to enhance disease resistance against fungal pathogens and viruses, in transgenic plants and address future challenges and prospect. The concept of programmed cell death (PCD) and phytoalexins played a special role in the molecular basis of inducible defense reactions
III-Usm-2 ZBORNIK ABSTRAKATA III KONGRESA GENETIÈARA SRBIJE 69 Subotica, 30. novembar - 4. decembar 2004. ANALIZA SKRAÆENOG KgmB PROTEINA IZ S. tenebrarius Sandra Markoviæ, Sandra Vojnoviæ i Branka Vasiljeviæ Institut za molekularnu genetiku i genetièko inenjerstvo, Beograd kgmB gen iz proizvoðaèa nebramicinskog kompleksa antibiotika, Streptomyces tenebrarius, kodira KgmB metilazu koja obezbeðuje rezistenciju na kanamicin i gentamicin. KgmB metilaza modifikuje ciljno mesto na 16S rRNK i na taj naèin štiti proizvoðaèa od sopstvenog toksiènog produkta. Ekspresija kgmB gena je autoregulisana na nivou translacije. Regulatorni model je najpre testiran in vivo korišæenjem kgmB-lacZ genskih fuzija. U cilju prouèavanja ovog procesa in vitro, KgmB protein je eksprimiran u E. coli i preèišæen pomoæu QIAexpress sistema. Nedavno je pokazano da inaktivirani kgmB gen (inaktivacija u BglII restrikcionom mestu koja dovodi do sinteze skraæenog KgmB proteina sa samo 66 originalnih aminokiselina) smanjuje aktivnost kgmB-lacZ genske fuzije. Ovaj rezultat ukazuje da je N-terminalni region KgmB metilaze zaduen za regulaciju t.j. za vezivanje za iRNK. Da bi se ispitale osobine i funkcionalnost N-terminalnog regiona KgmB metilaze inaktivirani kgmB gen je kloniran u pQE30 ekspresioni vektor. Eksprimirani protein obeleen histidinskim repiæem je preèišæen metal afinitetnom hromatografijom. Preèišæen skraæeni KgmB protein je analiziran elektroforetski i Western blotom. Funkcionalnost skraæenog KgmB proteina biæe ispitana eksperimentima RNK-gel šifta i uporeðena sa intaktnom KgmB metilazom. ANALYSIS OF TRUNCATED KgmB METHYLASE FROM S. tenebrarius The kgmB gene from Streptomyces tenebrarius, producer of nebramycin complex of antibiotics, encodes for kanamycin-gentamicin resistance methylase, KgmB. KgmB methylase modifies the target site on 16S rRNA and thus protect producer against its own toxic product. The kgmB gene is autogenously regulated at translational level. A regulatory model was first tested in vivo using kgmB-lacZ gene fusions. In order to study this process in vitro KgmB protein was overexpressed in E. coli and purified using QIAexpress System. Recent results showed that inactivated kgmB gene (inactivation in BglII restriction site which gives truncated protein with only 66 original amino acids) is able to repress activity of kgmB-lacZ gene fusions. This finding suggests that N-terminal region of KgmB methylase is responsible for regulation i.e. for binding to mRNA. With the intention of analyzing properties and functionality of N-terminal region of KgmB protein inactivated kgmB gene was cloned in pQE30 expression vector to make a construct that places the 6xHis tag at the N-terminus of the protein. For purification of 6xHis-tagged protein metal affinity chromatography was used. Purified truncated KgmB protein was analyzed electrophoreticaly and on Western blot. Functionality of truncated KgmB protein would be examined by RNA-gel shift experiments and compared with intact KgmB methylase.
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DRUŠTVO GENETIČARA SRBIJE SERBIAN
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ZAVRŠNO PREDAVANJE CLOSING LECTURE
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A Aksentijeviæ I. 194 Aleksiæ J.
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Stanojeviæ B. 223 Stanojeviæ J. 2
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Zbornik - Društvo genetičara Srbije

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