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Regulation of gene expression of metallothionein type 3 and buckwheat protein MT3 role in defending the plant tissues from stress induced by heavy metals

dc.contributor.advisorMaksimović, Vesna
dc.contributor.otherRadović, Svetlana
dc.contributor.otherDunđerski, Jadranka.
dc.creatorNikolić, Dragana B.
dc.date.accessioned2019-04-22T11:58:59Z
dc.date.available2019-04-22T11:58:59Z
dc.date.available2020-07-03T08:14:30Z
dc.date.issued2010
dc.identifier.urihttp://eteze.bg.ac.rs/application/showtheses?thesesId=6623
dc.identifier.urihttp://nardus.mpn.gov.rs/handle/123456789/10967
dc.identifier.urihttps://fedorabg.bg.ac.rs/fedora/get/o:19433/bdef:Content/download
dc.identifier.urihttp://vbs.rs/scripts/cobiss?command=DISPLAY&base=70036&RID=37014543
dc.description.abstractIz biblioteke cDNK semena heljde u srednjoj fazi sazrevanja prethodno su izolovana dva klona cDNK koja kodiraju metalotioneinu-sličan protein. Izolovan je i odgovarajući genomski klon koji obuhvata deo kodirajućeg regiona kao i 71 bp 5`UTR i 569 bp promotorskog regiona. U ovom radu, izolovani promotorski region gena FeMT3, kao i dve njegove 5` delecione varijante, funkcionalno su analizirani u stabilno transformisanom duvanu. Histohemijskim esejem GUS je detektovana intenzivna aktivnost promotorskih fragmenata u vaskulatnim elementima lista i u polenu, a slabija aktivnost uočena je i u tkivu korena. Kvantitativnim esejem GUS je pokazano značajno povećanje aktivnosti sva tri promotorska fragmenta (proporcionalno njihovim dužinama) u odgovoru na uslove kompleksnog stresa, odnosno u listovima potopljenim u MS medijum sa saharozom. Jaka indukcija promotorske aktivnosti je uočena i pod dejstvom jona Cu2+ i Cd2+. Navedeni rezultati sugerišu postojanje kompleksne transkripcione regulacije gena FeMT3 i učešće većeg broja različitih faktora. Povećanje nivoa transkripta FeMT3 u listu heljde pod uticajem jona Cu2+ i Cd2+ potvrđeno je metodom "Real-time RT-PCR". U cilju približavanja ulozi koju FeMT3 ima u biljnom tkivu, testirane su protektivne sposobnosti proteina kodiranog ovim genom u živim sistemima izloženim dejstvu teških metala. Odbrambene sposobnobnosti FeMT3 su potvrđene u prokariotskom sistemu E. coli i u tranzijentno transformisanim listovima duvana N. debneyii. Izlaganje visokim koncentracijama CdCl2 ili CuSO4, izazivalo je značajno sporiji razvoj oštećenja u tkivu koje je eksprimiralo FeMT3 u poređenju sa kontrolom. Unutarćelijska lokalizacija FeMT3 je praćena u fuziji sa fluorescentnim proteinom (YFP) u transformisanim ćelijama duvana. Proteinska fuzija YFP-FeMT3 je uočena u citoplazmi, a odsustvovala je u vakuoli i hloroplastima. Lokalizacija se nije promenila ni nakon tretmana teškim metalima, što sugeriše drugačiji mehanizam odbrambenog delovanja metalotioneina u odnosu na fitohelatine. Svojstva promotora FeMT3 i proteina kodiranog ovim genom koja su pokazana u ovom radu, ukazuju na potencijal gena FeMT3 za primenu u fitoremedijaciji.sr
dc.description.abstractTwo cDNA clones coding for metallothionein-like protein (FeMT3) were isolated previously from the cDNA library of developing buckwheat seeds. The corresponding genomic clone, containing part of the coding region, as well as 71 bp of the 5' UTR and 569 bp of the promoter region, was also isolated. To shed light on expression regulation of the metallothionein gene from buckwheat (FeMT3), herein functional promoter analysis was performed with a complete 5’ regulatory region and two deletion variants, employing stably transformed tobacco plants. Histochemical GUS assay of transgenic tobacco lines showed the strong signals in vascular elements of leaves and in pollen grains, while somewhat weaker staining was observed in the roots of mature plants. Quantitative GUS assay showed strong up-regulation of all three promoter constructs (proportional to the length of the regulatory region) in leaves submerged in liquid MS medium containing sucrose, after a prolonged time period, representing a complex stress situation composed of several synergistically related stress stimuli. Functional analysis of the promoter region also revealed extremely high inducibility upon Cu2+ and Cd2+ treatments. These findings suggest complex transcriptional regulation of FeMT3, requiring interaction of a number of different factors. FeMT3 transcription level increase under the influence of Cd2+ and Cu2+, in buckwheat leaves, was confirmed by Real-time RT-PCR. The protective role in vivo of FeMT3 during metal stress was examined. Increased tolerance to heavy metals of FeMT3 producing E. coli cells was detected. The defensive ability of buckwheat MT3 during Cd2+ and Cu2+ stresses was also demonstrated in Nicotiana debneyii leaves transiently expressing FeMT3. When exposed to CdCl2 or CuSO4, FeMT3 overexpressing leaf regions showed much slower development of tissue damage, compared with mock transformed regions. To reveal intracellular localization of FeMT3, a protein fusion with yellow fluorescent protein (YFP) was used. YFP-FeMT3 was localized in cytoplasm and was absent from vacuole and chloroplasts. In contrast to phytochelatins, the cytoplasmatic localization of FeMT3 was not altered under heavy metal stress. The confirmed defense ability of FeMT3 protein in vivo and the great responsiveness of its promoter during heavy metal exposure, make this gene a suitable candidate for biotechnological applications.en
dc.formatapplication/pdf
dc.languageen
dc.publisherУниверзитет у Београду, Биолошки факултетsr
dc.rightsopenAccessen
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/4.0/
dc.sourceУниверзитет у Београдуsr
dc.titleRegulacija ekspresije gena za metalotionein tipa 3 heljde i uloga proteina MT3 u odbrani biljnih tkiva od stresa izazvanog teškim metalimasr
dc.title.alternativeRegulation of gene expression of metallothionein type 3 and buckwheat protein MT3 role in defending the plant tissues from stress induced by heavy metalsen
dc.typedoctoralThesisen
dc.rights.licenseBY-NC-ND
dc.identifier.fulltexthttp://nardus.mpn.gov.rs/bitstream/id/3352/Disertacija.pdf
dc.identifier.rcubhttps://hdl.handle.net/21.15107/rcub_nardus_10967


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