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  • Juranić, Martina

    2013
    Web Resource

    Razmnožavanje u biljaka ovisi o pravilno razvijenom muškom i ženskom gametofitu. S obzirom na tešku dostupnost i vremensku ograničenost gametofita u životnom ciklusu biljke, proučavanje procesa koji dovode do razvoja gametofita predstavlja izazov. Usavršavanjem tehnika mikrodisekcije i novih metoda analize cjelokupnog transkriptoma, napravljen je korak prema razumijevanju molekularnih mehanizama uključenih u ove procese. Cilj ovog doktorskog rada je funkcionalna karakterizacija MATHBTB proteina iz kukuruza (ZmMAB1) čiji je gen višestruko zastupljen u cDNA bibliotekama jajne stanice i zigote kukuruza. Filogenetička analiza je ukazala da ZmMAB1 pripada proširenoj grupi gena u MATHBTB porodici koja broji 31 član i prethodno nije opisana u kukuruzu. Utišavanaje gena ZmMAB1 putem RNAi dovelo je do gubitka kromosoma i kraćeg diobenog vretena u mejozi tijekom mikrosporogeneze. Prijelazom iz mejoze u mitozu došlo je do nepotpunog razdvajanja dvaju jezgara na suprotne polove što je onemogućilo daljni razvitak muškog i ženskog gametofita u mab1 (RNAi) mutantima. Takav fenotip zajedno sa vrlo specifičnom ekspresijom ZmMAB1 tijekom prve mitotske diobe u mega- i mikrogametogenezi, te zigoti upućuje na ulogu proteina ZmMAB1 u asimetričnim diobama. Ta uloga je potvrđena i heterolognom ekspresijom gena ZmMAB1 u uročnjaku. Također, ZmMAB1 intereagira sa kulinom 3, te kao dio kompleksa ubikvitin ligaze E3 sudjeluje u procesu proteasomalne razgradnje što ujedno daje i naznaku o regulaciji mejoze i mitoze kroz selektivnu ubikvitinizaciju. Plant reproduction depends on proper development of the male and the female gametophyte. Due to the reduced size of the angiosperm gametophyte and especially inaccessibility of the female gametophyte, it is challenging to study these fundamental processes. Recent advances in microdissection techniques combined with powerful tools for transcriptome analysis have begun to shed a light on molecular mechanisms underlying the processes. The aim of this thesis was to functionally characterize a maize MATH-BTB (ZmMAB1) protein, which was encoded by a gene highly represented in cDNA libraries of isolated maize egg cells and zygotes. A phylogenetic analysis has shown that ZmMAB1 belongs to an expended group of a previously undescribed maize MATH-BTB gene family with 31 members. RNAi silencing of ZmMAB1 leads to chromosome segregation defects and short spindles during male meiosis. After meiosis-mitosis transition two identical nuclei are formed in mab1 (RNAi) mutants disrupting male and female germline progression. Cell division phenotypes of mutant plants together with its specific gene expression profile during the first mitotic division during male and female gametogenesis and in the zygote implied the importance of ZmMAB1 during asymmetric cell divisions. This hypothesis was further confirmed by heterologous expression of ZmMAB1 in Arabidopsis thaliana. Additionally, interaction of ZmMAB1 with cullin 3 suggests its role in proteasomal degradation as a part of an ubiquitin E3 ligase complex. These finding provide novel insights into the meiotic and mitotic regulatory network through selective ubiqiuitination.