Phylogenetic relationships within the genus Rana (Amphibia, Ranidae) based on a method RFLP and 16S, cytb, ITS2 sequence analysis

Abstract

Proteini MAB sadrţe dvije funkcionalno bitne interakcijske domene: MATH i BTB. Do sada je pokazano da su oni dio sustava eliminacije proteina koji stanici više nisu potrebni. Neki od istraženih proteina MAB (CeMEL-26, HsSPOP) dio su multiproteinskih kompleksa kulin 3 E3 ligaza i sluţe za prepoznavanje supstrata koje treba razgraditi. Tijekom razvoja pšenice ekspresija gena TaMAB2 prisutna je samo u oplođenoj jajnoj stanici, u zigoti te u dvostaničnom embriju. U ovom radu napravljena je heterologna ekspresija gena TaMAB2 iz pšenice u uročnjaku. Utvrđeno je da transgenične biljke proizvode cjeloviti protein TaMAB2 koji se u stanicama nakuplja. Sam protein TaMAB2 je ubikvitiniran, te vjerojatno stvara homo- i heterodimere. Rast i razvoj transgeničnih biljaka je značajno fenotipski promijenjen u odnosu na biljke divljeg tipa ali im fototropizam i gravitropizam nisu poremećeni. Analizom unutarstanične lokalizacije proteina TaMAB2 u stanicama BY-2 i pokožici luka utvrđeno je nakupljanje velikih agregata oko jezgrine ovojnice i u blizini stanične membrane. Ovi rezultati ukazuju da bi protein TaMAB2 mogao imati bitnu ulogu u eliminaciji proteina i uspostavljanju polarnosti tijekom rane embriogeneze pšenice.Water frogs (genus Rana, subgenus Pelophylax) present an interesting research subject for ecology, physiology and genetics. Rana kl. esculenta hybridogenetic complex, which consists of two parental species (Rana ridibunda and Rana lessonae) and their hybrid species (Rana kl. esculenta), is of special interest. Hybrids often show morphological traits similar to either parent, making them harder to identify on the field. Because of that, more relevance is given to molecular and phylogenetic methods in the identification of water frog species. Identification by morphological traits has shown that in central Croatia both parental populations of R. ridibunda and R. lessonae can inhabit the same location and thus lead to the formation of hybrids. The goal of this project was to determine the phylogenetic relationships of water frog populations from Crna Mlaka based on 16S rRNA, cytochrome b and ITS2sequence analysis, and to examine the efficiency of a simple RFLP method for identifying parental species and hybrids based on the digestion of a conserved ITS2 fragment with three restriction enzymes (KpnI, HaeII and SmaI). Results of this phylogenetic analysis have shown that both parental species R. ridibunda and R. lessonae, and thus potential hybrids, inhabit Crna Mlaka. Phylogenetic analysis of mitochondrial 16S rRNA and cytb genes proved to be the best method for species and haplotype identification, while the RFLP method and phylogenetic analysis of nuclear ITS2 regions, which could help differentiate hybrid samples, need further protocol optimization