Alternativno prekrajanje gena BPM1, BPM2 i BPM3 u uvjetima temperaturnog stresa

Članovi proteinske porodice MATH-BTB/POZ, koji sudjeluju u degradaciji proteina ovisnoj o ubikvitinu, utječu na odgovor biljke u stresnim uvjetima kroz interakciju s različitim transkripcijskim faktorima. Šest gena (BPM1-6) u uročnjaku (Arabidopsis thaliana L.) kodira za najmanje 16 različitih proteina BPM koji nastaju u procesu alternativnog prekrajanja gena. Novo istraživanje je pokazalo induciranu ekspresiju gena BPM1-3 te različiti intenzitet umnažanja varijanti gena BPM1 tijekom izlaganja biljke povišenoj temperaturi. U ovom radu je istražen učinak temperature na ekspresiju varijanti gena BPM1-3 pomoću lančane reakcije polimerazom. Osim toga nastojalo se ispitati postojanje novih ekspresijskih varijanti koje nastaju pod utjecajem temperaturnih varijacija. U tu svrhu dizajnirane su i optimizirane specifične početnice za umnažanje varijanti gena BPM1-3. Iz klijanaca uročnjaka izloženih različitim temperaturama izolirana je cDNA koja je korištena kao kalup u umnažanju, a fragmenti koji su se umnožili ugrađeni su u vektor pGEM®-T Easy tehnikom TA kloniranja. Kako bi se usporedila sličnost s postojećim varijantama gena BPM1-3, nova varijanta gena je sekvencirana. Potvrđeno je da su genska ekspresija i alternativno prekrajanje gena BPM1-3 inducirani temperaturom. Otkrivena je nova varijanta gena BPM2.6 koja se sintetizira u normalnim uvjetima rasta i u uvjetima povišene temperature. S obzirom na izostanak domene BTB/POZ koja je neophodna za interakciju s kulinom, otkriće ove varijante predstavlja osnovu za daljnja istraživanja u okviru uloga proteina BPM neovisnih o kulinu. Members of MATH-BTB/POZ protein family, which participate in the ubiquitin-dependent protein degradation, affect plant response under stress conditions through the interaction with various transcription factors. Six genes (BPM1-6) in Arabidopsis thaliana L. encode for at least 16 different BPM proteins in the process of alternative gene splicing. A new research has shown an induced expression of BPM1-3 genes and a differential expression of BPM1 gene variants during plant exposure to elevated temperature. Here, effects of temperature on the expression of BPM1-3 gene variants were analyzed by using PCR method. Furthermore, the existence of new splicing variants that could be generated due to temperature variations was examined in this work. For this purpose, gene-specific primers for amplification of BPM1-3 gene variants were designed and optimized. cDNA was generated from A. thaliana seedlings which had been exposed to different temperatures and used in amplification. During this analysis, fragments which have been nonspecifically amplified, were subsequently cloned into a vector pGEM®-T Easy using TA cloning. New variant was sequenced to compare its similarity with known BPM1-3 variants. Temperature-dependent regulation of gene expression and alternative splicing of BPM1-3 genes was confirmed. A new splicing variant BPM2.6 was discovered under normal conditions and conditions of elevated temperature. Due to the absence of BTB/POZ domain which is crucial for interaction with cullin, the discovery of new splicing variant provides the basis for further research regarding cullin-independent roles of BPM proteins.