Tagging of the chloroplast sensor kinase (CSK) with affinity epitopes HA and FLAG

U genomu biljke Arabidopsis thaliana (L.) Heynh identificiran je gen At1g67840 koji kodira za protein kloroplastnu senzorsku kinazu (CSK), dio „drevnih“ dvokomponentnih signalnih sustava. Analizom aminokiselinskog slijeda utvrđena je visoka sačuvanost i sličnost određenih motiva u svih vaskularnih biljaka i algi te je protein CSK tip bakterijske senzorske histidinske kinaze utvrđene u fotosintetskim plastidima tj. kloroplastima. Cilj ovog rada je obilježavanje CSK proteina afinitetnim biljezima HA i FLAG u svrhu funkcionalne i strukturalne karakterizacije senzorske kinaze i specifičnijeg utvrđivanja njene uloge kod biljke Arabidopsis thaliana. Lančanom reakcijom polimerazom fuzionirali smo biljege HA i FLAG na karboksi terminalni kraj CSK proteina. Koristeći „Gateway“ rekombinacijsku tehnologiju konstrukt CSK_HA_FLAG ugrađuje se u ishodišni pENTR (engl. entry) vektor te naknadno u binarni pH7WG2,0 vektor. Nadalje, stanice agrobakterija EHA105 transformirane su s konstruktom pH7WG2,0-CSK_HA_FLAG s ciljem daljnje ugradnje CSK_HA_FLAG komponente prisutne u T-DNA regiji binarnog vektora u genom biljke Arabidopsis thaliana. Ovim postupkom u transformiranim biljkama CSK-KO (engl. knock-out), inaktiviran je gen At1g67840.In plant genome of Arabidopsis thaliana (L.) Heynh gene At1g67840 that encodes chloroplast sensor kinase (CSK), which is a part of ancestral two-component signal systems has been identified. Sequence analysis showed high similarity of motifs in all major plant and algal lineages which indicates that CSK has evolved from a cyanobacterial sensor histidine kinase. The subject of this work has been tagging of the CSK protein with affinity epitopes HA and FLAG for the functional and structural characterization purpose of the sensor kinase and more specific deciphering of it’s role in Arabidopsis thaliana plant. For that purpose we fused epitopes HA and FLAG into the carboxy-terminal end of the CSK protein by polymerase chain reaction. Using “Gateway” recombinant technology CSK_HA_FLAG construct has been inserted into the entry and subsequently into the binary pH7WG2,0 vector. Futhermore, Agrobacterium EHA105 cells were transformed with a final construct pH7WG2,0-CSK_HA_FLAG with a goal of it further insertion in Arabidopsis thaliana plant genome, specifically into the CSK-KO plants