yeast two hybrid cdna screening of arabidopsis thaliana for seth4 protein interaction

seth4 coding sequence with 2013 bp is a member of gene family expressed in gametophytic tissues of arabidopsis thaliana. this fragment was pcr amplified using kod hi fi dna polymerase enzyme. this fragment was cloned into pgbkt7 bate vector and transformed e. coli dh5? cells containing vector were selected on lb medium containing kanamycin. finally, pgbkt7-seth4 bate was transformed into yeast strain y187 and were selected on sd ?leu ?trp medium. after mating of this strain with yeast strain ah109 containing arabidopsis thaliana cdna library as prey vector, diploid cells were selected on sd ?his ?leu ?trp medium. screening of cdna inserted into diploid cells indicated that, 35 colonies out of 240 showing x-gal staining. pcr amplified of cdna insertion fragments in diploid cells showed some colonies have a chance for seth4 protein interaction.