MODIFICATION OF SOME SYNTHETIC TRANSCRIPTION FACTORS FOR CONTROL OF GENE EXPRESSION IN PLANTS

Abstract

The aim of the present study is developing new synthetic transcription factor with modular DNA binding domain. This transcription factor aimed at controlling gene expression in plants. The origin of this transcription factor is based on non-pathogenic bacterial protein effector, namely TALE. Also, we aimed at constructing a new library for routine assembly of programmed transcription factors in plant. Sequence of Paraburkholderia transcription activation effector-like protein (TALE-like) was obtained from uniprot protein database (accession: E5AV36). SV40-type nuclear localization signal (PKKKRK) was added to N-terminus to facilitate nuclear transportation and Herpes simplex virus VP 16 activation domain (ADFEFEQMFTDALGIDELPQ) in C-terminus as transcription activation domain. The new protein sequence reverse translated to DNA sequence, which was modified by replacing DNA binding domain with two tail-to-tail BsmBI restriction site with 3´overhangs GGAC and 5´ overhang CTCT. Final designed DNA sequence was synthesized and cloned into plant expression vector (pRT103) under control of cauliflower mosaic virus promoter (CaMV 35S), and generated vector was named pdBAT scaffold. DNA binding domain of Paraburkholderia TALE-like protein consists of 33 amino acid motif tandem repeats. Each repeat is responsible for binding to one DNA base. Repeats varied in two variable diresidue determining the specificity to DNA base. In order to generate programmable DNA binding domain, we designed a library of two repeats (direpeat) encoding DNA fragment with 6 positional order (2 repeat multiplied by 6 positions resulting in 12 repeat domains). Each position has 16 combination of repeats covering possible 16 base