الفهرس 
Transcription Factors and Artificial Transcription Factors
Function of Natural TALEsOnly 14 pages are availabe for public view
 |  | from | 110 |  |  |
| | | from | 110 | | |
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 combinations. Our.repeats domain library was generated by site-directed PCR mutagenesis technique, then cloned to pGEM-T vector.
Custom DNA binding repeat array were generated. This array targets sequence ATATAAACCTAA of the minimal BS3 promoter (bacterial transcription activator dependent promoter). Six direpeats were selected by guidance of target sequence )positional and bases combination(
from our direpeats library. selected direpeats were mixed and digested with BsmBI restriction enzyme in one reaction. Digested DNA fragments were subsequently cloned into BsmBI digested pdBAT-scaffold plasmid, then transformed to E. coli with high efficiency. Positive clones were tested by PCR, and all PCR product sizes of the tested clones counted for 1438 bp. Final assembled designer transcription activator composed of nuclear localization signal, custom designed DNA binding domain and transcription activation domain.
In order to test the functionality of designed transcription activator
in this study, pdBAT HindIII fragment was sub-cloned to binary vector pCAMBIA 1300, then transformed to Agrobacterium tumefaciens strain GV3101. pCAMBIA-dBAT and mBS3::GUS introduced in Agrobacterum were used to transiently co-transform Nicotiana benthamiana using leaf agro-infiltration method. Two days post inoculation, leaf discs were stained with X-Gluc, where a strong signal was detected in discs co-transformed with dBAT and mBS3 and 35S::GUS (positive control), while no signal was detected in discs transformed with dBAT only, and a very week signal was generated when discs transformed with mBS3::GUS alone. The latter result is considered as leakage of mBS3 promoter.
Our results suggest that our designed synthetic transcription activation protein can positively function in any plant system. This system could be used as programmable DNA binding protein in fusion with any effector domain such as transcription activation, repression, methylation, de-methylation and nuclease domain. In this study, pdBAT scaffold and 96 direpeats library offer a new tool box for controlling gene expression in plants with high efficiency.