Cloning, Expression, Purification And Bioinformatics Analysis Of Trihelix Transcription Factor Gene In Cnidium Monnieri

Objective The Trihelix Transcription Factor(ttf) gene in a Chinese medicinal plant Cnidium monnieri(L.) Cuss. was subcloned to p ET-22b(+) vector. The recombinant plasmid was transformed into E.coli BL21 for protein expression. The target protein was purified by nickel column. The molecular characteristics and secondary structure of the TTF protein were analyzed using a series of bioinformatic tools.Method The total RNA of Cnidium monnieri was extracted and single-stranded RNA is reverse transcribed into complementary DNA(c DNA) by using total cellular RNA. The ttf gene was amplified by PCR from the c DNA of Cnidium monnieri, and plasmid p MD19-T-TTF was constructed by TA cloning. The c DNA fragment of ttf was subcloned to p ET-22b(+), and recombinant plasmid p ET-22b-ttf was verified by PCR reaction, restriction endonuclease analysis and DNA sequencing. Furthermore, the recombinant plasmid was transformed into E.coli BL21, and the fusion TTF protein was expressed with induction of IPTG at different concentration levels. The TTF protein was illustrated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis(SDS-PAGE), and purified by Ni-NTA column. Western-blotting assay revealed that fusion protein could be identified by His-tag antibody. The molecular characteristics such as physiochemical properties, conserved domain, and sub-cellular localization of the TTF protein were predicted using bioinformatics analysis.Results The Trihelix Transcription Factor(ttf) gene in a Chinese medicinal plant Cnidium monnieri(L.) Cuss was successfully sub-cloned into the prokaryotic expression vector p ET-22b(+). The recombinant plasmid p ET-22b-ttf was transformed into competent E.coli BL21 cells, and the target protein was expressed by induction with IPTG. The Western blot results show that the optimal inducing concentration of IPTG was 0.1 mmol/L, and the optimal inducing tempreture was 37 degree. Some characteristics of the deduced TTF protein such as amino acid composition, molecular weight and isoelectric point, hydrophilic and hydrophobic, and stability were analyzed by using the Protparam software. The full length c DNA of ttf was 540 bp in length and encoded the protein of 179 amino acids with a molecular weight of 21254.7 and an isoelectric point(PI) of 7.85. The various motif of the protein, including various active sites and structural domains, were analyzed by Scan Motif and PSORT. The secondary structure of the target protein was predicted by Predict Protein software. The TTF protein contains the putative coiled-coil motif(E116-R142? R144-A163). Phylogenetic analysis demonstrated that the ttf gene of Cnidium monnieri is clustered together with the three helix transcription factor of sesame.Conclusions The conclusion of this study was presented as follows.1, The Trihelix Transcription Factor(ttf) gene in a Chinese medicinal plant Cnidium monnieri Cuss was successfully cloned for the first time.2, The recombinant plasmid ttf-pMD19-T and recombinant plasmid ttf-p ET22 b were successfully reconstructed.3, The fusion TTF protein was expressed with induction of IPTG and the optimal inducing concentration of IPTG was 0.1 mmol/L, the optimal inducing tempreture was 37 degree.4, We performed a comprehensive bioinformatic analysis for the TTF protien, which provides a basis for its further structural and functional study.