Cloning and prokaryotic expression of transcription factor AvMYC4b in Amomum villosum / 中草药

ABSTRACT

Objective: The transcription factor AvMYC4b selected from transcriptome databases, which might be closely related to the terpene biosynthesis, was cloned from Amomum villosum for sequence analysis and prokaryotic expression. Methods: Based on the transcriptome data of A. villosum, specific primers were designed to obtain the AvMYC4b core sequence. In this study, the whole cDNA sequence of AvMYC4b was obtained by RACE method, then the GATEWAY TOPO cloning vector, and the express vector pDEST17 were constructed by ligation and LR method, respectively, and prokaryotic protein expression was performed. Induced by IPTG and arabinose, the recombinant protein AvMYC4b was successful expressed at the temperature of 16 ℃. Collected bacteria were processed through lysis, ultrasound and purification, and were used to determine the protein expression by SDS-PAGE. Results: AvMYC4b cDNA gene had 2 579 bp, including 165 bp 5'UTR, 1 995 bp ORF, and 389 bp 3'UTR, which encoded a deduce protein of 644 amino acid with a calculated molecular weight of 72 211. Bioinformatics analysis indicated that AvMYC4b had the conserved domain of transcription factor MYC family and predicted that AvMYC4b could be located in nucleus. The SDS-PAGE result showed that the AvMYC4b protein was expressed in Escherichia coil with a molecular mass of about 80 000, which was consistent with the predicted molecular weight. Conclusion: AvMYC4b gene of the bHLH family was cloned from A. villosum and had the whole ORF. The recombinant AvMYC4b protein also was successful expressed in Escherichia coil Rosetta (DE3). Therefore, this study could provide fundamental information for the function characterization of AvMYC4b in terpene biosynthesis pathway of A. villosum.