Abstract:

In order to further mine the enzyme gene related with methyl-eugenol synthesis and to elucidate the secondary metabolism pathway of Asarum heterotropoides. According to the transcriptome database information of Asarum heterotropoides,AhOMT1 gene was screened and cloned,and the corresponding bioinformatics analysis and prokaryotic expression analysis were carried out. The results showed that AhOMT1 contained an open reading frame of 1 089 bp,encoding 362 amino acids,with a theoretical molecular weight of 40.23 ku and an isoelectric point of 5.34. AhOMT1 protein was a hydrophilic protein without transmembrane structure and signal peptide sequence. AhOMT1 gene had dimerization domain and methyltransferase domain. AhOMT1 was close with the O-methyltransferase catalyzing flavonoids and eugenols. The prokaryotic expression vector pET-32b-AhOMT1 was successfully constructed and the recombinant protein of about 60 ku was successfully induced. The optimal induction condition was 0.2 mmol/L IPTG and induced at 16 ℃ for 16 h. Most of the recombinant proteins existed in the form of soluble proteins. AhOMT1 protein was separated and purified with Ni²⁺ resin,and the maximum amount of protein was obtained by eluting with 200 mmol/L imidazole. AhOMT1 gene of Asarum heterotropoides was cloned for the first time,prokaryotic expression vector was constructed,and the best induction conditions of the protein were screened.

Key words: Asarum heterotropoides, AhOMT1, Gene cloning, Bioinformatics, Prokaryotic expression