Cloning and analysis of a γ-tocopherol methyltransferase gene from Brassica oleracea and the function of its recombinant protein*

Abstract

Abstract The full-length cDNA (BoTMT) of γ-TMT is obtained from Brassica oleracea by 3′- and 5′-RACE methods. The 1265 bp cDNA contains an open reading frame of 1044 bp, which encodes a protein of 347 arnino acids with a predicted chloroplast transit peptide and two S-adenosylmethionine (SAM)-binding domains. Sequence analysis shows that the deduced protein shares 41.8% – 86.5% similarity to known γ-TMTs. Semi-quantitative RT-PCR reveals that BoTMT is expressed preferentially in flowers and leaves of B. oleracea. The recombinant γ-TMT protein is obtained by cloning its encoding region into the prokaryotic expression vector pET30a. The protein expressed in E. coli accounts for 22% of total bacterial protein. The enzyme activity assay indicates that the recombinant protein has relatively high activity to convert γ-tocopherol to α-tocopherol in vitro.