DOI: 10.3724/SP.J.1145.2013.00224

Chinese Journal of Appplied Environmental Biology (应用与环境生物学报) 2013/19:2 PP.224-230

Q78Molecular Cloning and Heterologous Expression of Putative Strictosidine Synthases from Arabidopsis thaliana

No complex alkaloids were proved to accumulate in Arabidopsis thaliana. However, many genes in the genome of A. thaliana were annotated as homologues to the genes involved in the biosynthetic pathway of terpenoid indole alkaloids (TIAs), for instance, 15 genes were annotated as strictosidine synthase (STR). To clarify the function of these genes, we selected 5 putative AtSTRs out of 15 genes on the basis of bioinformatic analysis of known functional RsSTR and AtSTRs. The five putative AtSTR genes were obtained by RT-PCR using total RNA as template. The plasmid containing AtSTRs were constructed, screened by the combination of X-gal and IPTG, enzymtic digestion and PCR. The sequences of these AtSTRs were confirmed by DNA sequencing. The expression plasmids were constructed, sequenced, and transformed into Escherichia coli BL21 (DE3) for protein overexpression. The fusion proteins were soluble and purified by nickel ion affinity chromatography. The putative catalytic activity of AtSTRs was assayed at four levels, including whole cells, cell lysates, supernatant, and purified enzyme, by adding the native substrates tryptamine and secologanin to the reaction mixture. No desired or new products were observed in the reaction mixture by HPLC-DAD detection, which suggested that more work is needed to clarify the possible function of these genes. Fig 8, Tab 3, Ref 22

Key words:terpenoid indole alkaloids,strictosidine synthase,Arabidopsis thaliana,RT-PCR,PCR,heterologous expression

ReleaseDate:2014-07-21 17:00:06