Recombination and Activity Assay of Betaine Aldehyde Dehydrogenase (BADH) Protein of Rice (Oryza sativa subs. japonica) and Spinach (Spinacia oleracea)
Abstract：This paper was to obtain the fusion proteins of betaine aldehyde dehydrogenases(OsBADH and SpBADH) from rice (Oryza sativa subs. japonica) and spinach (Spinacia oleracea) by means of gene cloning, prokaryotic expression and protein purification for measuring the enzyme activities of the two aldehyde dehydrogenases in vitro, proved that rice existed OsBADH with the function of aldehyde dehydrogenase. In this study, OsBADH and SpBADH full-length gene were cloned by RT-PCR from japonica rice and spinach, respectively. The amino acid sequences alignment showed 70.8% similarity. Recombinant plasmids of both OsBADH and SpBADH could be expressed in Escherichia. coli strain BL21 (DE3). SDS-PAGE analysis revealed that the fusion protein bands were observed at approximately 70 kD after inducing by 0.4 mmol/L IPTG. The proteins from E. coli strains BL21 were purified by TALON metal chelating resin and then the enzyme activities were determined. The result indicated that the purified recombination proteins exhibited the enzymatic activities of BADH. The catalytic activity of OsBADH was 74.87 nmol/min/mg with SpBADH (86.84 nmol/min/mg) as control, suggesting that OsBADH could catalyze the reaction effectively but no glycinebetaine(GB) production was detected by HPLC. These results image that little level of glycinebetaine in rice may not due to the activity deficiency of BADH. The study may provide basic information for further research of the molecular mechanisms of glycinebetaine synthesis in rice.
