Recombination and Activity Assay of Betaine Aldehyde Dehydrogenase (BADH) Protein of Rice (Oryza sativa subs. japonica) and Spinach (Spinacia oleracea) | 水稻、菠菜甜菜碱醛脱氢酶(BADH)重组蛋白表达及酶活性分析

中国人. 本研究目的是通过基因克隆、原核表达和蛋白纯化技术获得水稻(Oryza sativa subs. japonica)、菠菜(Spinacia oleracea)甜菜碱醛脱氢酶基因(OsBADH和SpBADH)的融合蛋白，体外测定两者的醛脱氢酶活性，说明水稻存在OsBADH且具有醛脱氢酶功能。通过RT-PCR成功克隆了OsBADH和SpBADH基因的全长序列，二者氨基酸序列同源性为70.8%。OsBADH和SpBADH的重组质粒均可在大肠杆菌(Escherichia coli)菌株BL21(DE3)中表达，SDS-PAGE分析表明，经0.4 mmol/L IPTG诱导的融合蛋白条带约70 kD。两者的表达产物经TALON金属螯合树脂纯化后进行酶活测定，结果显示都具有甜菜碱醛脱氢酶活性。OsBADH的比活力为74.87 nmol/min/mg，对照SpBADH比活力为86.84 nmol/min/mg，表明OsBADH能够有效行使醛脱氢酶功能，而高效液相色谱法未在水稻叶片中检测出甜菜碱。研究结果提示，水稻可能并非由于 BADH自身不具有酶活而不积累甘氨酸甜菜碱，该研究为进一步探讨水稻中甜菜碱合成的分子机制提供了研究基础。

英语. 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.