水稻乙醇酸氧化酶基因OsGLO1的原核表达及多克隆抗体制备

Prokaryotic Expression and Preparation of Polyclonal Antibody of Glycolate Oxidase Gene OsGLO1 from Rice

乙醇酸氧化酶是光呼吸代谢的关键酶.以水稻Oryza sativa叶片cDNA为模板,利用RT-PCR扩增水稻乙醇酸氧化酶基因(OsGLO1),并构建了该基因的原核表达载体pET23d-OsGLO1,转化E.coli BL21(DE3),经IPTG诱导表达融合蛋白.以纯化的OsGLO1融合蛋白为抗原免疫新西兰白兔,制备兔抗OsGLO1的多克隆抗体.Western-blot分析表明,制备的多克隆抗体能有效地检测水稻、菜心Brassica campestris、拟南芥Arabidopsis thaliana和菠菜Spinacia oleracea中乙醇酸氧化酶的表达,为进一步深入研究乙醇酸氧化酶在调控光呼吸及抗逆性等方面的作用奠定了基础.

Glycolate oxidase （GLO） is a key enzyme in photorespiratory metabolism. OsGLO1 was amplified by RT-PCR from rice leaf cDNA and cloned into a prokaryotic expression vector pET23d. The recombinant plasmid was transformed into Escherichia coli BL21 （ DE3 ） and the expression of recombinant protein was induced by IPTG. After purification, it was used as the antigen to immune a rabbit and then polyclonal antibody was obtained. Western-blot analysis confirmed that the polyclonal antibody was able to recognize the GLO proteins from different plant species, such as rice, cabbage, arabidopsis and spinach. This work laid a foundation for functional studies of GLO in regulating photosynthesis under stresses.