玉米ZmGAPDH和ZmACTIN的原核表达及抗体制备

Prokaryotic expression and antibody preparation of ZmGAPDH and ZmACTIN in maize

【目的】制备玉米三磷酸甘油醛(GAPDH)和肌动球蛋白(ACTIN)的多克隆抗体,检测热激条件下玉米B73叶片中ZmGAPDH和ZmACTIN的表达,为将ZmGAPDH和ZmACTIN作为非生物胁迫下的内参基因奠定基础。【方法】利用DNAMAN 7对ZmGAPDH和ZmACTIN与不同植物同源蛋白间的氨基酸序列进行比对。通过PCR克隆ZmGAPDH和ZmACTIN的CDS,将其构建到原核表达载体pET-28a中并测序;将重组质粒载体转化大肠杆菌BL21(DE3)和RG2,用异丙基硫代半乳糖苷(IPTG)诱导表达;将目的蛋白透析纯化后免疫家兔制备多克隆抗体。利用RT-PCR和Western blot检测42℃热激不同时间(0,2,4,8 h)下玉米B73叶片中ZmGAPDH和ZmACTIN mRNA及其蛋白的表达水平。【结果】克隆了ZmGAPDH和ZmACTIN基因的CDS序列,成功构建了原核表达载体pET-28a-ZmGAPDH和pET-28a-ZmACTIN。在大肠杆菌中表达出了玉米ZmGAPDH和ZmACTIN蛋白,将纯化的蛋白免疫家兔后获得了多抗血清。ZmGAPDH多克隆抗体能清晰检测到4 ng原核表达的ZmGAPDH抗原,ZmACTIN多克隆抗体同样能够清晰检测到4 ng原核表达的ZmACTIN抗原。热激不同时间下玉米B73叶片中的ZmGAPDH和ZmACTIN蛋白表达水平一致,mRNA丰度稳定不变。【结论】成功制备了ZmGAPDH和ZmACTIN蛋白多克隆抗体;ZmGAPDH和ZmACTIN可以作为内参基因,用于检测热激条件下目标基因的表达水平。

【Objective】The ZmGAPDH and ZmACTIN polyclonal antibodies were prepared and the accumulation of ZmGAPDH and ZmACTIN in maize B73 leaves under heat shock was determined to provide basis for using ZmGAPDH and ZmACTIN as internal reference genes under abiotic stress.【Method】The amino acid sequence alignment of ZmGAPDH and ZmACTIN in different plants was performed using DNAMAN 7.The CDS of ZmGAPDH and ZmACTIN were amplified by PCR and cloned into the prokaryotic expression vector pET-28a before being sequenced.The recombinant plasmid vector was transformed into E.coli BL21(DE3)and E.coli RG2 and induced by isopropyl thiogalactoside(IPTG).The polyclonal antibody was prepared by immunizing rabbits using the purified protein.RT-PCR and Western blot were performed to determine the mRNA and protein levels of ZmGAPDH and ZmACTIN in maize B73 leaves under heat shock at 42℃(0,2,4,8 h).【Result】The CDS sequences of ZmGAPDH and ZmACTIN genes were cloned and the prokaryotic expression vectors pET-28a-ZmGAPDH and pET-28a-ZmACTIN were constructed successfully.ZmGAPDH and ZmACTIN proteins were successfully expressed and purified from E.coli.The antibodies were prepared using the purified proteins to immunize into rabbits.ZmGAPDH polyclonal antibody detected 4 ng prokaryotic expressed ZmGAPDH antigen,and ZmACTIN polyclonal antibody also detected 4 ng prokaryotic expressed ZmACTIN antigen.ZmGAPDH and ZmACTIN protein expression levels and mRNA abundance were consistent in maize B73 leaves that were treated with different duration of heat shock.【Conclusion】The specific antibodies against ZmGAPDH and ZmACTIN proteins were prepared successfully in maize.ZmGAPDH and ZmACTIN can be used as internal reference genes to investigate the expression of target genes that were treated with heat shock.