ZmAGO18b的原核表达载体的构建与表达

Construction and Expression of a Prokaryotic Expression Vector of Zm AGO18b

AGO18是禾本科植物中特有的Argonautes(AGOs)蛋白的一个分支,在植物生殖器官发育和逆境胁迫过程中发挥着重要的调控作用。玉米中AGO18b(ZmAGO18b)的表达具有很强的组织特异性,在雄穗小孢子减数分裂时期特异富集。为了研究ZmAGO18b基因的功能,本研究从减数分裂时期的玉米雄穗cDNA中特异扩增Zm AGO18b的全长ORF(2760 bp),利用酶切连接的方法构建原核表达载体,通过大肠杆菌表达菌株(Transetta)进行IPTG诱导表达。结果显示,ZmAGO18b蛋白最佳诱导条件是IPTG终浓度0.10 mmol/L,诱导温度是37℃,诱导时间为3 h,此时体外表达的蛋白量达到最高,蛋白大小约为100 kD。本研究有助于后期对ZmAGO18b蛋白的分离与纯化,为进一步抗体制备及相关功能研究积累了有价值的资料。

AGO18 is a grass-specific AGO protein and plays an important regulatory role during reproductive organ development and response to stress in plants.ZmA GO18 b is very tissue-specific and specifically enriched in meiotic microspore of tassels in maize.In this study,the full-length ORP(2760 bp)of ZmA GO 18 b was specifically amplified from the cDNA of maize tassel at meiosis stage,and the recombinant vectors for prokaryotic expression were constructed by restriction enzyme digestion and ligation.The expression of ZmAGO18 b protein was induced by IPTG in E.coli Transetta(DE3)cells,and 0.10 mmol/L IPTG and 3 hours under 370 C induction as the optimal induction conditions.Under these conditions,the expression level of the protein was highest,and the molecular weight of ZmAG018 b was about 100 kD.This study might provide a theoretical help for the separation and purification of protein and for further antibody preparation and studying the function of ZmAGO18 b in maize.