玉米APX基因的克隆及其原核表达研究

Cloning and Prokaryotic Expression Analysis of APX Gene from Maize

为了研究冷胁迫处理下的玉米抗坏血酸过氧化物酶的作用,根据GenBank发表的APX基因的ORF序列设计引物,利用RT-PCR从冷胁迫后的玉米自交系(E28)叶片总RNA中经cDNA转化,pMD19-T载体的亚克隆,获得APX基因开放阅读框,长度为753 bp,编码250个氨基酸残基。生物信息学分析显示,APX相对分子质量和理论等电点依次为27.3 kDa和5.56。将携带完整的ORF序列连入表达载体pET-28a(+)中,APX基因经IPTG诱导后高效表达,SDS-PAGE电泳检测到目标蛋白的大小约为27.3 kDa,与预测结果一致。抗盐分析显示,重组大肠杆菌BL21(pET28-APX)的抗盐性明显高于对照菌株BL21(pET28)且其抗NaCl的临界浓度为0.6 mol/L,这为后期对作物的抗逆研究提供了科学依据。

Ascorbate peroxidase （ APX） is a key enzyme which eliminates oxygen free radicals and increases plant resistance in adverse circumstances .In order to study the function of ascrobate peroxidose in cold treated maize leves . The complete open reading frame of APX gene was cloned from cold-stressed （4 ℃） maize inbred line E28 using the method of reverse transcription PCR （ RT-PCR） .The primers were designed according to the published APX cDNA se-quences .The sequence of APX from E28 was 753 bp,encoding a protein of 250 amino acid residues with a predicted molecular weight of 27.3 kDa and a theoretical pI of 5.56.APX were constructed into the expression vector pET-28a （＋）with full-length ORF,then transferred into E.coli.After inducing by IPTG,the product proteins of APX were de-tected by SDS-PAGE and the proteins were 27.3 kDa as expected .Salt tolerance analysis showed that the recombinant exhibit strong tolerance to salt than the non-recombinant bacteria ,and the critical concentration of NaCl is 0.6 mol/L. Our researches could lay the foundation for the study of plant tolerance in the future .