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 sequences.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 detected 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.
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