CAJ | 학술논문

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。
위료연구랭협박처리하적옥미항배혈산과양화물매적작용，근거GenBank발표적APX기인적ORF서렬설계인물，이용RT-PCR종랭협박후적옥미자교계（ E28）협편총RNA중경cDNA전화， pMD19-T재체적아극륭，획득APX기인개방열독광，장도위753 bp，편마250개안기산잔기。생물신식학분석현시，APX 상대분자질량화이론등전점의차위27．3 kDa화5．56。장휴대완정적ORF서렬련입표체재체pET-28a（＋）중，APX기인경IPTG유도후고효표체，SDS-PAGE전영검측도목표단백적대소약위27．3 kDa，여예측결과일치。항염분석현시，중조대장간균BL21（pET28-APX）적항염성명현고우대조균주BL21（pET28）차기항NaCl적림계농도위0．6 mol/L，저위후기대작물적항역연구제공료과학의거。
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 .