小麦Fe超氧歧化酶基因的原核表达分析
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安徽省高等学校省级自然科学研究项目(KJ2012Z105)


Expression of Wheat Fe Superoxide Dismutase (FeSOD) Gene in Prokaryotic Cell
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    摘要:

    采用RT-PCR技术分离小麦Fe超氧歧化酶基因(FeSOD)的ORF全长cDNA,然后构建其原核表达载体,并对其表达的诱导时间、IPTG浓度、温度进行优化,以期获得较大量的重组蛋白。结果表明:实验获得了小麦FeSOD基因的ORF全长(600 bp),ORF全长与原核表达载体pET-Dute1相连接构建了原核表达载体pET-FeSOD,将pET-FeSOD导入宿主菌Rosetta(DE3)中,经SDS-PAGE电泳结果显示,可以高效表达融合蛋白且表达的蛋白均主要以包涵体的形式存在;重组质粒表达出25.8 kD的融合蛋白,除去载体pET-Duet1自身表达的3.0 kD蛋白后,与FeSOD编码的约为22.8 kD蛋白的大小一致;对诱导表达条件的优化结果显示,融合蛋白 pET-FeSOD最佳的诱导表达条件为:0.5 mmol/L的IPTG浓度,37 ℃诱导5 h。该研究结果为进一步深入研究该基因的特性与功能奠定了基础。

    Abstract:

    In order to construct prokaryotic expression vector and to study the recombinant gene expression in the host bacteria,we cloned complete gene of Triticum aestivum FeSOD ORF by the RT-PCR method followed by restriction enzymes cutting and connection.The induced time,IPTG concentration and temperature of prokaryotic expression were optimized using SDS-PAGE.The results showed that the full-length cDNA sequence of T.aestivum FeSOD ORF(600 bp) was cloned,then a recombinant of prokaryotic expression vector pET-SOD was constructed successfully.FeSOD protein in inclusion body can express effectively after the expression vector pET-FeSOD was transformed to Rosetta (DE3).The SDS-PAGE results indicated that pET-FeSOD fusion protein was expressed with molecular weight of 25.8 kD,with the pET-Duet1’s own induction produced 3.0 kD protein.The result is consistent with the 22.8 kD protein which encoded by FeSOD gene.The optimal expression condition was set as 0.5 mmol/L IPTG,induced temperature 37 ℃,and induced time 5 hours.These results are expected to lay a foundation for further studies on the properties and function of this gene.

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李 娟,王泽文,杨利新,等.小麦Fe超氧歧化酶基因的原核表达分析[J].西北植物学报,2013,33(11):2147-2152

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  • 在线发布日期: 2013-12-16
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