杜仲Fls基因的克隆及原核表达

Cloning and prokaryotic expression of Fls gene in Eucommia ulmoides Oliver

【目的】克隆杜仲黄酮醇合成酶基因(Fls)全长,对其开放阅读框(ORF)进行原核表达分析。【方法】以杜仲叶片为材料提取总RNA,采用反转录聚合酶链式反应(RT-PCR)和cDNA末端快速扩增(RACE)技术克隆杜仲Fls基因;Fls基因的ORF经限制性内切酶酶切,构建其原核表达载体pET-28a-Fls;最后利用IPTG诱导Fls基因在大肠杆菌BL21(DE3)中表达。【结果】获得了黄酮醇合成酶基因全长序列,长度为1 220bp,ORF为1 011bp,编码336个氨基酸;成功构建了原核表达载体pET-28a-Fls;利用IPTG诱导Fls在BL21(DE3)中表达,SDS-PAGE电泳结果显示,在约44ku处有特异性的蛋白条带出现。【结论】获得了杜仲Fls基因的全长和ORF,并成功对其进行了原核表达。

【Objective】The study aimed to clone the full cDNA sequence of Fls gene in Eucommia ulmoides,and analyze the prokaryotic expression of its open reading frame （ORF）.【Method】The reverse transcription-polymerase chain reaction （RT-PCR） and rapid amplification of cDNA end （RACE） methods were used to clone the Fls gene from the total RNA of E.ulmoides leaf tissue.The ORF of Fls gene was digested with restriction endonucleases to construct the vector pET-28a-Fls.At last,Isopropyl β-D-1-Thiogalactopyranoside （IPTG） was used to induce its expression in Escherichia coli BL21（DE3）.【Result】The full cDNA sequence of Fls gene was 1 220 bp including an ORF of 1 011 bp with 336 amino acids encoded.The prokaryotic expression system of recombined vector pET-28a-Fls was constructed.SDS-PAGE analysis showed that the expressed protein accumulated as inclusion bodies with molecular weight of 44 ku.【Conclusion】The full sequence and ORF of Fls gene in E.ulmoides were cloned and expressed in E.coli BL21 （DE3）.