苦瓜谷氨酰胺合成酶基因(McGS1)原核表达载体的构建及表达分析

Prokaryotic Expression and Analysis of McGS1 Gene of Bitter Gourd

以"热研3号"苦瓜为试材,根据苦瓜谷氨酰胺合成酶基因(McGS1)全长序列信息设计引物,提取"热研3号"苦瓜总RNA并反转录成cDNA作为模板,通过PCR扩增、产物测序、酶切等方法与pET-30a(+)原核表达载体相连,构建苦瓜McGS1基因原核表达载体,并在大肠杆菌BL21(DE3)中进行表达分析,研究不同IPTG浓度及诱导时间对苦瓜McGS1基因蛋白在大肠杆菌BL21(DE3)中表达的影响。结果表明:该研究成功构建了苦瓜McGS1基因的原核表达载体,37℃、0.2μmol/L IPTG诱导4h苦瓜McGS1基因蛋白在大肠杆菌BL21(DE3)中可高效表达,表达的蛋白质分子量约为35kDa。

In this study,prokaryotic expression vector expressing bitter gourd McGS1 gene was constructed and transformed into E.coli BL21（DE3）for its expression.A pair of primers was designed according to McGS1 gene sequence.Total RNA of the‘Re Yan No.3＇bitter gourd was extracted,and then McGS1 gene was amplified by RT-PCR.The recombinant expression vector was identified using enzyme digestion and PCR method.The expression of McGS1 gene was optimized by different concentration of IPTG and induction time.The expression product of McGS1 gene was analyzed by SDS-PAGE.The results showed that the optimal expression condition of McGS1 protein was 37℃,0.2μmol/L IPTG induction for four hours,and the molecular weight of protein product of McGS1 gene was about35 kDa.