摘要
利用RT-PCR技术获得大豆的过氧化氢酶基因家族的GmCAT3、GmCAT4、GmCAT5的cDNA片段,进化树分析表明GmCAT3和GmCAT1/2,GmCAT5和豌豆PsCAT、豇豆VrCAT的亲缘关系较近;将3个目的基因克隆到pMD19-T载体上,再连接到pET28a表达载体上,获得重组载体pET28a-GmCAT3、pET28a-GmCAT4和pET28a-GmCAT5,并转化大肠杆菌BL21(DE3)进行诱导表达,经SDS-PAGE电泳分析,在20℃条件下,0.1mmol/L的IPTG诱导12h表达量最佳,诱导的目的蛋白在60kD处以包涵体的形式存在,获得重组蛋白分别占总蛋白的35.7%、44.2%和42.5%。
GmCAT3、GmCAT4、GmCAT5 genes were amplified by RT-PCR.Phylogenetic analysis indicated that GmCAT3 and GmCAT1/2,GmCAT5 and PsCAT,VrCAT had close genetic relationship.The target genes were cloned into pMD19-T vector,and ligated to expression vector pET28a.The recombinant vectors pET28a/GmCAT3、pET28a/GmCAT4 and pET28a/GmCAT5 were transformed into E.coli BL21(DE3) and induced.The expression products were identified by SDS-PAGE.The optimal expression conditions of genes were 12h under 20℃ by 0.1mmol/L IPTG inducing.They were expressed at 60kD molecular weight specific protein as inclusion body respectively.The proportion of recombinant proteins in total bacterial proteins were as follows:35.7%,44.2% and 42.5% under this specific conditions.
出处
《中国油料作物学报》
CAS
CSCD
北大核心
2013年第4期384-388,共5页
Chinese Journal of Oil Crop Sciences
基金
河南省教育厅科技研究重点项目(12B180015)
关键词
大豆
过氧化氢酶
大肠杆菌
表达
Soybean
Catalase
Escherichia coli
Expression
