摘要
根据植物偏爱密码子优化设计、合成纳豆激酶基因sNK,利用重叠延伸PCR法在其中插入番茄果实特异性表达基因E8的第一内含子构成sNKi基因,通过农杆菌渗透法将这两种基因渗入到烟草NC89叶片中并实现瞬时表达。通过RT-qPCR法将两种基因在烟草叶片中转录水平的表达量进行比较,结果表明两种基因在烟草叶片中均表达,且sNKi基因的表达量显著高于sNK基因;通过纤维蛋白平板法在两种基因的瞬时表达样品中均能检测到纤溶酶活性,表明目的基因在烟草叶片中可正常翻译并表现出溶栓活性,且sNKi基因在翻译水平的表达量显著高于sNK基因。表明内含子对人工合成的纳豆激酶基因的瞬时表达具有显著的促进作用。
The nattokinase encoding gene（ s NK） was synthesized by modifying its sequence based on the optimized codon usage in the plant. The first intron of tomato fruit-specific expression E8 gene was inserted in s NK gene to construct s NKi gene by overla Pextension PCR method. These two synthetic nattokinase gene were transiently expressed in tobacco NC89 leaves by agroinfiltration. Real-time quantitative reverse transcription PCR（ RT-q PCR） result showed that higher expression level was detected in tobacco leaves which infiltrated with s NKi gene,compared with that infiltrated with s NK gene. Fibrinolytic activity was detected in transiently expressed samples of two synthetic genes by fibrin plate method,which indicated that the target genes can be normally translated and show thrombolytic activity in tobacco leaves. The translational expression level of s NKi gene was significantly higher than that of s NK gene. Those proved that intron can significantly improve the transient expression of s NK gene.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2015年第9期14-20,共7页
China Biotechnology
基金
内蒙古自治区科技创新团队和内蒙古自治区高等学校创新团队发展计划资助项目(NMGIRT1401)
关键词
纳豆激酶
密码子优化
瞬时表达
内含子
烟草
Nattokinase Codon optimization Transient expression Intron Tobacco
