密码子优化的纳豆激酶基因在番茄果实中的瞬时表达  

Transient Expression of Codon Optimized Nattokinase Genes in Tomato Fruits

作　　者：韩岚[1,2] 王欢[1] 王佳琪[1] 欧阳衫 哈斯阿古拉[1] 

机构地区：[1]内蒙古大学生命科学学院内蒙古自治区牧草与特色作物生物技术重点实验室,呼和浩特010021 [2]呼和浩特职业学院生物化学工程学院,呼和浩特010051

出　　处：《内蒙古大学学报（自然科学版）》2016年第1期73-79,共7页Journal of Inner Mongolia University：Natural Science Edition

基　　金：内蒙古自治区高等学校创新团队发展计划(No.NMGIRT1401)

摘　　要：根据植物偏爱密码子设计并人工合成了纳豆激酶基因sNK,在其中插入番茄内含子构成sNKi基因.将这两种基因通过农杆菌渗透法在不同成熟时期的番茄果实中实现了瞬时表达.通过实时荧光定量PCR法(RT-qPCR)比较两种基因在番茄果实中转录水平的表达量,通过纤维蛋白平板法检测两种基因表达产物的纤溶酶活性.结果表明,两种基因在番茄果实的转色期、成熟期和完熟期均有表达,其中成熟期表达量最高,破色期基本无表达.果实特异性E8启动子调控下的纳豆激酶基因的表达量较组成型35S启动子调控下的该基因表达量明显提高.插入内含子的sNKi基因的表达量高于没有内含子的sNK基因,表明内含子在提高纳豆激酶的表达量方面具有显著的作用.The nattokinase encoding gene （sNK） was synthesized according to preferential co- dons in plant, and sNKi gene was constructed by inserting an intron of tomato into sNK gene. These two genes were transiently expressed in tomato fruits of different maturity stages by agroin- filtration. The expression level of two genes in tomato fruits were measured by quantitative real- time reverse transcription-PCR （qRT-PCR） analysis,and fibrinolytic activity were detected through fibrin plate method. The results showed that sNK gene and sNKi gene can expressed in tomato fruit at veraison,maturation and full ripeness stage, but not at break stage. The expression level of re- combinant NK controlled by fruit-specific E8 promoter was higher than that controlled by constitu- tive 35S promoter. The expression level of sNKi gene,which was interrupted with an intron from tomato,was higher than that of the sNK gene. The results showed that the inserting of intron played a significant role in improving the expression of nattokinase gene.

关 键 词：纳豆激酶 密码子优化 番茄 瞬时表达 农杆菌渗透法 

分 类 号：Q786[生物学—分子生物学]