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当前位置: 首页 > 文章 > 大鳞副泥鳅β-actin基因近端启动子的克隆及启动活性分析 淡水渔业 2014 (4) 18-24
Position: Home > Articles > The cloning and activity analysis of proximal promoter of β-actin in Paramisgurnus dabryanus Freshwater Fisheries 2014 (4) 18-24

大鳞副泥鳅β-actin基因近端启动子的克隆及启动活性分析

作  者：
李相赫;徐琪;俞钦明;童一宇;先友成进;陈国宏
单  位：
扬州大学动物科学与技术学院;朝鲜金日成综合大学平壤农业大学;朝鲜金亨稷师范大学生命科学系
关键词：
大鳞副泥鳅(Paramisgurnus dabryanus);β-actin;启动子;绿色荧光蛋白;显微注射
摘  要：
采用PCR技术从大鳞副泥鳅(Paramisgurnus dabryanus)基因组中克隆了β-actin基因近端启动子片段DPRK1,并将该基因启动子片段DPRK1定向亚克隆到不含启动子的绿色荧光表达载体pEGFP-N1中,构建了重组荧光表达载体pDPRK1-EGFP。将该载体分别转染真核细胞293T、CEL及DF1,检测绿色荧光表达情况。重组载体经NdeⅠ酶切线性化后,显微注射到斑马鱼(Danio rerio)的受精卵中。序列分析显示该片段的长度为1 282 bp,含167 bp的5'侧翼序列近端启动子区和共1 115 bp的第1个外显子以及第1个内含子区。5'侧翼序列近端启动子含有CAAT box,CArG motif和TATA box,分别位于转录起始位点(+1)上游的-94、-64和-31处。在将重组载体pDPRK1-EGFP转染293T、CEL及DF1的实验中观察到3种细胞都有很强的绿色荧光,在显微注射到斑马鱼受精卵的实验中也观察到绿色荧光,该结果表明大鳞副泥鳅β-actin基因近端启动子片段DPRK1具有效的启动功能,为下一步创制转基因泥鳅新品系提供了技术支撑。
译  名：
The cloning and activity analysis of proximal promoter of β-actin in Paramisgurnus dabryanus
作  者：
LI Xiang-he;XU Qi;YU Qin-ming;TONG Yi-yu;XIANYOU Cheng-jin;CHEN Guo-hong;Life-Science Faculty,KimHyongJik University;Animal Science and Technology College,Yangzhou University;Life-Science Faculty,KimIlSung University Pyongyang Agricultural College;
关键词：
Paramisgurnus dabryanus;;β-actin;;promoter;;green fluorescent protein;;microinjection
摘  要：
By using PCR amplification techniques,β-actin gene proximal promoter fragment,called DPRK1,was cloned from Paramisgurnus dabryanus genome. The DPRK1 was inserted into pEGFP-N1,as a result,a fluorescent protein expression vector pDPRK1-EGFP was constructed. Those vectors were transfected into 293 T,CEL and DF1 to observe the green fluorescence expressions respectively. Meanwhile,the vector was line-typed by Nde Ⅰ enzyme and microinjected into the fertilized eggs of Danio rerio. The complete sequence of the promoter fragment DPRK1 was 1 282 bp including 5'flanking proximal promoter(167 bp) and another fragment consisting of first exon and first intron(1 115 bp). The 5'flanking proximal promoter sequence contained CAAT box,GArG motif and TATA box,located at- 94,- 64 and- 31 sites of upstream sequence respectively. Secondly,the recombined vector pDPRK1-EGFP using experiment in 293 T,CEL and DF1 cells showed strong green fluorescence in these cells. And the fertilized eggs of D. rerio microinjected line-typed vector pDPRK1-EGFP also expressed the green fluorescence. This experiment results showed that β-actin gene proximal promoter DPRK1 of P. dabryanus had positive translation initiation activity and lay laid foundation for producing new varie-ties of transgenic loach.

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