大鳞副泥鳅β-actin基因近端启动子的克隆及启动活性分析  

The cloning and activity analysis of proximal promoter of β-actin in Paramisgurnus dabryanus

作　　者：李相赫[1,2] 徐琪[2] 俞钦明[2] 童一宇 先友成进 陈国宏[2] 

机构地区：[1]朝鲜金亨稷师范大学生命科学系,平壤99903 [2]扬州大学动物科学与技术学院,江苏扬州225009 [3]朝鲜金日成综合大学平壤农业大学,平壤99903

出　　处：《淡水渔业》2014年第4期18-24,共7页Freshwater Fisheries

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。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 ex-pression vector pDPRK1-EGFP was constructed.Those vectors were transfected into 293T, celland DF1 to observe the green fluorescence expressions respectively .Meanwhile , the vector was line-typed by NdeⅠenzyme and microinjected in-to the fertilized eggs of Danio rerio.The complete sequence of the promoter fragment DPRK 1 was 1 282bp 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 pDPRK 1-EGFP using experiment in 293 T, celland 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 .

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 

分 类 号：S917.4[农业科学—水产科学]