唐鱼β-actin基因近端和远端启动子的鉴定及其启动活性分析  

Identification of Two Different Length of β-actin Promoters of Tanichthys albonubes and Comparison of Their Activity

作　　者：王海英[1,2] 叶星[1] 劳海华[1] 夏仕玲[1] 白俊杰[1] 简清[1] 

机构地区：[1]中国水产科学研究院珠江水产研究所中国水产科学研究院热带亚热带鱼类选育与养殖重点开放实验室,广州510380 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《生物工程学报》2008年第10期1768-1775,共8页Chinese Journal of Biotechnology

基　　金：广东省科技计划项目(No.2003C20310);广东省科技重点引导项目(No.2005B20301018);国家科技基础条件平台建设项目(No.2006DKA30470-008)资助~~

摘　　要：利用高保真PCR技术获得唐鱼(Tanichthys albonubes)长为1.3 kb的β-肌动蛋白(β-actin)近端启动调控序列(TA,1.3 kb)。在此基础上采用基因组步移技术(Genome walker)获得5′侧翼上游序列1.7 kb,再根据所获的近端启动子序列和上游序列设计引物,扩增获得全长为3.0 kb的唐鱼β-actin基因远端启动调控序列(TLA,3.0 kb)。此1.3 kb和3.0 kb启动调控序列均包含3个转录活性元件:CAAT Box(-89^-85),CArG Box(-59^-49),TATA Box(-26^-20)。利用启动子分析软件TRANSFAC 6.0分析,结果显示启动调控序列(-105~1261)中含有E-Box、NF-Y、Sp1等多个重要转录因子结合位点,在远端启动调控序列(-1719~1261)中还含有更多的重要转录因子结合位点。将这两个序列分别定向克隆到红色荧光蛋白(Red fluorescent protein,RFP)表达载体中,构建重组表达载体pTLA-DsRed和pTA-DsRed,并分别注射到唐鱼受精卵中,结果显示转pTLA-DsRed基因唐鱼的阳性率较转pTA-DsRed的高,且所发出的红色荧光的强度也比后者的强。采用RT-PCR检测孵化后第15天的转基因唐鱼中RFP的mRNA,结果显示转pTLA-DsRed基因唐鱼中RFP mRNA的表达量比转pTA-DsRed基因唐鱼高35.7%。结果表明两种长度的启动调控序列均能有效驱使外源基因在唐鱼体内表达,且长度为3.0 kb的启动子序列具有更强的驱动活性。Through PCR amplification, 1.3 kb of 5＇-proximal promoter （TA, 1.3 kb） of the β-actin gene of white cloud mountain minnow Tanichthys albonubes was obtained. Using Genome Walker, a 1.7 kb 5＇-upstream sequence from the proximal promoter of the β-actin gene was isolated, and a further promoter （3.0 kb in size） was amplified according to the isolated 5＇-proximal and upstream sequences （TLA, 3.0 kb）. Both the 1.3 kb and 3.0 kb promoters contain elements that were critical to the transcription activity of other species, including the CCAAT Box （-89--85）, CArG Box （-59--49）, TATA Box （-26--20）. Results of putative transcription binding sites analysis of the promoters by software TRANSFAC 6.0 revealed the presence of E-box, several transcript binding sites NF-Y, SP 1 （Stimulating Protein 1 ）, AP 1 （Activator Protein 1）, and some more transcription binding sites existing in the further promoter. The two promoter sequences were inserted into the expression vector to construct the recombinant expression vector, pTA-DsRed and pTLA-DsRed, respectively. The vectors were microinjected into the fertilized eggs of Tanichthys albonubes and higher positive rate was obtained and stronger red fluorescence was observed in pTLA-DsRed transgenic fish. RT-PCR analysis showed that RFP （Red fluorescent protein） mRNA level in pTLA-DsRed transgenic fish was 35.7% higher than that of the pTA-DsRed transgenic fish of 15-days-post-hatched. The present study showed that both the proximal and further promoter sequences have effective transcription activities and the 3.0 kb promoter possesses higher potent activity than that of the 1.3 kb promoter.

关 键 词：唐鱼 β-肌动蛋白启动子 红色荧光蛋白 显微注射 表达 转录活性 

分 类 号：Q953[生物学—动物学]