摘要
为获得快速生长的泥鳅(Misgurnus anguillicaudatus),研究了泥鳅β-actin基因启动子介导的革胡子鲶(Clarias gariepinus)生长激素(growth hormone,GH)重组基因。采用PCR和RT-PCR技术克隆泥鳅β-actin基因近端启动子、3′-UTR及革胡子鲶GH基因编码区,构建一个长为2 418bp的GH重组基因DPRK。首先,构建了以增强型绿色荧光蛋白(EGFP)基因作为报告基因的重组表达载体p AF(pβ-actin promoter-EGFP),然后转染DF1真核细胞,同时经酶切线性化后显微注射到斑马鱼(Danio rerio)和泥鳅的受精卵中,以评价泥鳅β-actin启动子的启动活性。其次,将重组基因DPRK显微注射到泥鳅受精卵中。结果显示:泥鳅β-actin启动子能在DF1细胞、斑马鱼和泥鳅的受精卵中启动绿色荧光蛋白的表达;泥鳅的荧光表达率(78.44%)显著高于斑马鱼(29.62%);泥鳅受精卵显微注射DPRK 20d后,在m RNA水平检测到GH基因的表达。这表明泥鳅β-actin基因近端启动子具有显著的启动活性,且重组基因DPRK能在泥鳅体内表达,为下一步泥鳅的基因工程育种奠定了理论基础。
β-actin gene proximal promoter fragment and 3'-UTR fragment of Misgurnus anguillicaudatus were obtained by homology cloning strategy, and north Africa Clarias gariepinus growth hormone gene CDS fragments were amplified using RT-PCR technology for producing fast-growing transgenic loach. Through the reactions of cutting by restriction enzyme and linking, the"all fish"growth hormone gene recombinant DPRK with a length of 2418 bp was constructed. And then the recombinant DPRK was inserted into non-CMV promoter pEGFP-N1 for cloning. Meanwhile, in order to assess M. anguillicaudatusβ-actin gene promoter function and the level of expression of DPRK, the recombinant vectors pAF, with EGFP as a report gene, were constructed. Strong green fluorescence was observed in DF1 cell. And the fertilized eggs of Danio rerio and M. anguillicaudatus also expressed the strong green fluorescence with transfection rate of nearly 29.62%and 78.44%, respectively. The expression of GH mRNA was detected in fertilized eggs 20 days after microinjection. The result showed that M. anguillicaudatusβ-actin gene promoter has significant function of gene expression, and that"all fish"Clarias gariepinus growth hormone gene recombinant DPRK expressed in the body of fish. This study will lay the theo-retical foundation for the further application of gene engineering to cultivate new varieties of loach.
出处
《水产学杂志》
CAS
2014年第6期1-7,共7页
Chinese Journal of Fisheries
基金
江苏高校优势学科建设工程(2011-137)
关键词
泥鳅
β-actin启动子
革胡子鲶鱼GH基因
显微注射
Misgurnus anguillicaudatus
β-actin promoter
growth hormone gene of Clarias gariepinus
microinjection
