摘要
高保真 PCR 克隆青鱼β-actin 基因开放阅读框和 5′端侧翼序列,DNA 测序结果表明:青鱼β-actin 基因开放阅读框编码一段含 375 个氨基酸的蛋白,与其他物种 actin 家族相比较具有高度保守性。青鱼β-actin 与鲤鱼、草鱼及斑马鱼的同源性均为 100%,而与人和 Norway 鼠β-actin 的同源性均为 99.2%,与鸡和 Kenyan 爪蟾β-actin 的同源性分别为 98.9%和98.1%。将青鱼β-actin 基因 5′端启动调控区插入不含启动子的 pEGFP1 载体构建青鱼β-actin 启动子/EGFP 表达载体,与第一次卵裂之前显微注射该重组质粒入泥鳅受精卵,同时也用该重组质粒转染 HeLa 细胞系。观察结果表明:GFP 在 50%的泥鳅胚胎和 2/3 的 HeLa 细胞有所表达。GFP 在泥鳅胚胎的各个部分均有表达,且在某些胚胎中 GFP 的表达遍布全身。因此,以 EGFP 为报告基因证实了青鱼β-actin 基因启动子为一种非特异性表达的启动子。
A 3 338 bp DNA fragment including the open reading flame and 5＇-flanking region of β-actin gene for black carp genome was obtained through PCR amplification. Analysis of the sequencing results indicated the ORF of black carp β-actin gene encoding a 375 amino acid protein that shares a high degree of conservation to other known actins. The black carp β-actin sequence showed 100% identity to common carp, grass carp, and zebrafish, 99.2% identity to human and Norway rat β-actin gene, 98.9% and 98.1% identity to chicken and Kenyan clawed frog β-actin gene, respectively. The promoter region of black carp β-actin gene was inserted into the promoterless pEGFP1 vector. The recombinant plasmid was microinjected into the fertilized eggs of mud loach before two-cell stage as well as transfected into HeLa cell line. GFP expression was found in 50% of mud loach embryos and 2/3 HeLa cells. The GFP expression could be observed in every part of the mud loach embryos, and in some embryos, the GFP was expressed in the whole body. Thus, the usefulness of black carp β-actin promoter as a ubiquitous expression promoter was confirmed using the EGFP as a reporter gene.
基金
This work was supported by the National 973 Program (No.2001CB109006), the Scientific Funds of the Educational Bureau of Hunan Province (No.04C378), and the Ph.D Programs Foundation in Hunan Normal University (No. 24030610)
