斑马鱼基因打靶载体的构建及基因打靶的初步研究生物化学与分子生物学专业论文.docxVIP

AbstractThe Abstract The zebrafish foanio rerio)growth hormone疆fG}羚gene was isolated and sequenced following ampliflcation of its genomic DNA by the nest polymerase chain reaction (PCR)．The drGH span 5．5kb，comprised of five exons and foor introns．and was perhaps the longest GH gene up to date．A11 the introns started witll GT dinueleotide and ended with an AG．The intron 11 was shorter than that of the other cyprinids．and the intron III and intron IV were five times longer than the other carps．The deduced coding sequence was identical to the reported cDNA sequence of drGH gene and hi曲ly conserved among all the fishes．The reading frame encoded a deduced amino acid sequence of 210 amino acids lncluding the signal sequence of 22 amino acids。 霹lo predicted aminO acid sequence of drGH shared 85％homology wi氆other carp species．A typical t凇box located砒一28nt and no CAAT box was detected．There were five putative Pit一1／GHFl binding sites in the drGH promoter region．which they were at一68／一76，一332／一341，一396／_404，一418／一426，-466／一474．弧e 916 bp(一886／+3∞ and 272 bp(一24掣+3筑sequences of 5-flanking sequence of the蠢G珏gene proximal promoter region were cloned and their functional expressions in zebrafish embryo were analyzed．The GFP expression pattern indicated that the 272bp sequence is enough to control the onset of the transcription of the drGH gene．The investigation of the structure of zebrafish GH gene may be exploited to study the physiology regulation of gene expression。structure—to—function relationships and gene evolution． Base on the reported DNA sequence of zebrafish B．actin gene．PCR was employed to clone the promotor of t3-actin gene．The activity of the promotor was analyzed in the CHO cells via the green fluorescence protein report system． According to the principles of positive and negative selections撂NS)for gene targeting，two gene target vectors with double fluorescent protein selection cassette were designed and constructed． Gene targeting was extensively studied in zebrafi