作　　者： ; ; ; ; ; ;

机构地区： 河南农业大学牧医工程学院

出　　处： 《Acta Genetica Sinica》 2004年第7期688-694,共7页

摘　　要： 从家蝇基因组文库中分离到含约 1 7kb 5′上游区的家蝇卵黄蛋白 1基因组基因序列 ,根据其 5′上游序列 ,PCR扩增出大小不同的 4个启动子片段 ,分别插入到切除了CMV启动子的 pCMV GFP质粒中的绿色荧光蛋白报告基因上游 ,构建了 pMYP1 GFP、pMYP2 GFP、pMYP3 GFP和 pMYP4 GFP 4个重组质粒。另将 +6 84 /+7、+116 5 /+7这两个启动子片段用SpeⅠ和HindⅢ双酶切 ,去除包含CAAT/TATA盒的 +30 2 /+7序列区后 ,分别构建了pMYP5 GFP和 pMYP6 GFP两个重组质粒。通过电转移实验和荧光检测表明 ,+6 84 /+7、+116 5 /+7、+16 16 /+7这 3个启动子片段具有转录活性 ,而 +6 84 /+7启动子片段的转录活性最强 ,+2 96 /+7、+6 84 /+30 2、+116 5 /+30 2这 3个启动子片段无转录活性。上述实验结果表明 ,+30 2 /+7序列区为启动子的核心部分 ,+30 2到 +16 16之间存在调控性启动子或增强子等其他一些顺式元件。细胞转染实验证实 ,6种启动子片段在BHK-2 1和Sf9细胞中都未表现出可检测的转录活性 ,说明家蝇卵黄蛋白基因启动子具有组织或细胞特异性。 A partial Musca domestica genomic library was constructed.It was consisted of 1.2×10~5 recombinants with insert length ranging from 10 kb to 23 kb(15 kb average).High molecular weight genomic DNA with more than 50 kb size was extracted from the larva hatched 36 h and digested with unfrequently cutting restriction enzyme BclⅠ.DNA fragments of 10～23 kb were recovered by agarose gel electrophoresis and ligated with EMBL3 BamHⅠ Arms CIPase treated.Then the products of ligation were packed in vitro using packing protein.The cloning efficiency of the genomic library was 5×10~4 pfu/mL.The genomic library was screened by hybridization using a probe of a 768 bp partial cDNA fragment of Musca domestica yolk protein 1(mdYP1) gene obtained by PCR and the probe was labeled with Digoxigen.A positive plaque was chosed and purified by in situ hybridization.A genomic DNA fragment about 4.0 kb mdYP1 was isolated from purified positive plaque by southern blotting analysis.Sequence analysis revealed that mdYP1 genomic gene was composed of 5′-upstream region about 1.7 kb with typical CAAT/TATA box.The promoter of the mdYP1 gene was characterized by examining the ability of 5′-upstream fragments to regulate expression of green fluorescent protein (GFP) in Musca domestica larva.Four fragments of the promoter region,P1(+296/+7),P2(+684/+7),P3(+1165/+7) and P4(+1616/+7),were obtained by PCR specific amplification using template of recombinant λ-DNA containing mdYP1 gene sequence.Then the four fragments were respectively subcloned into pCMV-GFP reporter vector deleted CMV promoter.All the fragments showed no promoter activity when the four recombinant vectors were transfected into Sf9 and BHK (-21) cells respectively,but three of them,P2,P3 and P4,showed significant promoter activity when they were respectively introduced into Musca domestica larva by electroporation.The two fragments,P5(+684/+302) and P6(+1165/+302),obtained by digesting P2 and P3 with SpeⅠ and HindⅢ,,were also subcloned into pGFP vector,and they showed

关 键 词： 家蝇 卵黄蛋白 基因 启动子 克隆 绿色荧光蛋白

领　　域： [生物学] [生物学]