[Cloning and characterization of the promoter region of Musca domestica yolk protein-1 gene].
A partial Musca domestica genomic library was constructed. It was consisted of 1.2 x 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 Bcl I. DNA fragments of 10 approximately 23 kb were recovered by agarose gel electrophoresis and ligated with EMBL3 BamH I Arms CIPase treated. Then the products of ligation were packed in vitro using packing protein. The cloning efficiency of the genomic library was 5 x 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 A-lambdaNA 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 -2 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 (+165/+302), obtained by digesting P2 and P3 with Spe I and Hind III, were also subcloned into pGFP vector, and they showed no promoter activity in Sf9 cells, BHK -21 cells and Musca domestica larva. The results demonstrated that the core promoter spanned 302bp and contained a CCAAT box and a TATA box upstream translation initiation codon (ATG), but itself had no transcriptional activity, and that regulatory promoters or enhancers and other cis-elements presented from +302 to +1616 were necessary to maintain the specific expression.
Xia
Ping-An
PA
College of Biology, China Agricultural University, Beijing 100094, China.
Liu
Wei-Quan
WQ
Jiang
Yu
Y
Sun
Shao-Guang
SG
Yang
Shu-Yan
SY
Wang
Ji-Gui
JG
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004527:Egg Proteins / Q000235:genetics*; D006793:Houseflies / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D018411:Spodoptera
D004527:Egg Proteins