Preliminary Study Of Transcriptional Regulation In Duck ALB Gene Promoter

Albumin(albumin,ALB)was serum protein only expressed in liver,accounting for 5%to 10%of newly synthesized proteins.The early study found that ALB gene was closely associated with duckling hepatitis,expressed specifically in liver tissues.Thus,it seemed that ALB gene can be used as a resistance gene marker.At present,the study of ALB gene mainly concentrated on the relationship between ALB gene and disease and regulatory mechanism of its efficient and specific expression in rat or human.Although the technology was mature and widely used on transgenic animals,little research was done about poultry,especially in its transcriptional regulation.For revealing the molecular mechanism of the specific expression of ALB gene,the paper carried out a study on transcriptional regulation of ALB gene.Transcription activity of ALB promoter was identified in different cell lines and then the core promoter and important transcription regulatory elements in liver were identified using transcription system in vitro1.Analysis of expression activity of duck ALB gene promoter in different cell linesIn order to investigate the molecular mechanism of ALB gene's specific expression in liver,the study analyzed the obtained promoter in the early stage with highly specific promoter prediction software.After a comprehensive analysis,it found that the promoter region of ALB gene may be initially located near-1000bp of upstream of the transcription start site,therefore the region fragment-1037 to +42bp(+1 transcription initiation site)was employed in the study.Used as the template obtained by previous genomic walking,ALB gene promoter sequence was amplified and replaced the CMV promoter in pEGFP-N1 vector,which lead to the construction of the vector pALB-EGFP,pEGFP-N1 and pEGFP-N1-CMV-(removal of the complete CMV promoter).The plasmids above were transfected into chicken embryo liver cell lines(CEL),rat liver cell lines(BRL-3A),chicken fibroblast cell lines(DF-1),mouse spermatogonial celllines(GC-1)and human renal epithelial cell lines(293T)mediated by lipofectamine.Then we detected the expression of GFP in above cells under the inverted fluorescence microscope.The results showed that pEGFP-N1 was transiently transfected into the 5 cells above.Strong green fluorescence was detected after 24h transfection and the fluorescence intensity was steady;pEGFP-Nl/CVM-had not been detected;we detected a small amount of GFP expression in the cell lines of DF-1,GC-land 293T;there was visible green fluorescence in transfected cells CEL and BRL-3A after 24h,lasting to 72h.The preliminary results showed that the ALB promoter's transcription was activated only in liver-oriented cells,and there was no species specificity.2.For the promoter region-1037 to +42bp,6 ALB promoter deletions were constructed to determine the core promoter region with the luciferase reporter gene vectors.In order to further locate the core region,a total of 6 deletion mutants were constructed with duck genomic DNA used as template by missing part of the 5' end sequence and were as follows:M1(pGL3-Enhancer-1037bp/+42bp),M2(pGL3-Enhancer-581 bp/+42bp),M3(pGL3-Enhancer-450bp/+42bp),M4(pGL3-Enhancer-190bp/+42bp),M5(pGL3-Enhancer-70bp/+42bp)and M6(pGL3-Enhancer-51bp/+42bp).The results showed that the 6 deletion mutants had different promoter activity in CEL and BRL-3A cells with the same trend,proving ALB promoter had no species specificity once again.All constructions were compared with pGL3-Control,the activity of M6 was lowest,M4 had high activity with insignificant difference from M2 and M3,but significantly higher than Ml,M5.M1,M5 had about 60%activity of pGL3-Control.The region of-190 to +42bp was the core area,the region-70 to-51bp may play a vital role on promoter activity,in which there were important regulatory elements,and there may be negative regulatory elements in the region-1037 to-581bp.3.The analysis of important regulatory elements of the core promoter region with site directed mutagenesis.Two important transcription factor binding sites in liver,HNF-1(hepatocyte nuclear factor-1)and C/EBPa(CCAAT/Enhancer binding protein),were revealed in the promoter region-70 to-51 bp by bioinformatics analysis and forecasting.It had been confirmed that the sites HNF-1 and C/EBPa played an important role on regulation of ALB gene promoter in mammals.The binding sites above within the scope of-70 to-51bp were subjected to site mutation in this research(firstly each site mutation,then both sites mutation).The activity of M4 was significant different from M5,the region-190 to-70bp contained 3 spaced nuclear factor binding sites predicted by bioinformatics software:HNF-3p(hepatocyte nuclear Factor-3p),NF1(nuclear factor 1),and C/EBP?.Site-directed mutageneses of 3 binding sites were carried out respectively.The obtained vectors above were transfected into CEL cell lines.It indicated that the activation of sites HNF-3? and NF1 were weak;while the sites HNF-1 and C/EBPa had a role in transcription regulation of promoter,the activation of HNF-1 was stronger after exploring the effect of these sites mutation on promoter activity,...