Cloning And Preliminary Research Of Porcine CREBs Family

The CREB(cAMP response element binding protein)/ATF(activating transcription factor) family transcription factors are a kind of basic transcript factors in eucaryote, which all contain the typical bZIP motif.CREB/ATF proteins are crucial for a variety of cellular processes including cell proliferation,differentiation,apoptosis(Persengiev et al., 2002),extra-stimuli and stress response.CREB2,CREB3 and CREB3L4 are three important members of ATF/CREB family.In this study,we first reported the gene cloning,chromosome mapping and expression analysis of CREB2,CREB3 and CREB3L4 in pigs.The cDNA and genomic sequences of the three genes had been successfully cloned and analysed.Radiation Hybrid(IMpRH) panels and semi-quantitative RT-PCR method had been used to obtain the information of chromosome mapping and tissue transcription profiles.Additionally, we cloned the promoter region of CREB3 and further constructed pGL3 Luciferase Reporter vector.Real time qRT-PCR was used to detect the reletive expression of CREB3 mRNA after stimuli of lipopolysaccharide(LPS),and Dual luciferase assay system was used to detect the promoter activity after LPS stimulation.The main results are as follows:1.The cDNA sequences including full coding region sequences of CREB2 and CREB3 had been cloned and analysed in pigs.The coding region of CREB2 and CREB3 was 1047 bp which encoded 348 amino acids and 1098bp which encoded 365-amino acid, respectively.The coding region of CREB3L4 varant1 and varant2 was 1188bp and 816bp which encoded 395-amino acid and 271 amino acid,respectively.The deduced amino acid sequence of porcine CREB2 shared 85.8%and 71.7%similarity with its human and mouse counterparts,respectively.The predicted amino acid of porcine CREB3 shared 72.6%and 65.0%similarity with its homologous in human and mouse, respectively.The deduced amino acid sequence of porcine CREB3L4 shareed 82.5%and 61.1%similarity with its homologs in human and mouse,respectively.2.The genomic sequences of porcine CREB2,CREB3 and CREB3L4 had also been obtained.We further characterized their genomic structures.The genomic region of porcine CREB2 covered 2069bp consisted of three exons and two introns;Porcine CREB3 spanned approximately 5.3 kb of the genomic DNA and comprised nine exons and eight introns;The genomic sequence of porcine CREB3L4 spans approximately 6 kb consisted of ten exons and nine introns;Moreover,the splice pattern of each intron conformed to the GT-AG rule.Base on the obtained genomic sequences of the three porcine genes and method of Radiation Hybrid(IMpRH) mapping,porcine CREB2, CREB3 and CREB3L4 localized at chromosome 5p,1q28 and 4q,respectively.3.Tissue expression patterns were analyzed by the method of semi-quantative RT-PCR in 11 porcine(Meishan) tissues including adipose(subcutaneous back fat),spleen,lung, kidney,liver,small intestine,brain(cerebellum),stomach,muscle(longissimus dorsi), heart,lymph(neck lymph node).Tissues expression analysis revealed that porcine CREB2,CREB3 and CREB3L4 mRNA were ubiquitously expressed in all examined tissues.CREB2 and CREB3 are highly co-expressed in several tissues including white adipose tissue,stomach,lung,cerebellum,liver and lymph.The relatively high expression level of CEEB3L4 variant1 was found in stomach,liver,cerebellum,moderate in testis, small intestine,followed by lymph,white adipose,spleen,kidney,lung and heart,and extremely low in skeletal muscle.As to CREB3L4 variant2,weak signals were detected in all porcine tissues.Therefore,the normal CREB3L4(variant1) should be the predominant form in pigs.4.5' flanking region of porcine CREB3 was cloned and sequenced to investigate its transcriptional regulation,we identified four consensus sequences for SP1(stimulating protein 1),a canonical NF-κB binding site,two consensus recognition sequence for activator protein-1(AP-1) and an activator protein-2(AP-2) recognition site in the predicted promoter,we then constructed pGL3 Luciferase Reporter vector containing our predicted CREB3 promoter region,and this region was proved to have high promoter activity.5.The expression of CREB3 mRNA was detected using real-time qRT-PCR under stimuli of lipopolysaccharide(LPS) with a dose of 1μg/ml in IBRS and mouse RAW264.7 cells. As a result,CREB3 mRNA was not changed under the stimuli of LPS.Moreover,we further observed whether the promoter activity of CREB3 was affected under the treatment of LPS in 293T cells using the Dual luciferase assay system.The promoter activity of CREB3 also showed no significant change,which was consistent with the results of qRT-PCR.