The Initial Research On Promoter Activity In Porcine AGL

AGL encoding glycogen debranching enzyme is a multifunctional enzyme acting as 1,4-a-D-glucan 4-a-D-glycosyltransferase and amylo-1,6-glucosidase in glycogen degradation. Genetic deficiency of AGL activity causes glycogen storage disease typeⅢ.Currently, the AGL gene structure and function of research has focused on men and horses.studies about porcine AGL has been reported rarely.In this study,the research was carried out to examine the promoter activity of the 5'flanking sequence. The main results are as follows:1 Study on the promoter activity of the corresponding to porcine AGL 5'UTRpGL3-5'UTR were constructed by substitution of SV40 promoter with AGL 5'UTR cDNA.Then pGL3-5'UTR were transfected into LO2 cells,and dual-luciferase reporter gene detection system and reverse transcription polymerase chain reaction detection were detected.The results indicate that, AGL 5'UTR had an obvious luciferase activity,and able to start the expression of downstream genes.2 Identification of the promoter domain in porcine AGL 5'flanking sequenceWe found that the existence of the TATA box sequence, CAAT box, GC box and several potential transcription factor binding sites, such as Smad3, Smad4, C/EBPβ, MAZF, USF, SP1, AP-2, MEF2, MyoD and HNF-3β/foxa2 and so on in the sequence of AGL 5'UTR by bioinformatics analysis. As a template to construct pGL3-5'UTR, a series of deletion constracts were constracted, Then they were transfected into LO2 and C2C12 cells. dual-luciferase reporter gene detection system and reverse transcription polymerase chain reaction detection were detected.It was found that 218bp sequence of translation start site upstream started with basal promoter activity; sequence of-411～-228bp and -685--602bp in AGL 5'UTR cDNA may be have a very important positive components.Bioinformatics analysis carried on sequence of-411-228bp,it was found that the HNF-3βbinding sites conserved. pGL3-690 was used as template,construct pGL3-△-690 was constructed by using overlap extension PCR. Then they were transfected into LO2 and C2C12 cells. Compared with pGL3-690, pGL3-△-690 activity decreased 98% and 84%, indicating that HNF-3βplay an important role in the AGL transcriptional regulation.3 Tissue-specific of porcine AGL 5'flanking sequence promoter activityWe selected high transcriptional activity of pGL3-690 and pGL3-5'UTR vectors which were transfected into LO2 and C2C12 cells. The results showed that, the activity of pGL3-5'UTR and pGL3-690 in C2C12 cells higher than in LO2 cells, but the difference was not significant.The transcriptional activity of these two sequences do not have tissue-specific, tissue-specific of the transcriptional activity of AGL5'flanking sequence needs further study.