Abstract
This study aims to provide a theoretical basis on the regulatory mechanism of the α-l,2-fucosyltransferase (FUT1) gene in pigs by analyzing the transcriptional activity of its promoter region. On the basis of the previously obtained promoter sequence, primers upstream and downstream of the gene were designed using the restriction endonucleases KpnI and HindIII respectively, and the recombinant plasmids of the pGL3-promoter were constructed by inserting promoter sequences with partially missing regions. The resultant mutants were observed by transient transfection assay into HEK293 cells, and the transcriptional activity of the promoter region was determined by luciferase activity. The 5′-flanking region of the FUT1 gene (−1150 to +50 bp) exhibited promoter activity. The −1150-bp to −849-bp region showed negative regulation of the gene. The recombinant plasmid pGL3-898 showed the strongest luciferase activity, and the activity showed a decreasing trend when the deleted region was increased. Recombinant plasmids were successfully constructed, verified, and the positive and negative regulation areas and core promoter region were detected, providing a deeper insight into the transcriptional regulatory mechanism of the FUT1 gene.
Highlights
Porcine post-weaning diarrhea (PWD) and porcine edema disease (ED) are two diseases caused by enterotoxigenic Escherichia coli F18 (ETEC F18)
The α-l,2-fucosyltransferase (FUT1) gene can affect the expression of the ETEC F18 receptor gene ECF18R and an M307 mutant locus in the FUT1 gene was a genetic marker for selecting new pig cell lines resistant to ETEC F18 [2]
The sequence-deleted fragment in the regulatory region was subcloned into the firefly luciferase plasmid pGL3-basic and transiently co-transfected into human embryonic kidney 293 (HEK293) cells with the internal reference plasmid pRL-TK containing sea pansy luciferase
Summary
Porcine post-weaning diarrhea (PWD) and porcine edema disease (ED) are two diseases caused by enterotoxigenic Escherichia coli F18 (ETEC F18). These diseases are responsible for tremendous damage and economic losses in the pig industry [1]. Other studies that analyzed the polymorphism at the M307 locus discovered that over 20 Chinese native pig breeds had only the GG genotype, and the distribution of these breeds in China was extremely skewed [4,5,6,7] These findings suggested that differences other than the coding region in the FUT1 gene may exist between native Chinese and commercial western pig breeds. Further studies are required to verify and validate the regulatory mechanism of ETEC F18 resistance in the FUT1 gene
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