Cloning of porcine LPAR3 gene and its expression in endometrial cells

Objective  To obtain the complete mRNA sequence and structure of porcine LPAR3gene, study the gene promoter activity, and explore the transcriptional regulation of LPAR3 gene in endometrium and the mechanisms which may affect sow farrowing.

Method  The 5'RACE and 3'RACE techniques were used to obtain the complete LPAR3mRNA sequence. The potential promoter transcription factor binding sites and CpG islands in the 5' regulatory region were predicted. Recombinant vectors with different length of promoters and dual-luciferase reporter gene were constructed, then were transfected with pRL-TK plasmid into pig endometrial cells, and the promoter activities were detected. RT-qPCR was used to compare the relative expression of LPAR3 gene in Erhualian(ER) and Landrace×Large White (LL) pigs on the 12th day of gestation. Sodium bisulfite modified sequencing was used to compare the methylation status of LPAR3 gene in endometrial tissues of ER and LL pigs on the 12th day of gestation.

Result  The full length of pigLPAR3 mRNA was 2 127 bp, the 5'UTR and 3'UTR were 202 and 860 bp respectively, and the CDS region was 1 065 bp. The 5' regulatory sequence, including 3 080 bp (–2 430/+650 bp) upstream of the LPAR3gene, was cloned. The online prediction results of the 5' regulatory region showed that there was no TATA box in the LPAR3promoter, but there were GC element and binding sites for CPBP and other regulatory factors. Two potential CpG islands were found at –190/–84 and –44/+651 bp. Nine recombinant vectors with 5' deletions of the promoter were constructed and transfected into pig endometrium cells. The dual-luciferase assay showed that the activity of promoter P4 (+454/+80 bp) was the highest, followed by P6 (–123/+80 bp). RT-qPCR results showed that the expression of LPAR3 gene in the endometrium of ER pigs on the 12th day of gestation was higher than that in other tissues, and it was extremely significantly higher than that in the endometrium of LL pigs on the 12th day of gestation. The methylation status of LPAR3 gene was low in the endometrium of two breeds and had no significant difference between two breeds.

Conclusion  The full length of pigLPAR3mRNA is 2 127 bp. The endometrial expression level of LAPR3 gene in ER pigs is higher than that in LL pigs on the 12th day of gestation, indicating that LPAR3 gene may be involved in early gestation of pigs and affects litter size.