Regulation Of Porcine EXOC4 Gene Expression And Its Correlation With Reproductive Traits

EXOC4(Exocyst complex component 4)gene is one of the members of Exocyst complex gene family.Exocyst complex gene family is mainly involved in the biological process of vesicle mediated transport.Previous studies showed that there was a significant correlation between the number of nipples in the sow and the two SNPs loci which were located in the second intron of the EXOC4 gene.Early studies also showed that the number of nipples had significant positive correlation with reproductive performance of sows,especially the weight of litters and weaning weight.In addition,our previous work of the GWAS showed that the SNP in the seventh intron of the EXOC4 gene was significantly correlated with the reproductive traits of Duroc.Here,we anazyled the effect of SNP of EXOC4 gene on the reproductive traits of sows,and explored the regulation of EXOC4 gene expression,which will provide the data for the molecular regulation mechanism of reproductive traits.In this study,the genetic diversity of EXOC4 gene in the Duroc,Yorkshire,Landrace and Landrace × Yorkshire were analyzed by PCR-PRLP technique,and the relations of different genotypes with reproductive traits of sows were calculated with SPSS19.0 software.q RT-PCR was used to quantify the expression level of m RN A in different tissues of different genotypes of EXOC4 gene.The core promoter region was determined by double luciferase activity analysis,and the potential transcription factor binding sites were predicted by bioinformatics in porcine ovarian granulosa cells.The results are as follows:(1)There is a rs81471943 locus(16079181-16079820)in the seventh intron of cloned EXOC4 gene,and T> C mutation was found at +149706bp;(2)In the population 387 samples including Duroc,Landrace populations and Yorkshire,the detected C allele frequency was 0.864,which was much higher than the frequency of the T allele(Q(T)=0.136),and SNP rs81471943 of EXOC4 gene exhibited Hardy-Weinberg equilibrium;(3)In the Duroc and Landrace populations,the EXOC4 gene showed three different genotypes.In the Duroc group,the numbers weaned litter and weanling piglets of CC type were sign significantly higher than that of TC type(P<0.05),but there was no significant difference in litter size and primary weight between the two genotypes(P<0.05).In the Landrace population,the numbers of primary and weaned piglets of CC type were significantly lower than those of TC(P<0.05),but there was no significantly in litter size and weaning weight between the two genotypes(P>0.05).There was no significant difference in the litter size,litter weight at birth,weaning weight and number of piglets weaned among the three types(P>0.05).Only two genotypes of CC and TC were detected in the Yorkshire population,and the litter size,birth weight,weaning weight and weaning piglets were not significant betweent the two types(P>0.05).and there were not significant for these reproductive traits(P> 0.05).(4)In CC and TC individuals,the EXOC4 m RN A was detected in different tissues had different expressed.In genotype TC,the higher m RNA of the gene was expressed in liver,followed by cerebellum,kidney and large intestine.In genotype CC,the higher expression was in large intestine,followed by cerebellum,brain,lungs.Second,in the liver,kidney,and cerebellum tissues,the m RNA of TC type was significantly higher than in the CC type,while in the lung and overian tissue,its expression in TC type was significantly lower than the CC type.(5)The fragment of 2791 bp of EXOC4 gene promoter was cloned,there were seven SNPs in the promoter region of EXOC4 gene with SNP rs81471943,which were-544C> G,-832G> A,-904G> A,934G> A,-1646C> T,-1665 A> G and-1781 G> A.(6)Five haplotypes of the SNP rs81471943 and EXOC4 gene promoter region SNP association were found in the population.The haplotype of WZ-2(AGTGAAG-T)is with rare frequency,while the other are with higher frequencies.The transcriptional activity of WZ-1(GAC GGGC-C)was significantly higher than that of WZ-4(AAC AGGC-T)(P<0.5).There was no significant difference between the two groups(P <0.5).It is speculated that the mutation in-1781 G/A and-934 G/A may be the reason for the difference in transcriptional activity between WZ-1(GACGGGC-C)and other haplotypes in ovarian granulosa cell.(7)The fragment of EXOC4 gene promoter was cloned,and was linked into the vector.Then the constructed vector was transfected into ovarian granulosa cells.The results showed that EXOC4-P2 activity was higher than the control,while EXOC4-P3 and EXOC4-P4 activity were lower than the control(P <0.05).The activity of EXOC4-P2 was higher than EXOC4-P1 significantly(P <0.05).Conclusion:The difference genotypes of the rs81471943 influence LWW and NW of foreign pig,it may be the results of the mutation of EXOC4 in-1781.