Association Between Polymorphisms Of SLC30A Gene Family And Reproductive Traits Of Yorkshire And Landrace Sows

The reproductive traits of sows were low heritability quantitative traits affected by multiple factors such as breed,environment,and age.Since zinc plays an important role in animal reproduction,the zinc transporter gene family（SLC30A,Zn T）which contribute to the cytoplasmic zinc balance by exporting zinc out to the extracellular space or by sequestrating cytoplasmic zinc into intracellular compartments was selected as the candidate genes,and a variety of bioinformatics software was used to analyze the SLC30A gene family of pigs,including motif and conserved domain analysis.The different expression of SLC30A gene family in the heart,liver,spleen,lungs,kidneys,ovaries and mammary glands from Yorkshire were determined by using fluorescence q RT-PCR.SPSS,DMU and other software were used to estimate the phenotypic and genetic correlation coefficient between the total number born（TNB）,the number of born alive（NBA）,the number of born soundness（HLS）and litter birth weight（LBW）.The SNP loci in SLC30A gene family from 285 Yorkshire and 183Landrace sows were detected by flight mass spectrometry,and reproductive traits-related SNPs in SLC30A gene families were screened.The results of the study are as follows:1.It was found that the proteins encoded by SLC30A gene family belongs to the transmembrane structural protein,and the basic order of motif contained in these proteins was consistent,and Cation efflux was a conserved domain shared by the SLC30A proteins.The strongest interaction was found between SLC30A1with SLC30A3 protein,and SLC30A8 showed weakest interaction with other family members.All genes,except SLC30A2,showed high expressions in the lungs and relatively low expression in the heart,and except for SLC30A5 and 7,the SLC30A gene family had low expression levels in muscle.High expressions of SLC30A2,3,5,9 and 10 could be detected in breast tissue.2.The worst reproductive performance was found in the 1st parity.The TNB,NBA,HLS and LBW showed an increasing trend from the 2nd to 5th parity.The genetic and phenotypic correlation coefficient of TNB with NBA,HLS and LBW were 0.975,0.911,0.621 and 0.893,0.741,0.489,respectively in Yorkshire,and 0.902,0.864,0.693,and 0.829,0.648,0.546,respectively,in Landrace sows.3.Twenty-three SNPs were detected in SLC30A gene family and 9 SNPs which located respectively in SLC30A2,SLC30A5,SLC30A6 and SLC30A9 gene were found to significantly associated with at least one or more reproductive traits in Yorkshire and Landrace sows.The SNP of rs328358158 C>T which located in the SLC30A2 3’UTR region was found to significantly relate with TNB,NBA,HLS and LBW in Yorkshire and TNB,NBA and LBW in Landrace sows（P<0.05）.Yorkshire sows with CC or CT genotype had significantly higher TNB、NBA、HLS、and LBW compared to TT genotype（P<0.05）.It was also found that TNB,NBA and LBW in Landrace sows with CC genotype were significantly higher than those in CT or TT genotype sows（P<0.05）.The SNP of rs342307573 A>G which located in the SLC30A2 coding sequence was also found to significantly relate with NBA、HLS and LBW in Yorkshire and Landrace sows（P<0.05）,It was significantly correlated with TNB of Yorkshire（P<0.05）.Yorkshire sows with AA or AG genotype had higher TNB、NBA、HLS and LBW than those with GG genotype（P<0.05）,and significantly higher NBA and HLS were also found in Landrace sows with AA or AG genotype compared to sows with GG genotype（P<0.05）.4.Micro RNA prediction of rs328358158 C>T of SLC30A2 was performed by bioinformatics,and this site was found to bind to mi R-345-3p.The targeting relationship between mi R-345-3p and SLC30A2 gene was verified by Dual-Luciferase Reporter assay.Luciferase activity was significantly down-regulated by co-transfection of mi R-345-3p mimic and SLC30A2-3’-UTR wild-type vector（SLC30A2-3’UTR-WT）into PGC cells（P<0.001）,while luciferase activity was significantly up-regulated by co-transfection of SLC30A2-3’UTR-WT and mi R-345-3p inhibitor into PGC cells（P<0.001）.Luciferase activity was not significantly affected by co-transfection of mi R-345-3p mimic or mi R-345-3p inhibitor with SLC30A2-3’UTR-mutant vector into PGC cells（P>0.05）.The results confirmed that SLC30A2 gene was the target gene of mi R-345-3p and the3’-UTR mutation of SLC30A2 gene（rs328358158 C>T）was a loss-of-function mutation resulting in mi R-345-3p missing its function targeting to SLC30A2 gene.5.Overexpression of mi R-345-3p significantly down-regulated the expression of SLC30A2,Stat5a and GLUT1 in PGC cells and inhibited the proliferation of PGC cells（P<0.05）.It was speculated that mi R-345-3p affected the state of zinc in cells by regulating SLC30A2 expression and then through the JAK/STAT signaling pathway to regulate PGC cell proliferation.6.Based on the rs342307573A>G mutation in the SLC30A2 gene coding sequence,the wild-type vector（p EGFP-N1-SLC30A2-WT）and mutant-type vector（p EGFP-N1-SLC30A2-MUT）of SLC30A2 gene coding sequence were constructed.The expression of GLUT1 in PGC cells transfected with p EGFP-N1-SLC30A2-WT was significantly increased（P<0.001）.However,AKT1 expression was not significantly affected（P>0.05）.Transfection of p EGFP-N1-SLC30A2-MUT lead to GLUT1 and AKT1 expression in PGC cells significantly decreased（P<0.001）.It was also found that the serum Zn²⁺concentration of sows with AA or GA genotype of SNP rs342307573A>G was significantly higher than those with GG genotype（P<0.05）.These results suggested that the rs342307573A>G mutations in SLC30A2 gene coding sequence may affect SLC30A2 to transport Zn²⁺in the cells.In conclusion,9 SNPs detected in SLC30A gene family of Yorkshire and Landrace sows were found to be significantly associated with one or more reproductive traits,which suggest that they can be used as genetic markers affecting reproductive traits of Yorkshire or Landrace pigs.Mutation of SLC30A2gene（rs328358158 C>T）resulted in mi R-345-3p missing its function targeting to SLC30A2 gene.The rs342307573A>G mutation in encoding sequence of SLC30A2 gene may lead to the change of SLC30A2 protein function to transport Zn²⁺in cells.The changes of SLC30A2 protein translation or protein function may be one of the mechanisms that both mutation affected on the reproductive performance of Yorkshire and Landrace sows.