Tissue Expression Pattern And Association Analysis Of Porcine MMP7 And MMP9 Genes With Diarrhea In Suckling Piglets

The matrix metalloproteinases 7(MMP7)and matrix metalloproteinases 9(MMP9)are members of the matrix metalloproteinases(MMPs)gene family.Enterotoxigenic Escherichia coli(ETEC)F18 is one of the main pathogens contributed to piglet diarrhea.Studies have shown that after ETEC F18 infection,the m RNA expression of MMP7 and MMP9 genes in duodenum of resistant individuals was higher than that of ETEC F18 sensitive individuals.In this study,the MMP7 and MMP9 genes were selected as candidate genes for piglet diarrhea,and the expression patterns of MMP7 and MMP9 genes in gastrointestinal tract and other tissues of weaned piglets were detected by q RT-PCR;the p EGFP-C1-MMP7 and p EGFP-N1-MMP9 eukaryotic expression plasmids were constructed to study the fusion protein subcellular localization;association analysis between SNPs in MMP7 and MMP9 genes and piglets diarrhea and growth traits was performed in Min and Landrace populations.The promoter region of MMP9 gene was identified using 5’deletion analysis.The results are as follows:(1)Ten tissues of 28-day-old weaned piglets,including heart,liver,spleen,lung,kidney,stomach,duodenum,jejunum,ileum and colon were collected.The total RNA was extracted.After reverse transcription,the m RNA expressions of porcine MMP7 and MMP9 genes were detected by q RT-PCR.The results show that the MMP7 and MMP9 genes were expressed in all the tissues including the stomach,duodenum,jejunum,ileum and colon.(2)The recombinant plasmids p EGFP-C1-MMP7 and p EGFP-N1-MMP9 were constructed and transfected into porcine kidney cells and porcine intestinal epithelial cells line.The MMP7 and MMP9 fusion proteins were found in the cytoplasm using fluorescence microscope.(3)The genetic variation in MMP7 gene was identified by sequencing and the Hae III PCR-RFLP assay was established to detect SNP rs327380117.Statistic analysis between this Single nucleotide polymorphism and diarrhea scores and growth traits was conducted in Min and Landrace population.In the Landrace population,the diarrhea score of the TT genotype was extremely significantly lower than that of the TC genotype(P<0.01);the 14-day-old body weight of the TT genotype was significantly lower than that of the TC genotype(P<0.05).Association analysis between SNPs in MMP9 and diarrhea scores and growth traits were also performed in these two populaitons.The results showed that the diarrhea scores of the AA genotype(g.48178429G>A)was lower than that of the GA genotype(P<0.05)whereas GG had higher day-35 body weight and average daily gain(ADG)than AA in the Landrace population(P<0.05).At the rs336583561 locus,Min piglets with the GG genotype have lower diarrhea score than AG piglets(P<0.05).At g.48184777C>T,the birth weight and body weight of 3th,7th,14 th,21th,28 th,and 35 th day of CC piglets were higher than TC in the Landrace population(P<0.05 or P<0.01).(4)The bioinformatics analysis found that SNPs in the coding region of the MMP7 gene are synonymous mutations.The SNP g.48178429G>A of the MMP9 gene is located in the 5’flanking region of the MMP9-203 transcript,but this region has no promoter activity.In summary,this study found that porcine MMP7 and MMP9 genes were expressed in the heart,liver,spleen,lung,kidney and gastrointestinal tissues of weaned piglets;the EGFP-MMP7 and EGFP-MMP9 fusion proteins were localized in the cytoplasm of PK15 and IPEC-J2 cells.SNP rs327380117 in MMP7 gene is associated with diarrhea score in Landrace piglets.The SNP g.48178429G>A and rs33658361 in porcine MMP9 gene are associated with diarrhea in Min population;SNP g.48178429G>A and g.48184777C>T are associated with growth traits in Landrace population.The promoter region of MMP9 gene is located in the 5’ flanking region of the MMP9-201 transcript.