The Relationship Of Gut Microbiota With Body Fat And ANGPTL4 Gene In Different Breeds Of Pigs

TaqMan real—time PCR and pig ileum epithelial cells in primary culture were selected to evaluate the relationship of gut microbiota with body fat and ANGPTL4 Gene in Different Breeds of Pigs,six experiments were conducted as follows:EXP.1 Establishment of fluorescence quantitative 16SrDNA TagMan-PCR for detecting swine bacteroides—prevotella populationBased upon multiple sequence alignment of the 16S rDNAs of 30 bacteria in pig tract,The fluorescence quantitative specific primers and probe were designed for deceting bacteroides-prevotella. The DNA of B.thetaiotaomicron(CDC1404-A) was used as template, the target 16S rDNA fragment was amplified by PCR with specific primers, and used as standard sample, the dilution gradients of which were subjected to the standard quantitation curve in FQ-PCR assay.Diferent concentrations of Bacteroides thetaiotaomicron genomic DNA were detected by FQ-PCR in a 25ul reaction system with TaqMan probe. At the same time, various genomic DNAs of lactobacillus,escherichia,bifidobacterium were used as negative controls to confirm specificity of the FQ-PCR detection assay.The results showed that:1) The targret 16SrDNA fragment of B.P respectivly had 92% and 94% similarity with Bacteroides thetaiotaomicron(Genebank number: M58763) and Bacteroides intermedia(Genebank number: AF414822). 2) In a 25ul reaction system, the best working concentration of B.P probe and Mg²⁺ respectivly was 0.24μM and 5.0mM, The sensitivity of B.P probe was 3.73 cells/ul;3) The standard curve of B.P had good linear dependence when the reactinon system contained 2.67×10²-2.67×l0⁶copies/mL, at the same time,the best linear correlation occured between amplification CT and the logarithm of copy number.EXP.2 The Relationship of Intestinal Flora in Different Breeds of Pigs with body fatThese pigs were fed with the same diet and slaughtered at 0,1,2,3,4 and 5 month,and determined for backfat thickness,fat percentage,adpiose cell diameter, intramusculary fat, the number of total bacterial number, gram positive bacteria(G⁺), gram negative bacteria(G^-), G⁺/G^-and B.P in the contents of ileum and cecum in two breeds pig.The results showed that:1) The ileum and cecum of Taihu pigs had greater(p<0.05) G^-and and B.P, but lower(p<0.01) the proportion of G⁺/G^-and had no difference in total bacterial number and G⁺(p>0.05) than those of Landrace pigs.2) The positive correlation exsited of G^-and B.P with backfat thickness,fat percentage and adpiose cell diameter,and intramusculary fat, but the negative correlation of G⁺/G^-with body fat.EXP.3 ANGPTL4 Gene Cloning and Establishment of Real-Time Fluorescence Quantitative TagMan-PCR for detecting its ExpressionThe targeted DNA fragments of ANGPTL4 andβ-actin were first cloned, According to the mRNA sequences of ANGPTL4 andβ-actin genes in Landrace and Taihu pigs, the fluorescence quantitative specific primers and probes were designed. The cDNA of ileum was used as template, the target ANGPTL4 and p-actin fragment was amplified by PCR with specific primers, and used as standard sample, the dilution gradients of which were subjected to the standard quantitation curve in FQ-PCR assay. In a 25ul reaction system, optimized the working concentration and detected the sensitivity of TaqMan probe.The results showed that: 1) The targeted DNA fragments of ANGPTL4 and P-actin was cloned and sequenced.The sequences of ANGPTL4 gene in Taihu and Landrace pigs had 92% and 98% similarity With canonical sequence(AY751522),and p-actin had all 98% similarity With canonical sequence(U07786).2) In a 25ul reaction system, the working concentration of ANGPTL4 andβ-actin probes respectivly was 0.35μM and 0.40μM, The sensitivity of ANGPTL4 andβ-actin probes were 13.7and 83.1copies/ul.3) The standard curve of ANGPTL4 and p-actin genes had good linear dependence when the reactinon system contained 1.37×10⁴-1.37×10⁹ ANGPTL4 copies/mL and 8.31×10⁵-8.31×10¹⁰β-actin copies/mL, at the same time,the best linear correlation occured between amplification CT and the logarithm of copy number.EXP.4 ANGPTL4mRN A Expression in Tissues of Different Breeds of PigsFour Landrace pigs and four Taihu pigs fed with the same diet was slaughtered at 4 month Semi-quantitative RT-PCR was applied to determine ANGPTL4 gene expression difference in liver,fat,heart, jejunum,ileum,cecum and colon of Landrace and Taihu pigs.The results showed that:1) ANGPTL4 gene was expressed in liver,fat,heart,small intestine and large intestine. Furthermore,it was most abundance in adipose tissue,followed by liver, large intestine, heart and small intestine.2) Compare with those of Landrace pigs, Taihu pigs had extremely higher(p<0.01)in the ANGPTL4mRNA of liver and fat,extremely lower(p<0.01) ANGPTL4 expression in jejunum and ileum, no difference(p>0.05) in heart, cecum and colon.EXP.5 The Relationship of Intestinal Flora in Different Breeds of Pigs with ANGPYL4 Gene ExpressionTwenty-four Landrace neborn pigs and twenty-four Taihu neborn pigs was randomly divided into 2 treatment according to two breeds,each treatment has 6 replications.These pigs were fed with the same diet and slaughtered at 0,1,2,3,4 and 5 month,and determined for backfat thickness,fat percentage,adpiose cell diameter, intramusculary fat, ANGPTL4RNA and Bacteroides-Prevotella group.The results showed that:1) From 1 to 5 month,the B.P contents of ileum in Taihu pigs were higher, especially at 4 and 5 month,was significantly higher(p<0.01) than those of Landrace pigs. The B.P contents of ileum in bothe breeds of pigs had the tendency of increasion-decrease-incresion again with ages increased,the decrease occurred at the second month.2) From 0 to 5 month, ANGPTL4mRNA of ileum in Taihu pigs were lower, especially at 1, 2and 5month,was significantly lower(p<0.01) than those of Landrace pigs, However, there was no difference at newborn pigs. ANGPTL4mRNA of ileum in both breeds of pigs had the tendency of decrease-increasion-decrease again with ages increased,the increasion occurred at the second month.3) The negative correlation exsited between ANGPTL4mRNA with G^-and B.P,but the positive of correlation ANGPTL4mRNA with the proportion of G⁺/G^-.EXP.6 The Influence of Bacteroides thetaiotaomicron and Metabolite on ANGPTL4 gene expression of pig ileum epithelial cells in primary culturePig intestinal epithelial cells in primary culture were selected to evaluate the influence of Bacteroides thetaiotaomicron(BT) and its metabolite on ANGPTL4mRNA in ileum epith-elial cells.The experiment were designed 7 treatments:BT1(10⁸Cfu/ml) BT2(10⁷ Cfu/ml), BT3(10⁶Cfu/ml), metabolite1(10⁸dilution),metabolite2(10⁷dilution),metabolite 3(10⁶)dilution. Control,BT1(10⁸Cfu/ml) and metabolitel(10⁸dilution) respectivly had 20 replications. other treatments had 4 replications.Each replication had one hole. These pig intestinal epithelial cells were pre-incubated for 96h at 37℃, then transferred to the same medium containing different concentrations of BT and its metabolite, The incubation was continue-d for a further 1,3,7,12 and 26h in BT+10⁸Cfu/ml and metabolite+10⁸ dilution, others was incubated for 7h, these cells was collected to investigate their influence on ANGPTL4.The results showed that:1) Bacteroides thetaiotaomicron inhibited ANGPTL4 gene expression in dose-and time-dependent manner. When the ration of BT and cell number was 1:1, ANGPTL4 gene expression could be inhibited. The higher concentrion(10⁸Cfu/ml) had higher inhibition than lower concentration(10⁶Cfu/ml).2) The metabolite of BT did not influenceANGPTL4 gene expression in dose-dependent manner, but increased ANGPTL4 gene expression with the time prolongation.Through the above five experiments,we concluded that:1) The ileum and cecum of Taihu pigs had greater G^-and and B.P, but lower the proportion of G⁺/G^-and had no difference in total bacterial number and G⁺ than those of Landrace pigs. With ages increased,there was increasing tendency in total bacterial number, gram positive bacteria(G⁺), gram negative bacteria(G^-) and B.P,but had the decreasing tendency of G⁺/G^-in the contents of ileum and cecum in two breeds pig.2) ANGPTL4mRNA of ileum in Taihu pigs were lower than those of Landrace pigs, ANGPTL4mRNA of ileum in both breeds of pigs had the tendency of decrease-increasion-decrease again with ages increased.3)Bacteroides inhibited ANGPTL4 gene expression but its metabolites did not influence it.4) The positive correlation exsited of G^-and B.P with body fat but the negative correlation of G⁺/G^-with body fat..