【作者】 刘静波；

【导师】 陈代文； 余冰；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2010， 硕士

【摘要】 宫内发育迟缓显著影响后代个体出生后的生长发育,本研究应用RT-PCR和蛋白质组学技术,考察了叶酸对母猪繁殖性能和宫内发育迟缓仔猪肝脏基因表达和蛋白质组的影响。试验采用单因子试验设计,选择36头纯种大约克初产母猪,分为3个处理,每个处理12个重复,每个重复1头母猪。将配种后的母猪随机分到基础日粮组(叶酸含量1.3mg/kg)、基础日粮+叶酸10mg/kg和基础日粮组+叶酸含量30mg/kg。试验从母猪配种后开始至仔猪断奶,母猪妊娠第46,60和74d采血,记录各处理组每头母猪的繁殖性能指标,21 d断奶时称量仔猪体重。分娩时在基础日粮组和添加叶酸30mg/kg组中分别选取8头正常体重(NBW，BW＞1.4kg)和宫内发育迟缓(IUGR,0.8<BW<0.9kg)仔猪屠宰收集血清和肝脏。用放射免疫分析法测定血清叶酸浓度,用RT-PCR方法研究甲硫氨酸腺苷转移酶(MAT)、胱硫醚合成酶(CBS)、亚甲基四氢叶酸还原酶(MTHFR)、DNA甲基化转移酶-1(DNMT-1)、过氧化物酶体增生物激活受体(PPAR)、糖皮质激素受体(GR)、瘦素受体(ob-R)和乙酰辅酶A氧化酶(AOX)基因mRNA水平在肝脏中的表达差异,荧光标记差异电泳(DIGE)结合质谱串联质谱(MS/MS)技术获得处理间蛋白表达差异图谱以及目的蛋白鉴定。试验结果表明：1、母猪饲粮中添加叶酸可显著提高新生仔猪和妊娠不同阶段(妊娠46d、60d和74d)母猪血清叶酸含量(P<0.05),但对仔猪初生重、产仔数、产活仔数、仔猪存活率以及断奶体重无显著影响(P>0.05)。2、母猪叶酸营养影响新生仔猪肝脏中与一碳单位代谢和氧化应激相关基因的mRNA表达量。IUGR+C组仔猪MAT1A、MAT2A和DNMT-1基因mRNA的相对表达量显著低于IUGR+F组、NBW+C组和NBW+F组,而PPARγ、GR、AOX基因表达量高于其他各组(P<0.05)。IUGR组和IUGR+F组CBS及ob-R表达量低于NBW+C组和NBW+F组(P<0.05)。各处理间MTHFR和PPARα基因表达量无显著差异(P>0.05)。3、母猪饲粮中添加叶酸可显著上调宫内发育迟缓仔猪肝脏热激蛋白90、脂水解酶、亚胺甲基转移酶环脱氨酶、顺乌头酸酶和琥珀酸脱氢酶表达量,下调不均一核糖核蛋白、遍在蛋白缀合酶、胰蛋白酶、肌动蛋白和输出蛋白表达量(P<0.05)。综上可见,本试验发现妊娠母猪日粮添加高剂量叶酸显著提高母猪和新生仔猪血清叶酸水平,但对母猪繁殖性能无显著影响。高剂量叶酸可缓解宫内发育迟缓对仔猪肝脏中与一碳单位代谢和能量代谢相关基因表达的负面影响,改变宫内发育迟缓仔猪肝脏中许多蛋白的表达量,进而一定程度上缓减宫内发育迟缓对新生仔猪生长发育的负面影响。更多还原

【Abstract】 Intrauterine growth restriction (IUGR) significantly influenced the postnatal growth performance of the offspring. To gain insights into the changes of gilts reproductive performance, hepatic gene expression patterns and proteomics in IUGR piglets after maternal folic acid supplementation, RT-PCR and proteomics technology were used in this study.Single factorial experimental design was used in the present study. Thirty-six gilts were randomly allocated into three groups fed three dietary folate level immediately after mating until weaning, basal diet group (folic acid 1.3mg/kg), basal diet+folic acid 10 mg/kg and basal diet+folic acid 30 mg/kg with 12 replicates per group. The blood samples of the gilts were collected at gestation d 46, d 60 and d 70, weanling weight of the piglets at 21-day-old, and reproductive performance of the gilts were recorded. Eight normal body weight (NBW, BW> 1.4kg) and intrauterine growth restriction (IUGR,0.8<BW<0.9kg) piglets from control and treatment 2 were used to collect serum and liver. Radioimmunoassay (RIA) and RT-PCR were used to determine the serum folic acid content and hepatic mRNA expression of methionine adenosyltransferase (MAT), cystathionine-(3-synthase (CBS), methylenetetrahydro-folate reductase (MTHFR), DNA methyltransferasel (DNMT1), peroxisomal proliferator-activated receptor (PPAR), glucocorticoid receptor (GR), obesity receptor (ob-R) and Acyl-CoA oxidase (AOX) in the piglets. DIGE combined with MS/MS technology were used to obtain the differentially expressed protein and target protein identification.The results showed that:1. Folic acid supplementation could increase serum folic acid content in the newborn piglets and gilts at different phrase(d 46, d 60 and d 74 of gestation) during the gestation (P＜0.05), but had no effects on birth weight, litter size, born alive number, survival rate and d 21 weaning weight(P＞0.05).2. Maternal folic acid supplementation altered mRNA expressions of hepatic gene associated with one-carbon metabolism and energy metabolism in newborn piglets. Real-Time PCR indicated that gene expression pattern of MAT1A, MAT2A and DNMT1 were the lowest in IUGR group (P<0.05). Transcriptional expression level of PPARy, GR and AOX were the highest in IUGR group (P<0.05). The expression abundance of CBS and ob-R were lower in IUGR and IUGR+F groups than in NBW and NBW+F groups (P<0.05). There were no significantly difference in gene expression levels of MTHFR and PPARa among groups (P>0.05).3. Proteomic analysis indicated that folic acid supplementation increased the content of heat shock protein 90kD, aconitase 1, succinate dehydrogenase, formiminotransferase cyclodeaminase and abhydrolase, but down-regulated the expression of heterogeneous nuclear ribonucleoprotein, exportin cselp, ubiquitin-conjugating enzyme, trypsin and actin-related protein in the liver of the piglets (P<0.05).The results indicated that maternal high dose folic acid addition increased serum folic acid levels of gilts and newborn piglets, but had no effects on gilts reproductive performance. Maternal folic acid supplementation reduced the negative effects of IUGR on hepatic expression levels of genes involving in one-carbon metabolism and energy metabolism and altered several proteins expression profiles. The changes of the proteins expression profiles may alleviate the negative effects of IUGR on piglet growth performance.更多还原