【作者】 张丽萍；

【导师】 王康宁；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2007， 博士

【摘要】 本研究以实时荧光定量TagMan-PCR和原代培养猪回肠上皮细胞技术，研究了不同品种猪肠道微生物与体脂、ANGPTL4基因的关系。研究包括六个试验。试验一猪拟杆菌属-普雷沃氏菌属16SrDNATagMan-PCR实时荧光定量PCR方法建立通过对猪肠道30种细菌的16SrDNA序列进行比对分析，设计拟杆菌属—普雷沃氏菌属荧光定量的特异引物；以多形拟杆菌菌株（CDC1404-A）基因组的DNA为模板，用常规PCR引物扩增16SrDNA靶片断，将PCR产物纯化后作为DNA标准品，按1.19×10²—1.19×10⁶copies／ul 5个稀释梯度的标准品作模板，建立定量标准曲线：用TaqMan探针建立25ul反应体系，对不同浓度的多形拟杆菌DNA样品进行检测，检测探针的敏感性；并以乳酸杆菌、大肠杆菌和双歧杆菌的基因组DNA模板作阴性对照，检测拟杆菌属—普雷沃氏菌属的特异性。结果表明：1)通过克隆测序，得到了拟杆菌属-普雷沃氏菌属细菌的16SrDNA部分共有片段序列，该序列与Genbank中发表的多形拟杆菌（基因序列号为M58763）和中间普雷沃菌（基因序列号为AY323522）16SrDNA基因序列相比，该片段为多形拟杆菌的792～1003和中间普雷沃菌的763—974位碱基的片段，同源性分别为92％和94％。2)25ul PCR反应体系中Mg²⁺的最佳工作浓度均为5.0mM，探针最佳工作浓度为0.2μm，Taqman探针敏感性为3.73个／ul；3)本试验所制作的标准品稀释度在1.19×10²—1.19×10⁶copies／ul范围线性关系良好，标准品可用于后续试验的研究。试验二不同品种猪肠道微生物与体脂的关系试验通过选用在同一条件下饲养的0、1、2、3、4和5月龄的长白和太湖猪各4头，屠宰取后回肠和盲肠内容物，检测1、3和5月龄长白和太湖猪回肠和盲肠内容物的总细菌、革蓝氏阳性细菌（Gram positive bacteria，G⁺）、革蓝氏阴性细菌（Gram negativebacteria，G^-）、革蓝氏阳性细菌／革蓝氏阴性细菌（G⁺／G^-）、拟杆菌属-普雷沃氏菌属（Bacteroides-Prevotella, B. P）细菌数量、背膘厚度、脂肪率、脂肪细胞直径和肌内脂肪。结果表明：1)太湖猪的回肠和盲肠内容物的G^-及B．P细菌数量在不同年龄阶段均显著（p＜0.05）高于长白猪，G⁺／G^-极显著低于（p＜0.01）长白猪，总细菌、G⁺与长白猪无显著差异（p＞0.05）。2)猪回肠和盲肠的B．P和G^-数量都与背膘厚度、脂肪率、脂肪细胞直径和肌内脂肪都有正相关关系，而G⁺／G^-比例与背膘厚度、脂肪率、脂肪细胞直径和肌内脂肪有负相关关系。试验三猪ANGPTL4基因的克隆及其实时荧光定量TaqMan-PCR检测方法的建立为了对ANGPTL4基因进行更为准确定量，本研究首先克隆长白和太湖猪的ANGPTL4和β-actin的DNA序列。根据所测定的序列，分别设计引物和探针，以回肠基因组的cDNA为模板，用常规PCR引物扩增，将PCR产物纯化作为DNA标准品，ANGPTL4和β-actin基因标准品分别按1.37×10⁴－1.37×10⁹和8.31×10⁵－8.31×10¹⁰copies／mL的稀释梯度建立定量标准曲线，以TaqMan探针建立25ul反应体系，优化探针的最佳工作浓度，并检测探针的灵敏度。结果表明：1)应用克隆测序方法获得了长白和太湖猪的目的基因ANGPTL4和内参基因；β-actin的部分序列，所获得的长白和太湖猪的ANGPTL4的片断与参照序列（AY751522）的同源性分别为98％和92％，而B-actin与参照序列（U07786）的同源性都为98％。2)ANGPTL4和β-actin探针在25ul体系中的最佳工作浓度分别为0.35μM和0.40μM，探针的灵敏度分别为13.7和83.1拷贝／ul；3)所构建的ANGPTL4和β-actin的标准曲线良好，反应体系中分别含有1.37×10⁴－1.37×10⁹和8.31×10⁵－8.31×10¹⁰copies／mL时，扩增反应ct值与拷贝数的对数呈线性关系，所得标准品可以用于后续试验。试验四ANGPTL4基因在猪不同组织器官中的表达差异试验通过选用在同一条件下饲养的4月龄长白和太湖猪各4头，屠宰后取肝脏、脂肪、心脏、空肠、回肠、盲肠和结肠。根据试验三克隆的长白和太湖猪的ANGPTL4基因的序列，采用半定量RT－PCR技术检测ANGPTL4在不同品种组织中的表达差异。结果表明：1)猪ANGPTL4基因在肝脏、脂肪、心脏、小肠和大肠四种组织中都有表达，其中在脂肪组织的表达最丰富，其次是肝脏、大肠、心脏、小肠。2)太湖猪的脂肪和肝脏组织ANGPTL4基因相对表达量都极显著（p＜0.01）高于长白猪，回肠和空肠中ANGPTL4基因表达量极显著低于（p＜0.01）长白猪，盲肠、结肠、心脏中ANGPTL4基因表达量与长白猪无差异（p＞0.05）。试验五不同品种猪回肠微生物与ANGPTL4基因的关系选择0日龄健康正常的太湖猪和长白猪各24头进行150天的试验。每个品种设为一个处理，每个处理按体重一致原则设4个重复，公母各半，每个重复6头猪。在同一环境条件下饲予相同的饲粮，分别在0、1、2、3、4、5月龄，每种猪各屠宰4头，测定回肠中ANGPTL4基因表达量和拟杆菌属-普雷沃氏菌属细菌数量。结果表明：1)1-5月龄的太湖猪回肠拟杆菌属-普雷沃氏菌属数量均高于长白猪，特别是在2、3和5月龄，极显著（p＜0.01）高于长白猪。两品种猪的拟杆菌属-普雷沃氏菌属数量，都是随着年龄增加，呈现先增加，2月龄下降，然后再上升的规律。2)1-5月龄太湖猪回肠ANGPTL4基因表达量均低于长白猪，特别是在1、2和5月龄极显著（p＜0.01）低于长白猪，但在0日龄与长白猪的差异不显著（p＞0.05）；两品种猪ANGPTL4基因表达量与拟杆菌属-普雷沃氏菌属的变化成相反趋势，即先下降，后增加，再下降。3)猪回肠拟杆菌、G^-数量都与回肠ANGPTL4基因表达存在显著（p＜0.01）的负相关关系，而G⁺／G^-比例与回肠ANGPTL4基因表达存在极显著（p＜0.01）的正相关关系。试验六多形拟杆菌及代谢产物对猪回肠上皮细胞ANGPTL4表达的影响本试验以原代培养猪回肠上皮细胞为研究模型，研究了多形拟杆菌及其代谢产物对猪回肠上皮细胞ANGPTL4表达的影响。试验设7个处理，多形拟杆菌-1（108Cfu／ml），多形拟杆菌-2（10⁷Cfu／ml）、多形拟杆菌-3（10⁶Cfu／ml）、代谢产物-1（10⁸稀释度）、代谢产物-2（10⁷稀释度）、代谢产物-3（10⁶稀释度）及对照组。10⁸Cfu／ml多形拟杆菌和其相应代谢产物组都设20个重复，其它处理组设4个重复，每个重复1孔，在37℃培养96h后分别换为含多形拟杆菌10⁸、10⁷、0⁶Cfu／mlDMEM培养液和相应的代谢产物，分别于1、3、7、12和26小时收集10⁸Cfu／m多形拟杆菌和其相应代谢产物处理组的细胞，观察随浓度和作用细胞时间的变化，多形拟杆菌和其代谢产物影响ANGPTL4基因的表达情况。试验结果表明：1)多形拟杆菌与肠细胞数目为1:1时。即可抑制回肠ANGPTL4基因表达，这种抑制作用随多形拟杆菌的浓度增加和作用时间延长越明显。2)多形拟杆菌产生的代谢产物不影响ANGPTL4基因表达，但随着时间的延长，ANGPTL4基因表达有提高的趋势（p＞0.05）。综合上面六个试验，可得出如下结论：1)太湖猪回肠和盲肠内容物G^-及B．P的数量高于长白猪，而G⁺／G^-低于长白猪，总细菌数及G⁺与长白猪的差异不显著；随年龄的增加，总细菌数、G⁺、G^-的数量有增加的趋势，G⁺／G^-有下降的趋势，B．P的数量呈现上升-下降-再增加的趋势；2)太湖猪回肠ANGPTL4基因表达量低于长白猪，随年龄增加即先下降，后增加，再下降；3)多形拟杆菌可抑制回肠上皮细胞ANGPTL4基因表达量，而其代谢产物无明显作用；4)回肠和盲肠的拟杆菌属—普雷沃氏菌属及G^-与体脂有正相关关系，G⁺／G^-与体脂有负相关关系。更多还原

【Abstract】 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..更多还原