【作者】 郭秀兰；

【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， 2009， 博士

【摘要】 硬壁菌门(Firmicutes division,FD)和拟杆菌门(Bacteroidetes division,BD)是哺乳动物肠道中的主要细菌,它们的相对丰度与人和鼠机体脂肪含量相关。本研究在成功建立检测肠道拟杆菌门和硬壁菌门16S rRNA基因的实时定量PCR方法的基础上,研究了猪肠道硬壁菌门、拟杆菌门、拟杆菌属(Bacteroides spp.,BS)的丰度及硬壁菌门/拟杆菌门(FD/BD)和硬壁菌门/拟杆菌属(FD/BS)比例与脂肪沉积的关系。研究包括以下五个部分:试验一建立定量PCR检测肠道硬壁菌门和拟杆菌门16S rRNA基因的方法通过对猪肠道中200多个菌株的16S rRNA基因序列进行比对分析,找到硬壁菌门和拟杆菌门16S rRNA各自保守又特异的碱基位点,并针对这些位点进行引物的设计。再用8种代表细菌作常规和定量PCR以检验这些引物的特异性,并用标准品等梯度稀释以检验这两个实时荧光定量PCR体系的定量情况。结果表明这两对引物能够特异地检测目的细菌,并得到预期长度的产物,熔解曲线分析也表明为特异性扩增;标准品扩增的Ct值与目标细菌DNA的拷贝数间呈线性相关。这些表明这两个体系能够特异地对硬壁菌门和拟杆菌门菌群作定量的检测。试验二肥胖和瘦的版纳微型近交系猪肠道硬壁菌门、拟杆菌门及拟杆菌属的差异运用定量PCR技术对胖的和瘦的云南版纳微型近交系猪粪便中的硬壁菌门、拟杆菌门、拟杆菌属及总细菌16S rRNA基因的拷贝数进行检测,并计算各类细菌占总细菌的比例(相对丰度),以研究猪肠道微生物与脂肪沉积的相关关系。结果表明胖猪的拟杆菌门丰度比瘦猪低～68%(P＜0.001),而拟杆菌属丰度也低～65%(P＜0.001);拟杆菌属和拟杆菌门在胖猪和瘦猪中数量变化趋势一致,拟杆菌属在一定程度上可代表拟杆菌门,且都与体重呈显著的负相关关系(P＜0.01);胖猪的硬壁菌门丰度略高于瘦猪,但差异不显著,而FD/BD和FD/BS显著高于瘦猪(P＜0.05),且FD/BS与猪体重呈正的线性关系(P＜0.05)。试验三梅山和长白猪肠道硬壁菌门、拟杆菌门和拟杆菌属丰度与脂肪沉积的相关关系为在商用品种猪上进一步验证猪肠道微生物与脂肪沉积的相关关系,本试验选40kg左右的脂用型梅山猪和瘦肉型长白猪饲喂同一日粮。体重达90 kg时每品种选6头进行屠宰,测定背膘厚度和脂肪细胞直径,并取盲肠内容物以测定硬壁菌门、拟杆菌门、拟杆菌属的拷贝数和丰度。结果表明梅山猪的背膘厚度比长白猪高48%(P＜0.001),脂肪细胞直径和面积也显著大于长白猪;长白猪盲肠内容物中有更高的总细菌、硬壁菌门、拟杆菌门及属拷贝数(P＜0.05);梅山猪有低的拟杆菌门(P＜0.01)和拟杆菌属(P=0.013)丰度,硬壁菌门的丰度也略高于长白猪(P=0.290),但差异没有达到显著;且梅山猪肠道菌群中FD/BD(P=0.055)和FD/BS(P＜0.05)较长白猪高,FD/BD和FD/BS与背膘厚度呈正相关(P＜0.05);拟杆菌门和属的数量趋势也一致,并且梅山猪的拟杆菌属占拟杆菌门的比例低于长白猪(P=0.059),它们的丰度都与猪的背膘厚度呈负相关(P＜0.05)。这些结果证明了梅山猪的确是脂肪型,而长白猪为瘦肉型;并再次证实猪的体脂沉积与肠道拟杆菌门及属的相对丰度呈负相关,与FD/BD和FD/BS呈正相关。试验四脂肪型和瘦肉型猪在不同日龄肠道的硬壁菌门、拟杆菌门和拟杆菌属的变化为了研究猪脂肪沉积的动态变化对肠道菌群的影响,考察了3、4、5、7月龄梅山(脂肪)和DLY(瘦型)猪粪便中的总细菌、硬壁菌门、拟杆菌门和拟杆菌属数量情况。结果表明,梅山猪硬壁菌门丰度在各阶段都略比DLY猪高,其中五月龄时达到显著水平;随着月龄增加两种猪拟杆菌属的数量逐渐下降(P＜0.05);随月龄的增加,猪硬壁菌门丰度都逐渐增加(梅山P＜0.05,DLY P＜0.01),拟杆菌门丰度也有逐渐下降的趋势(梅山P=0.104,DLY P＜0.05),拟杆菌属的丰度在两个品种中都显著或极限著地下降,FD/BD(梅山P=0.101,DLY P＜0.01)和FD/BS(P＜0.01)也逐渐增加。这表明随月龄增加肠道菌群的变化是与脂肪沉积增加相适应的。而拟杆菌属丰度随脂肪沉积增加而下降幅度高于拟杆菌门,这说明拟杆菌属是一个很好的脂肪沉积生物标识。试验五日粮碳水化合物和蛋白质比例对猪脂肪沉积、肠道微生物及相关基因表达的影响为了进一步研究动态的脂肪沉积与肠道微生物的关系,采用不同碳水化合物/蛋白质(CHO/CP)日粮饲喂相同日龄的脂肪型和瘦肉型猪,考察皮下脂肪、肌内脂肪、肠道菌群及脂肪沉积相关基因表达的情况。选5月龄梅山猪和DLY猪各12头,将其分别分成两组(处理),一组饲喂高碳水化合物/蛋白质(简称高CHO,CP:10.1-11.2%)日粮,一组饲喂低碳水化合物/蛋白质(简称低CHO,CP:22.7%)日粮,试验期为3个月。试验开始、两个月时测定活体背膘和粪便菌群,试验结束后每组选5头试猪屠宰,测定背膘厚度、板油重、肌内脂肪含量、盲肠内容物的菌群、回肠ANGPTL4(angiopoietin-like protein 4)、脂肪和肌肉中PPARγ(peroxisome proliferator-activatedreceptorsγ)和H-FABP(heart-type fatty acid binding proteins)基因的表达情况。结果表明,高CHO增加了脂肪沉积(P＜0.05),其中DLY猪肌内脂肪含量增加尤为显著(P＜0.01),并提高了DLY猪肌肉组织中的PPARγ(P=0.093)和H-FABP(P=0.095)表达。在试验开始时,梅山猪的硬壁菌门比例显著高于DLY猪(P＜0.05),并有较高FD/BD(P=0.127)和FD/BS(P=0.129)。两个月时,梅山猪的硬壁菌门比例仍有较高的趋势(P=0.106),FD/BS显著高于DLY猪(P＜0.05):高CHO日粮提高了硬壁菌门比例(P=0.076)和FD/BS(P＜0.01),降低了拟杆菌属在总细菌(P=0.094)和拟杆菌门(P＜0.05)中的比例。而三个月后各细菌占总细菌的比例差异均不显著。这表明改变日粮碳水化合物/蛋白质影响了猪的脂肪沉积及相关基因的表达和肠道微生物的组成,且肠道微生物的变化与脂肪沉积相适应。综上所述,本研究结果表明:1)建立的检测猪肠道拟杆菌门和硬壁菌门16S rRNA基因的定量PCR方法能特异地对目的细菌作定量检测;2)猪肠道拟杆菌门和硬壁菌门的丰度与脂肪沉积存在相关关系,综合表现为脂肪沉积高的硬壁菌门/拟杆菌门较高;3)拟杆菌属可代表拟杆菌门也与猪脂肪沉积呈负相关;4)随着猪日龄增加,拟杆菌属的数量和丰度逐渐下降,硬壁菌门丰度、硬壁菌门/拟杆菌门和硬壁菌门/拟杆菌属比值逐渐增加;5)提高日粮碳水化合物水平增加了猪的脂肪沉积和肠道硬壁菌门/拟杆菌属的比值。更多还原

【Abstract】 The gut microbiota of mammals is mainly consisted of the Firmicutes and Bacteroidetes divisions,and their relative abundance is closely related to the body fat in humans and mice.The aim of this study was to investigate the correlation between the body fat storage and the relative abundance of Firmicutes,Bacteroidetes,Bacteroides,and the ratios of Firmicutes/Bacteroidetes(FD/BD) and Firmicutes/Bacteroides(FD/BS) of the gut microbiota in pigs.This study consists of five parts of experiments as follows:Exp.1 Development of a Real-Time PCR Method for detecting 16S rRNA gene of Firmicutes and Bacteroidetes in the gut microbiotaBased on the multialignment analysis of 200 16S rRNA gene sequences,two pairs of 16S rRNA gene-targeted group-specific primers were designed for the gut Bacteroidetes and Firmicutes divisions.The specificity of group-specific primers was confirmed by amplifying genomic DNAs from 8 representative bacterial strains with each of the two pairs of primers by conventional PCR and real-time PCR.The equal-fold dilutions of standards were used to test the two real-time PCR assays.The primer pairs were specific for their target strains and yielded the PCR products of the expected size only from their targets,and melting curves were analyzed to confirm the specific amplification.There was a linear relationship between the log of the standard DNA copy number and the calculated Ct value across the specified concentration range.So the real-time PCR assays developed for Firmicutes and Bacteroidetes could use to investigate the composition of gut microbiota.Exp.2 Differences of Firmicutes,Bacteroidetes and Bacteroides in the gut microbiota of obese and lean pigs from Banna mini-pig inbred lineTo investigate the relation between body fat and gut microbiota in pigs,the specific primers for Firmicutes and Bacteroidetes were used together with the previously developed group-specific primers to detect the copy numbers of the 16S rRNA gene of all Bacteria,Bacteroidetes division,Firmicutes division and Bacteroides spp.in the feces of obese and lean pigs from Banna mini-pig inbred line(BMI) by real-time PCR analysis,and their percentages based on all Bacteria(relative abundance) were calculated.The obese pigs had a～68%fewer percentage of Bacteroidetes division(P＜0.001) and a～65%fewer proportion of Bacteroides spp.(P＜0.001) than the lean pigs.The trends of quantitative changes of both Bacteroidetes and Bacteroides were similar in obese and lean pigs,and their percentages had a negative correlation with the body weight(P＜0.01).The percentage Firmicutes was not different significantly,although it was higher in numerical value in obese pigs than in lean pigs.The ratios of FD/BD and FD/BS in obese pigs were higher than in lean pigs(P＜0.05),and the ratio of FD/BS had a positive correlation with the body weight(P＜0.05).Exp.3 Correlation between the relative abundance of Firmicutes, Bacteroidetes and Bacteroides in the gut microbiota and fat deposit in Meishan and Landrance pigsTo determine whether body fat is relative to the gut microbial communities in the common breeds,the～40 kg Landrace and Meishan pigs were chosen and had ad libitum access to the same diet in this study.When body weight reached to 90 kg,6 pigs from each breed were selected randomly and subjected to exsanguinations to measure backfat depth, diameter and area of fat cells,copy numbers and abundance of Firmicutes,Bacteroidetes, Bacteroides in cecal content.Results indicated that:Meishan pigs had higher backfat depth, diameter and area of fat cells comparing with the Landrace pigs.Landrace pigs had higher copy numbers of all Bacteria,Firmicutes,Bacteroidetes,Bacteroides in their cecal contents than Meishan pigs(P＜0.05).Lower percentages of Bacteroidetes(P＜0.01) and Bacteroides spp.(P=0.013) were in Meishan pigs than in Landrance pigs,and Firmicutes percentage of Meishan was higher than Landrance(P=0.290),although the difference was not significant.The ratios of FD/BD(P=0.055) and FD/BS(P＜0.05) in Meishan pigs were higher than in Landrance pigs,and these ratios had a positive correlation with the backfat depth(P＜0.05).The trends of quantitative changes of both Bacteroidetes and Bacteroides were similar,their percentages had a negative correlation with the backfat depth(P＜0.05), and the ratio of Bacteroides/Bacteroidetes in Meishan was lower than in Landrance (P=0.089).These results confirmed that Meishan pigs are typical genetically obese and Landrace pigs are lean,the percentages of Bacteroidetes and Bacteroides had a negative correlation with the body fat,and the ratio of FD/BS had a positive correlation with the body fat.Exp.4 Changes of Firmicutes,Bacteroidetes and Bacteroides in the gut microbiota of obese and lean pigs in different phasesTo investigate the effect of dynamic changes of body fat on the gut microbial communities in pigs,we detected the populations of all Bacteria,the Bacteroidetes,the Firmicutes and the Bacteroides spp.in the feces of Meishan(obese) and DLY(lean) pigs at 3,4,5,7 months old.The results indicated that:the abundance of Firmicutes in Meishan was higher than in DLY at different phases,and the difference reached a significant level at 5 months old.With the age increasing,the copy numbers of Bacteroides were reduced (P＜0.05).With the age increasing,the abundance of Firmicutes in the both breeds raised (Meishan P＜0.05,DLY P＜0.01),the proportion of Bacteroidetes trended to diminish (Meishan P=0.104,DLY P＜0.05),the Bacteroides percentage was also reduced(Meishan P＜0.05,DLY P＜0.01),and FD/BD(Meishan P=0.101,DLY P＜0.01) and FD/BS(P＜0.01) correspondingly increased.These data suggested that the changes in gut microbiota were fit in with the dynamic changes of body fat;the decreased amplitude of the Bacteroides percentage was bigger than the Bacteroidetes percentage accompanied with increase of body fat,so the Bacteroides spp.may be a good biomarker correlative closely with body fat of host.Exp.5 Ratio of carbohydrate and protein in diets on fat deposit,gut microbiota and expression of gene related to fat storage in pigsTo further investigate the relation between body fat and the gut microbiota in pigs with dynamic changes of body fat,the diets with different carbohydrate/protein(CHO/CP) were used to feed obese and lean pigs with the same ages,and detected the content of subcutaneous fat and intramuscular fat,gut microbial communities and gene expression relative to fat deposit.Twelve Meishan(obese) and twelve DLY(lean) pigs at 5 months old were assigned randomly to high carbohydrate/protein(HCHO,CP:10.1-11.2%) or low carbohydrate/protein(LCHO CP:22.7%) diet for 3 months.The backfat depth in vivo and fecal microbial communities were detected at the start and 2 months of experiment.When the experiment was end,5 pigs from each treatment were selected randomly and subjected to exsanguinations to measure backfat depth,abdominal fat weight,intramuscular fat content,the microbial communities in cecal content,ANGPTL4(Angiopoietin-like protein 4) gene expression in ileum,PPARγ(peroxisome proliferator-activated receptorsγ) and H-FABP(heart-type fatty acid binding proteins) gene expression in fat and muscle tissues. The results indicated that:HCHO diet made the fat deposit increased(P＜0.05),and especially increased the intramuscular fat content(P＜0.01).The gene expression of PPARγ(P=0.093) and H-FABP(P=0.095) was higher in HCHO than in LCHO treatment in DLY pigs.Meishan pigs had more Firmicutes proportion(P＜0.05) and higher ratios of FD/BD (P=0.127) and FD/BS(P=0.129) comparing with DLY pigs at the start,and Firmicutes proportion(P=0.106) and FD/BS(P＜0.05) in Meishan were still higher after the 2 months. Feeding HCHO diet for 2 months increased the abundance of Firmicutes(P=0.076) and the ratio of FD/BS(P＜0.01),and reduced the percentages of Bacteroides based on all Bacteria (P=0.094) and Bacteroidetes(P＜0.05) in the feces.But there was no difference in the abundance of bacterial groups between HCHO and LCHO treatments after the 3 months.It is concluded that the diets with different carbohydrate/protein had a significantly effect on fat storage,relative gene expression and gut microbial communities,and the changes in gut microbiota were consistent with the dynamic changes of body fat.In summary,data reported here demonstrated that:1) The Real-Time PCR assays for detecting the 16S rRNA gene of Firmicutes and Bacteroidetes in the gut microbiota were specific for their target strains;2) It is similar to in mice and humans that the fat storage is relative to the abundance of Firmicutes and Bacteroidetes of the gut microbiota in pigs.In general,there is a higher ratio of Firmicutes/Bacteroidetes in obese individual than in lean individual;3) The Bacteroides spp.may represent the Bacteroidetes division,and had a negative correlation with the body fat in pigs; 4) With the age increasing,the copy numbers and abundance of Bacteroides were reduced, the abundance of Firmicutes and the ratios of Firmicutes/Bacteroidetes and Firmicutes/Bacteroides increased;5) Feeding diet with higher carbohydrate increased the deposit of body fat and the ratio of Firmicutes/Bacteroides of the gut microbiota in pigs.更多还原