SLA-DQA基因在F18大肠杆菌抗性和敏感性断奶仔猪间的差异表达分析

运用荧光定量PCR方法分析SLA-DQA基因在苏太猪F18大肠杆菌病抗性和敏感性资源群体中各个组织的分布和表达水平,以及在抗性组和敏感组中的表达差异,以探讨SLA-DQA基因在断奶仔猪抗大肠杆菌F18菌株感染中发挥的作用。试验结果显示,SLA-DQA基因在所检测的11个组织中均有表达,并呈现相似的表达规律,在肺、脾和淋巴结中的表达量较高,在空肠、十二指肠和胸腺中也有中度的表达。SLA-DQA基因在抗性组个体各个组织中的表达量普遍高于敏感性个体,而且在肺、脾、淋巴结、空肠和十二指肠5个组织中,SLA-DQA基因在抗性组个体中的表达量显著高于敏感性个体(P<0.05)。由此可见,当断奶仔猪受到大肠杆菌毒素侵扰后,SLA-DQA基因较高的表达量有利于SLA-II类抗原分子的合成,SLA-DQA基因虽然不是针对由大肠杆菌F18菌株造成的断奶仔猪腹泻和水肿病的直接免疫因子,但是在断奶仔猪受大肠杆菌F18菌株病原菌侵袭后引起的一系列生理变化和应答过程中发挥着重要的作用。     

LTβR基因在大肠杆菌F18抗性型和敏感型苏太仔猪间的差异表达分析

旨在探讨LTβR基因在苏太断奶仔猪各组织间的mRNA表达谱,并比较分析该基因在大肠杆菌F18抗性/敏感型群体间的表达差异,为进一步探讨该基因的功能及筛选断奶仔猪大肠杆菌F18抗性遗传标记提供一定的指导和依据。试验分别挑选35日龄大肠杆菌F18抗性/敏感型苏太断奶仔猪各4头,利用实时荧光定量PCR方法比较分析LTβR基因在11个组织(心、肝、脾、肺、肾、胃、肌肉、胸腺、淋巴结、十二指肠、空肠)间的表达差异。结果:LTβR基因在检测的11个组织中均有表达,其中在肝、肺、肾、胃、十二指肠和空肠呈现高水平表达,在脾和淋巴结中呈现中等水平表达,在心脏、肌肉和胸腺组织中呈现低水平表达。除肝脏外,LTβR基因在抗性型个体中表达量普遍高于敏感型个体,而且在肠系膜淋巴结中的表达量极显著高于敏感型个体(P0.01)。由此推测,LTβR基因的表达上调有利于断奶仔猪抵御大肠杆菌F18的感染,且可能是通过在淋巴结组织中的高表达维持和提高了肠道的免疫水平,从而提高了断奶仔猪抵御大肠杆菌F18感染的能力。     

TNF-α基因在F18大肠杆菌抗性型和敏感型断奶仔猪间的差异表达分析

本研究运用Real-time PCR方法分析TNF-α基因在苏太猪F18大肠杆菌抗性型和敏感型断奶仔猪群体中11个组织中的表达谱,以及在抗性型和敏感型个体之间的差异表达,以探讨TNF-α基因在断奶仔猪抗F18大肠杆菌感染中的作用。结果表明:TNF-α基因在抗性型和敏感型个体所检测的11个组织中均有表达,其中在脾脏、肺、肾脏、胸腺和淋巴结组织中表达量较高,在十二指肠和空肠组织中呈中度表达;TNF-α基因在F18大肠杆菌抗性型个体各个组织中的表达量普遍高于敏感型个体,且脾脏、肾脏、胸腺和十二指肠组织中TNF-α基因在抗性型个体中的表达量显著高于敏感型个体(P0.05)。由此推测,TNF-α基因的较高表达可能有利于提升仔猪对F18大肠杆菌的抵抗能力,在F18大肠杆菌侵袭后引起的一系列免疫应答过程中发挥着重要的作用。     

F18大肠杆菌抗性型与敏感型苏太断奶仔猪十二指肠组织比较转录组分析

旨在揭示培育品种—苏太猪(杜洛克×梅山猪)F18大肠杆菌抗性的调控通路和重要候选基因,同时进一步探究中外猪品种F18大肠杆菌抗性调控遗传基础的差异。本研究以课题组前期获得的苏太猪和梅山猪F18大肠杆菌抗性型与敏感型断奶仔猪全同胞个体为研究对象,通过转录组测序筛选苏太猪F18大肠杆菌抗性相关的调控通路以及重要候选基因,并在细胞水平,利用qPCR和Western blot分析F18大肠杆菌刺激小肠上皮细胞IPEC-J2后重要候选基因(蛋白)的表达变化,同时利用qPCR检测重要候选基因在苏太和梅山断奶仔猪F18大肠杆菌抗性型与敏感型个体十二指肠组织中的差异表达情况。结果显示:1)在苏太断奶仔猪F18大肠杆菌抗性型与敏感型个体中筛选出238个差异表达基因,主要参与Toll样受体信号通路(toll-like receptor signaling pathway)和糖脂类通路(glycosphingolipid biosynthesis-lacto and neolacto series),其中TLR5、IL-1β、FUT2为重要候选基因;2)不同血清型F18大肠杆菌(F18ac、F18ab)菌体分别刺激小肠上皮细胞IPEC-J2后,FUT2、IL-1β、TLR5基因mRNA表达水平显著上调(P0.05),并且其蛋白表达水平也表现为明显的上调,由此表明,TLR5、IL-1β和FUT2在断奶仔猪F18大肠杆菌感染过程中发挥重要的调控作用;3)组织差异表达分析显示,TLR5、IL-1β和FUT2在苏太猪抗性型与敏感型个体十二指肠组织中表达差异均达到显著水平(P0.05),而梅山猪中TLR5、IL-1β表达差异达到极显著水平(P0.01),FUT2表达水平差异不显著(P0.05)。结合课题组前期关于梅山猪及外来引进品种F18大肠杆菌抗性相关分子机制研究及报道,本研究进一步证明中外猪品种F18大肠杆菌抗性调控的遗传基础确实存在差异,Toll样受体信号通路及CD14、TLR5等基因可能在中国地方品种—梅山猪抵抗F18大肠杆菌感染过程中发挥着免疫调控作用,而鞘糖脂合成通路及FUT2等基因可能在外来猪品种F18大肠杆菌受体形成过程中起关键作用。     

MUC4和MUC13基因在ETECF18抗性型和易感型梅山断奶仔猪间的差异表达分析

MUC4和MUC13基因作为产肠毒素大肠杆菌(enterotoxigenic Escherichia coil,ETEC)F4的重要抗性候选基因,可能在断奶仔猪抗ETEC F18菌株的感染过程中发挥重要作用,因此本试验运用实时荧光定量PCR法分析了MUC4和MUC13基因在梅山猪ETEC F18抗性型和易感型个体11个组织(心脏、肝脏、脾脏、肺脏、肾脏、胃、肌肉、胸腺、淋巴结、十二指肠和空肠)中的表达水平及差异。结果显示,MUC4和MUC13基因在检测的11个组织中均有不同程度的表达。其中,在胸腺和淋巴组织中,MUC4基因在抗性型个体中的表达水平显著高于易感型个体(P0.05);在肺脏组织中,MUC13基因在抗性型个体中的表达水平显著高于易感型个体(P0.05),且MUC13基因在抗性群体肠道中的表达水平较高。由此推测,MUC4基因在免疫组织、MUC13基因在肠道组织的高表达可在一定程度上提高了机体的免疫能力,保护和润滑肠道,从而抵抗ETEC F18菌株对机体的感染。     

猪TLR4基因在F18大肠杆菌抗性型和敏感型资源群体间的差异表达分析

本研究旨在检测TLR4基因mRNA在猪各个组织的分布以及在F18大肠杆菌抗性型和敏感型群体间差异表达水平,为探讨该基因在免疫识别和F18大肠杆菌抗性中发挥的作用提供理论依据。本试验运用SYBR GreenⅠ实时荧光定量PCR技术进行定量测定。首先,各取8头35日龄F18大肠杆菌抗性型和敏感型苏太仔猪组织样,包括心脏、肝脏、脾脏等11个组织,提取总RNA,以GAPDH基因为内参基因,进行实时荧光定量PCR。对TLR4基因mR-NA进行均一化处理,利用荧光阈值(Ct值)计算TLR4基因mRNA在各个组织以及F18大肠杆菌抗性型和敏感型群体间表达量。结果显示,TLR4 mRNA在猪各种组织中广泛表达,在肺、淋巴结、肾脏和脾脏等免疫器官中表达量较高,F18大肠杆菌敏感型的TLR4基因表达量普遍高于抗性型个体的表达量,在各个组织中(除胃外)TLR4表达量差异倍数从1.083到2.980不等;在肺、淋巴结、肾脏和胸腺组织中,F18大肠杆菌敏感型的TLR4基因表达量显著高于抗性型个体的表达量(P<0.05)。本研究结果表明,TLR4基因通过天然免疫识别机制对猪F18大肠杆菌病等革兰氏阴性疾病有一定的影响,TLR4基因表达量...     

HSP27基因在苏太猪F18大肠杆菌抗性型和敏感型个体间的差异表达分析

热休克蛋白27(heat shock protein 27,HSP27)是哺乳动物普遍存在的小休克蛋白家族成员之一,在参与调解细胞的增殖、分化和细胞凋亡过程中起到了重要的作用。为探讨猪HSP27基因的表达水平及其与F18大肠杆菌抗性的关系,本研究运用荧光定量PCR方法检测HSP27基因在苏太猪F18大肠杆菌病抗性型和敏感型资源群体中各组织的表达水平,并分析在E.coli F18抗性组和敏感组中的表达差异。结果表明:HSP27基因在所有检测个体的11个组织中均有表达,其中肺中表达量最高,其次在肝、脾、肾、十二指肠和空肠中表达较高,而在肌肉、胸腺和淋巴结中表达较低;抗性组和敏感组差异表达显示,在肝脏和淋巴结2个组织中,HSP27基因在抗性组个体中的表达量极显著高于敏感性个体的表达量(P0.01),在脾脏、胸腺和空肠3个组织中,HSP27基因在抗性组个体中的表达量显著高于敏感性个体的表达量(P0.05)。HSP27基因在E.coli F18抗性组免疫组织和肠道组织中的高水平表达提示HSP27基因可能在机体抵抗F18大肠杆菌感染过程中发挥了重要的免疫调控作用。     

不同日龄苏太仔猪LTβR基因组织表达谱及发育性表达规律分析

试验旨在探讨LTβR基因在仔猪出生至断奶期间的mRNA表达变化,为进一步研究该基因与F18大肠杆菌致病的相关性提供理论依据。本试验选取从初生到断奶的4个日龄(8、18、30和35日龄)苏太仔猪各4头,利用实时荧光定量PCR方法分别比较分析了LTβR基因在各个体11个组织(心脏、肝脏、脾脏、肺脏、肾脏、胃、肌肉、胸腺、淋巴结、十二指肠及空肠)间的表达规律。结果表明,LTβR基因在肝脏、脾脏、肺脏、肾脏、胃、淋巴结、十二指肠及空肠组织中呈现较高水平的表达,并且表现出明显的发育性表达差异。LTβR基因在8日龄仔猪淋巴、十二指肠和空肠组织中的表达极显著高于其他日龄(P<0.01);在8日龄仔猪肺脏组织中的表达极显著高于35日龄(P<0.01),且显著高于30日龄(P<0.05);在35日龄仔猪肝脏组织中的表达极显著高于其他日龄(P<0.01);在35日龄仔猪胃组织中的表达显著高于18日龄(P<0.05)。由此推测,8日龄左右为仔猪肠道免疫屏障快速形成期,LTβR基因的较高表达可能有利于仔猪对F18大肠杆菌抗性。     

BPI基因在梅山猪初生断奶以及成年阶段的组织表达谱分析

为探讨杀菌/通透性增强蛋白(BPI)基因在梅山猪从初生到成年各个时期不同组织中的表达规律,利用qPCR检测初生、断奶、性成熟、体成熟4个重要发育时期(即1、35、134、158日龄)梅山猪的心脏、肝脏、脾脏、肺脏、肾脏、胃、肌肉、胸腺、淋巴结、十二指肠、空肠和回肠12个组织中BPI基因的表达水平。结果表明:4个不同发育阶段BPI基因的组织表达谱在心脏、肝脏、脾脏、肌肉和胸腺中均表现出相对一致的规律,即BPI基因的表达量一直处于极低水平,而在肠道组织中从初生到成年各时期均高度表达;BPI基因在胃中的表达程度随着日龄增长而显著提高;在小肠组织中,35日龄断奶仔猪BPI基因的表达水平极显著高于其他3个日龄。综上,推断肠道中BPI基因的高度表达是仔猪从初生就具有的抵抗大肠杆菌等病原感染属固有免疫的一部分,而在胃中的表达很可能是其后天为了抵御不断侵染的大肠杆菌等病原的结果。     

不同日龄苏太仔猪FUT2基因的组织表达谱和表达差异分析

目前有研究表明FUT2基因是仔猪大肠杆菌F18受体形成的关键候选基因,探讨FUT2基因在仔猪出生至断奶期间的m RNA表达变化,可为进一步研究该基因对F18大肠杆菌致病的相关性提供一定的理论依据。本试验挑选4个日龄(8、18、30日龄和35日龄)苏太仔猪各4头,通过利用Real-time PCR方法分别比较分析了FUT2基因在各个体11个组织(心、肝、脾、肺、肾、胃、肌肉、胸腺、淋巴结、十二指肠、空肠)中的表达规律。结果表明:FUT2基因在不同日龄苏太猪11个组织中均有表达,其中在肺、肾、胃、胸腺、淋巴和十二指肠中呈现较高表达,在肝、脾、空肠中呈现中度表达,在心和肌肉中呈现低表达。FUT2基因在8日龄个体空肠组织上的表达水平显著的高于其他3个日龄(P0.05),而不同日龄仔猪十二指肠中的FUT2基因表达水平间没有显著差异(P0.05)。由此推测,8日龄左右FUT2基因的高表达可能有利于仔猪空肠组织中F18大肠杆菌受体的快速形成,需进一步对仔猪8日龄至18日龄间FUT2基因的表达变化做进一步细致的检测。     

不同猪种ESR基因多态性的PCR-RFLP分析

运用PCR-RFLP方法,检测了杜洛克、约克夏、长白猪、皮特兰、梅山猪、二花脸猪、枫泾猪、荣昌猪、苏太猪等9个国内外猪种的ESR基因PvuⅡ位点多态性。结果表明,在约克夏猪、二花脸猪、枫泾猪和苏太猪中检测到了AA、AB、BB 3种基因型,在杜洛克猪、皮特兰猪中检测到AB、BB 2种基因型,在长白猪、梅山猪、荣昌猪中只检测到了AB基因型。在本研究中不同品种的A、B基因频率差异显著,总体上在国外猪群体中A等位基因为优势等位基因,而地方猪种中B等位基因为优势等位基因。     

Tissue Expression Profile and Differential Expression of LTβR Gene in Sutai Piglets of Different Ages

Dynamic changes of LTβR expression levels in 11 tissues (heart, liver, spleen, lung, kidney, stomach, muscle, thymus, lymph node, duodenum and jejunum) of Sutai piglets ranging from newborn to post-weaning days 8, 18, 30, and 35 were compared and analyzed by the Real-time PCR method, which aimed to provide theoretical basis for further investigate the relationship between LTβR gene and pathogenicity of E.coli F18.The results revealed that the LTβR expression levels were higher in the liver, spleen, lung, kidney, stomach, lymph node, duodenum and jejunum, and showed obvious age-dependent expression differentiation.The LTβR expression levels in the lymph node, duodenum, and jejunum were extremely significant higher in 8 days old piglets than in the other age stages (P<0.01), and the expression levels were extremely significantly higher in the lungs of 8 days old piglets than in 35 days old piglets (P<0.01) and significantly higher than 30 days old piglets (P<0.05).In the liver tissue, the expression level was extremely significant higher in 35 days old piglets than in other age stages (P<0.01).In the stomach tissue, the expression level was significantly higher in 35 days old piglets than in 18 days old piglets (P<0.05).The results speculated that intestinal immune barrier of piglets formed rapidly around 8 days old and the higher LTβR expression could contribute to the resistance to E.coli F18.     

Differential Expression Analysis of MUC4 and MUC13 Genes Between Resistant and Sensitive Weaned Meishan Piglets to ETEC F18

MUC4 and MUC13 genes as important candidate genes for enterotoxigenic Escherichia coil (ETEC) F4 resistance,may play an important role in the process of against ETEC F18 infection in weaned piglets. In this study,ETEC F18-resistant and -sensitive weaned Meishan piglets were used,and the expression levels of MUC4 and MUC13 genes in 11 tissues (heart,liver,spleen,lung,kidney,stomach,muscle,thymus,lymph nodes,duodenum and jejunum) were determined by quantitative Real-time PCR. The results showed that MUC4 and MUC13 genes were broadly expressed with different expression levels in all the 11 tissues. In the thymus and lymph tissues,the expression of MUC4 gene in resistant piglets was significantly higher than that in sensitive piglets (P<0.05);In the lung tissue,theMUC13 gene expression level in resistant individuals was significantly higher than that in sensitive individuals (P<0.05),and in the intestinal tissues of duodenum and jejunum, the expression level of MUC13 gene was relatively higher in resistant individuals. Thus we speculated that the high expression of MUC4 gene in immune tissues and MUC13 gene in intestinal tissues might improve the immune ability of piglets,protect and lubricate the intestinal tract, and resist ETEC F18 infection.     

Investigation of the relationship between SLA-1 and SLA-3 gene expression and susceptibility to Escherichia coli F18 in post-weaning pigs

Porcine post-weaning diarrhea and edema disease are principally caused by Escherichia coli strains that produce F18 adhesin. FUT1 genotyping and receptor binding studies divided piglets into E. coli F18-resistant and -sensitive groups, and the roles of SLA-1 and SLA-3 were investigated. SLA-1 and SLA-3 expression was detected in 11 pig tissues, with higher levels of SLA-1 in lung, immune tissues and gastrointestinal tract, and higher levels of SLA-3 also in lung and lymphoid tissues. Both genes were expressed higher in F18-resistant piglets, and their expression was positively correlated in different tissues; a negative correlation was observed in some tissues of F18-sensitive group, particularly in lung and lymphatic samples. Gene ontology and pathway analyses showed that SLA-1 and SLA-3 were involved in 37 biological processes, including nine pathways related to immune functions. These observations help to elucidate the relationship between SLA class I genes and E. coli F18-related porcine gastrointestinal tract diseases.     

Genetic variation at the alpha‐1‐fucosyltransferase (FUT1) gene in Asian wild boar and Chinese and Western commercial pig breeds

Escherichia coli F18 bacteria producing enterotoxins and/or shigatoxin (ETEC/STEC) are main pathogens that cause oedema disease and postweaning diarrhoea in piglets, and alpha‐1‐fucosyltransferase (FUT1) gene has been identified as a candidate gene for controlling the expression of ETEC F18 receptor. The genetic variations at nucleotide position 307 in open reading frame of FUT1 gene in one wild boar breed and 20 western commercial and Chinese native pig breeds were investigated by polymerase chain reaction–restriction fragment length polymorphism. The results showed that the genetic polymorphisms of the FUT1 locus were only detected in western pig breeds and the Chinese Taihu (including Meishan pig, Fengjing pig and Erhualian pig), Huai and Lingao pig breeds; only Duroc and Pietrain possessed the resistant AA genotype, while the wild boar and other Chinese pig breeds only presented the susceptible genotype GG. The results indicated that Chinese native pig breeds lack genetic factors providing resistance to ETEC F18 bacteria. The resistant allele to ETEC F18 might originate from European wild boar. It was inferred that oedema and postweaning diarrhoea caused by ETEC F18 have close relationship with the growth rate, which can explain why on the contrary Chinese native pig breeds have stronger resistance to oedema and postweaning diarrhoea in piglets compared with western pig breeds.     

IL1RN基因在晋汾白猪仔猪和新山西黑猪仔猪不同组织和细胞中的表达特征分析

白细胞介素1受体拮抗剂（interleukin 1 receptor antagonist,IL1RN）是一种重要的宿主免疫调节因子,是天然存在的IL-1拮抗剂。为了解IL1RN基因在断奶仔猪不同组织和细胞中表达的特征,利用Trizol法提取晋汾白猪仔猪和新山西黑猪仔猪的心脏、肝脏、脾脏、淋巴细胞、巨噬细胞等18种组织和细胞的总RNA,采用RT-PCR方法检测IL1RN基因在不同组织和细胞中的表达情况,应用SPSS 19.0软件分析IL1RN基因在晋汾白猪仔猪和新山西黑猪仔猪4种免疫组织和细胞（脾脏、淋巴结、巨噬细胞、白细胞）中的表达差异情况。结果显示：IL1RN基因在晋汾白猪仔猪和新山西黑猪仔猪的脂肪组织中均不表达,在其余各组织和细胞中均表达;IL1RN基因在晋汾白猪仔猪的巨噬细胞与淋巴结、脾脏、白细胞之间的表达差异极显著（P<0.01）,且在淋巴结与脾脏、白细胞之间的表达差异极显著（P<0.01）;IL1RN基因在新山西黑猪仔猪的脾脏、淋巴结、巨噬细胞、白细胞的表达两两之间均呈极显著差异（P<0.01）;IL1RN基因在2个猪种的脾脏、巨噬细胞、白细胞之间的表达呈极显著差异（P<0.01）,在淋巴结中的表达呈显著差异（P<0.05）。该研究结果为进一步揭示2个猪种的IL1RN基因在疾病抵抗方面的作用及相关机制提供了依据。     

ITGB5和MUC13基因在产肠毒素大肠杆菌抗性和易感大白仔猪间的差异表达分析

为分析仔猪腹泻抗性候选基因在大白仔猪抗性和易感个体间的差异表达情况及组织表达特异性,实验利用荧光定量PCR技术检测整合素β5(ITGB5)基因和黏蛋白13(MUC13)基因在仔猪小肠、脾脏、肺脏、胸腺、肝脏和淋巴6种组织中的表达水平以及在产肠毒素大肠杆菌(ETEC F4)抗性和易感仔猪个体间的差异表达情况。结果表明:大白仔猪ITGB5基因在6种组织中均有一定的表达,在抗性个体小肠组织中的表达量低于易感个体(P>0.05);MUC13基因在小肠中高度表达,在其他组织中表达量较低,且抗性个体的表达量显著高于易感个体(P<0.05)。由此可知,小肠组织中ITGB5基因的低表达和MUC13基因的高表达可能有助于降低致病性大肠杆菌黏附到小肠上皮细胞上,进而实现对致病性大肠杆菌的抗性。ITGB5基因和MUC13基因可能与仔猪腹泻抗性存在密切关系,都可以作为抗性候选基因用于标记辅助选择,应用于抗腹泻仔猪的选育。     

Diversity and community pattern of sulfate-reducing bacteria in piglet gut

Background: Among the gut microbiota, sulfate-reducing bacteria(SRB) is a kind of hydrogen-utilizing functional bacteria that plays an important role in intestinal hydrogen and sulfur metabolism. However, information is lacking regarding diversity and community structure of SRB in the gut of piglets. Middle cecum contents were collected from 6 Yorkshire and 6 Meishan piglets at postnatal days(PND) 14, 28 and 49. Piglets were weaned at PND28. Real-time quantitative PCR was performed to detect the number of SRB in the cecum based on dissimilatory sulfite reductase subunit A(dsrA) gene. Prior to real-time PCR, plasmid containing the dsrA gene was constructed and used as external standard to create a standard curve, from which the gene copies of dsrA were calculated. H_2S concentration in the cecal contents was measured. Illumina PE250 sequencing of dsrA gene was used to investigate SRB diversity in cecum contents.Results: The qPCR results showed that the number of SRB at PND49 was significantly higher than that at PND28 in Meishan piglets. The concentration of H_2S has no significant difference between piglet breeds and between different ages. The Illumina sequencing analysis revealed that the Chao1 richness index was significantly higher at PND49 than that at PND14 and PND28 in Yorkshire piglets. Based on dsrA gene similarities, Proteobacteria, Actinobacteria, and Firmicutes were identified at the phylum level, and most sequences were classified as Proteobacteria. At the genus level, most of sequences were classified as Desulfovibrio. At the species level, Desulfovibrio intestinalis was the predominant SRB in the piglet cecum. The relative abundance and the inferred absolute abundance of Faecalibacterium prausnitzii at PND49 were significantly higher than that at PND14 in Yorkshire piglets. Pig breeds did not affect the dsrA gene copies of SRB, diversity index and community pattern of SRB.Conclusions: Sulfate-reducing bacteria are widely colonized in the cecum of piglets and D. intestinalis is the dominant SRB. The age of piglets, but not the pig breeds affects the diversity and community pattern of SRB.