【作者】 叶兰；

【导师】 王金玉； 吴圣龙；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2013， 博士

【摘要】 断奶仔猪腹泻和水肿病是规模猪场危害最为严重的疾病之一,产肠毒素大肠杆菌(Enterotoxigenic Escherichia coli, ETEC)是造成断奶仔猪腹泻的主要病原菌,产Vero细胞毒素大肠杆菌(Verotoxigenic Escherichia coli, VTEC)则与仔猪水肿病密切相关。国外学者前期研究表明,α-(1,2)岩藻糖转移酶基因1(FUT1)与E.coliF18受体基因相连锁,可以作为控制E.coliF18粘附的候选基因。FUT1基因M307处存在G/A突变位点,并且G对A为显性,即AA基因型猪对E.coliF18表现为抗性,GG型和AG型表现为敏感性,并据此在国外猪种中实现了抗病育种。然而,在国内地方猪种中均未检测到FUT1基因M307AA和AG基因型个体,全部为GG基因型,呈极端偏态分布,由此可见,中外猪种在抗E.coliF18感染过程中具有不同的遗传机理。课题组在前期研究中利用在苏太猪群体中检测到的少量FUT1基因AG型个体进行适当选种选配,通过多年选育,已在苏太猪中建立了大肠杆菌病抗性(AA型)和敏感性(AG和GG型)资源家系群体。此外,课题组还构建了基于表达E.coli F18黏附素的V型分泌系统,并结合受体黏附试验技术,对资源家系群体E.coli F18抗性／易感性进行进一步的分析和验证。本研究基于建立的苏太猪E.coli F18抵抗性(AA型)和敏感性(AG和GG型)资源家系,利用基因表达谱芯片、蛋白质组学等相关技术对猪E.coli F18抗性候选基因进行了分析和筛选,以期揭示中国地方猪种抗E.coli F18的遗传基础,解决国内地方猪种E.coli F18抗性育种的关键科学问题。主要研究结果如下：1.运用表达谱芯片筛选大肠杆菌F18敏感型和抗性型两对比组间差异基因。(1)以Fold change绝对值大于2倍作为差异标准,在敏感型(GG基因型)对抗性型(AA基因型)配对组中,差异基因共13个,其中上调6个,下调7个；在另外敏感型(AG基因型)对抗性型(AA基因型)配对组中,共筛选出差异基因6个,其中上调4个,下调2个。(2)经GO分析可知,上述差异表达的基因涉及免疫应答、胞外区修饰(如糖基化)、细胞黏附、信号转导、转录调控、代谢等多个方面。通过KEGG数据库查询敏感型对抗性型差异基因所在通路以及文献挖掘,确定了与受体形成相关的鞘糖脂生物合成—球系列通路和炎症免疫通路对F18大肠杆菌病抗性具有调控作用,并将其作为下一步研究的重点。。(3)差异基因SLA-1、SLA-3、ST3GAL1、A基因,以及FUT1、TAP1和SLA-DQA基因通过荧光定量验证后与表达谱芯片结果一致,说明芯片结果可靠。2,对芯片结果炎症免疫通路中两个差异基因SLA-1与SLA-3在E.coli F18抗性组和敏感性组间进行表达比较及分析。(1)运用3个内参基因对SLA-1与SLA-3基因表达水平进行均一化。组织表达谱结果表明,SLA-1与SLA-3基因在11个组织中均表达；SLA-1在肺、免疫器官如脾、胸腺、淋巴结以及消化器官胃、十二指肠、空肠中的表达量相对较高；SLA-3基因在肺和淋巴结中的表达量较高,在心、肝、脾、肺、胃、肌肉、胸腺、十二指肠和空肠中的表达量较低。(2)SLA-1与SLA-3基因在E.coli F18抗性组大部分组织,如脾、肺、胃、胸腺、淋巴结、十二指肠和空肠中的表达量相对较高,但是与敏感性组之间的表达差异不显著。(3)线性相关关系分析显示,SLA-1与SLA-3基因在E.coliF18抗性组脾、肺、胃、胸腺、淋巴结、十二指肠和空肠等表达较高组织中的表达量呈现弱的正相关。而在E.coliF18敏感组,SLA-1与SLA-3基因在组织淋巴结中的表达量呈现弱的负相关；在组织胃、胸腺、十二指肠和空肠中呈现强烈的正相关,在组织脾和肺中呈现弱的正相关。(4)经GO和Pathway功能分析显示,SLA-1与SLA-3基因均参与了37个生物学过程,主要涉及抗原递呈和免疫应答的调控,并涉及9个通路,其中5个通路与免疫功能相关。3,对芯片结果中鞘糖脂生物合成—球系列通路中所有基因在个体不同组织中的表达,尤其针对肠道组织进行全面的分析。(1)组织表达谱结果显示,FUT1、FUT2、ST3GAL1、HEXA、HEXB、B3GALNT1和NAGA7个基因在所检测的11个组织中均表达,总体来看,所有基因在肝、肺、肾、胃中表达量较高,其次为十二指肠和空肠,在脾、胸腺和淋巴结等免疫组织中的表达量较低,在心和肌肉中的表达量最低。(2)7个通路基因在E.coli F18抗性组和敏感组个体十二指肠和空肠组织之间表达进行比较显示,在十二指肠中,FUT1和NAGA基因在E.coli F18抗性型个体的平均表达量高于敏感型个体；其余5个基因则在E.coli F18敏感性组中的表达量相对较高,差异倍数达2倍以上的有ST3GAL1基因。在空肠组织中仅FUT1基因在E.coli F18抗性型组中的表达量高于敏感组；其余6个基因均在敏感组中的表达量较高,差异倍数达2倍以上的有FUT2、HEXA和NAGA基因。(3)7个通路基因在苏太猪E.coli F18抗性个体十二指肠组织和空肠组织相关性分析表明,在苏太猪E.coli F18抗性个体十二指肠组织中,FUT1基因与HEXA、HEXB和NAGA基因,FUT2基因与ST3GAL1基因,ST3GAL1基因与HEXA、HEXB和B3GALNT1基因之间均表现为弱的负相关；其余基因之间均表现为弱的正相关。在苏太猪E.coli F18抗性个体空肠组织中,FUT1基因与ST3GAL1、HEXA和NAGA基因,FUT2基因与ST3GAL1基因,ST3GAL1基因与HEXA、HEXB和B3GALNT1基因之间均呈现弱的负相关,其余基因之间均表现为弱的正相关。(4)7个通路基因在苏太猪E.coli F18敏感性个体十二指肠组织和空肠组织相关性分析表明,在苏太猪E.coli F18敏感性个体十二指肠组织中,NAGA与另外6个基因FUT1、 FUT2、ST3GAL1、HEXA、HEXB以及B3GALNT1均呈现弱的负相关；另外,FUT1基因与FUT2、HEXB、B3GALNT1呈弱的负相关。除FUT2与B3GALNT1两基因间呈显著正相关外(P<0.05),其余基因之间均呈现弱的正相关。通路基因在苏太猪E.coli F18敏感性个体空肠组织中,NAGA基因仅与FUT1、FUT2基因呈现弱的负相关。其中,FUT1与FUT2、 HEXB, FUT2与HEXB、B3GALNT1, HEXB与B3GALNT1间均表现为显著正相关。4,运用差异蛋白质组学筛选大肠杆菌F18菌株敏感型和抗性型两对比组间差异蛋白。(1)在E.coli F18敏感型和对抗性型配对组中,发现20个蛋白点在表达量上有显著变化,其中10个点在E.coli F18抗性组中高表达,10个点在E.coli F18敏感组中高表达。(2)对两组之间呈现的20个差异蛋白质进行ESI-MS/MS分析,成功对16个蛋白斑点进行了肽指纹图谱的测定,共确定为12种蛋白质,其中2287、2589和2484,2074、1967和1912斑点分别呈现同一种蛋白,即actin, alpha2(ACTA2)和albumin (ALB)。另外10个有意义的差异蛋白分别为在E.coli F18抗性组中上调的Transferrin (TF)、similar to collapsin response mediator protein-2A (LOC100151886)、ribosomal protein SA (RPSA)和similar to AGAP005293-PA (LOC100153507)4种蛋白,以及在E.coli F18敏感组中上调的vinculin (VCL)、aconitase2(ACO2)、actin, alpha, cardiac muscle1(ACTC1)、actin beta (ACTB)、 heat shock protein27kDa (HSP27)和smooth muscle protein22-alpha (SM22A)6种蛋白。(3)对差异蛋白进行GO分析可知,在已知功能的基因中,显著性功能包括了肌肉收缩、细胞表面蛋白定位、胞外刺激应答、激活MAPKK等多个方面。通过KEGG数据库查询差异蛋白对应基因的所在通路,结果中包括有肌动蛋白细胞骨架的调控、黏着连接、白血球经内皮迁移、黏着斑等。(4)基于KEGG数据库现有信息,结合所有显著性Pathway中所属的蛋白,构建了差异蛋白间相互作用网络图。由于本实验结果获得的差异蛋白数量较少,以及数据库中信息的有限性,网络图中只包含5个差异蛋白(其余为连接蛋白),分别为：ACO2、 LOC100151886、ACTC1、VCL、HSP27,并且以上5个蛋白都处于网络的最下游,有着特殊重要的地位。结合对差异蛋白的功能分析,表明ACTB、VCL、转铁蛋白和热应激蛋白(HSP)与断奶仔猪F18大肠杆菌的抗性调控密切相关。更多还原

【Abstract】 Porcine post-weaning diarrhea (PWD) and porcine edema disease (ED) are two of the worst diseases resulting in tremendous damage and loss to the pig industry. Enterotoxigenic Escherichia coli (ETEC) is the main pathogen causing PWD, while Verotoxigenic Escherichia coli (VTEC) is related to ED. It has been reported that the (1,2) fucosyltransferase1gene (FUT1) is linked to the receptor of E.coli F18and can be considered as the candidate gene for controlling the adhesion of E.coli F18. An M307G/A point mutation occurs in FUT1, and the G allele is phenotypically dominant over the A allele. Pigs of genotype AA are resistant to E.coli F18, and pigs of genotypes GG and AG are sensitive to E.coli F18. Based on this principle, the researchers initiated E.coli F18-resistant breeding in foreign pigs. However, many Chinese scientists discovered an extremely skewed distribution in Chinese domestic pig breeds. Except for GG genotype, all Chinese pig breeds don’t carry the AA and AG genotype. This clearly suggests that the molecular mechanism between foreign and Chinese native pig breeds is different and the FUT1genetic marker, although suitable for foreign pig breeds, does not work well for Chinese domestic breeds.In previous studies, we identified a few FUT1AG animals in a Sutai pig population. After several years of continuous selection and breeding, two pig resource populations were established with one carrying the E.coli F18-resistant AA genotype and the other harboring E.coli F18-sensitive AG or GG genotypes. Simultaneously, we also constructed a type V secretion system to express E.coli F18adhesin. The display of functional adhesin through the type V secretion system was combined with receptor binding experiments to further analyze and verify the resistance/sensitivity to the E.coli F18strain among these pig resource populations. This study utilized the established E.coli F18-resistant (genotype AA) and-sensitive (genotypes AG and GG) pig resource populations to analyze and screen for candidate pig genes and differential proteins conferring E.coli F18resistance using gene expression profiling microarrays proteomics and bio informatics techniques.The identification of relevant genes and differential proteins may shed light on the genetic basis of E.coli F18-resistance in Chinese domestic pig breeds, and it could solve the crucial scientific issues in the breeding of E.coli F18-resistant lines for domestic Chinese pig breeds.The main results were as follows:1. The differential genes were identified by microarray screening between E.coli F18-resistant and-sensitive groups.(1) Using a two-fold change minimum threshold, we found13differentially expressed genes, with6up-regulated and7down-regulated in the pairs of GG and AA animals. And there were6genes had a fold-change>2in the pairs of AG and AA group with4up-regulated and2down-regulated. The FUT1gene was found to be differentially expressed between E.coli F18-sensitive (AG) and-resistant (AA) animals.(2) Our Gene Ontology (GO) analysis revealed that the differential genes with known functions are involved in a plethora of processes including immune responses, extracellular modification (such as glycosylation), cell adherence, signal transduction, transcriptional regulation, and metabolism. Subsequently, according to the KEGG database and the referances, we focused on the following two kinds of pathways:immune-related pathways paticipating in resistance to E.coli F18and the Glycosphingolipid biosynthesis-globo series related to the formation of E.coli F18receptor.(3) The expression validation results of7genes (SLA-1, SLA-3, ST3GAL1, A, FUT1, TAP1and SLA-DQA gene) between resistant and sensitive pairs to E.coli F18duodenal tissues matched the results of the microarrays, which showed that the microarray results was reliable.2. The relationship between the expressions of differential genes SLA-1and SLA-3and E.coli F18infection in post-weaning pigs was detected here.(1) The expression profiles showed that the expression levels of SLA-1and SLA-3measured by3different inner genes were found to be almost the same. SLA-1and SLA-3transcripts were expressed in all tissues. SLA-1was expressed at relatively high levels in lung, immune tissues such as spleen, thymus, lymph node and tissues of the digestive system (stomach, duodenum and jejunum). However, SLA-3was expressed at relatively low levels in piglet heart, liver, spleen, kidney, stomach, muscle, thymus, duodenum and jejunum compared to its expression in lung and lymphoid tissues.(2) The difference of SLA-1and SLA-3expression between E. coli F18-resistant and-sensitive groups was not significant, both genes expressed comparatively higher in the resistant group in the tissues including spleen, lung, stomach, thymus, lymph node, jejunum and duodenum.(3) The linear correlation result showed that a weak positive correlation was observed in the tissues of spleen, lung, stomach, thymus, lymph node duodenum and jejunum analyzed between the expression of SLA-1and SLA-3and disease resistance. Furthermore, on the whole, SLA-3was expressed at higher levels than SLA-1in the resistant group. However, in the sensitive group, a weak negative correlation was observed in lymph node, a relatively strong positive correlation in the stomach, thymus, duodenum and jejunum, and a weak positive correlation in the spleen and lung.(4) Gene ontology and pathway analysis was used to determine the potential SLA-1and SLA-3interactions. Both SLA-1and SLA-3can be included in37potential gene ontology biological processes, mainly relating to antigen processing and presentation, and regulation of immune responses. Results showed potential participation in nine pathways, five of which were related to immune function. Such information regarding these genes represents the basis for further study.3. The expression analysis of key genes belong to Glycosphingolipid biosynthesis-globo series in E.coli F18-resitant and-sensitive post-weaning pigs has been detected here, especially in the tissues of intestinal tract.(1) The expression profiles showed that the expression levels of FUT1, FUT2, ST3GAL1, HEXA, HEXB, B3GALNT1and NAGA were found to be almost the same in11tissues. On the whole, all the7genes expressed at relatively high level in liver, lung, kidney and stomach, followed by duodenum and jejunum. The expression was lower in immune tissues such as spleen, thymus, lymph node, and they expressed lowest in heart and muscle.(2) The expression result of those7pathway genes between E. coli F18-resistant and-sensitive groups in the tissues of intestinal tract was compared here. It indicated that in jejunum FUT1and NAGA genes expressed comparatively higher in the resistant group. Genes FUT2, ST3GAL1, HEXA, HEXB and B3GALNT1all expressed higher in E. coli F18-sensitive groups, in which ST3GAL1gene had a fold-change>2. While in the tissue of duodenum, except for the FUT1gene, other6genes expressed higher in the E. coli F18-sensitive group also with FUT2、 HEXA and NAGA genes had a fold-change>2.(3) The linear correlation among7genes in jejunum and duodenum tissues in E. coli F18-resistant group has been analysed in this research. The result showed that in jejunum in E. coli F18-resistant group there was a weak negative correlation observed in the genes of FUT1and HEXA, HEXB, NAGA, genes between FUT2and ST3GAL1, and genes of ST3GAL1and HEXA, HEXB, B3GALNT1. The correlation among other genes all showed a weak positive relation. In addition, in the tissue of duodenum in E. coli F18-resistant group a weak negative correlation was detected in the genes of FUT1and HEXA, ST3GAL1, NAGA, genes between FUT2and ST3GAL1, and genes of ST3GAL1and HEXA, HEXB, B3GALNT1. Also, the correlation among other genes all showed a weak positive relation.(4) The linear correlation among7genes in jejunum and duodenum tissues in E. coli F18-sensitive group has been studied in this research. The result showed that in jejunum in E. coli F18-sensitive group there was a weak negative correlation observed in the genes of NAGA and FUT1, FUT2, ST3GAL1, HEXA, HEXB, B3GALNT1, as well as genes of FUT1and FUT2, HEXB, B3GALNT1. The linear correlation between genes FUT2and B3GALNT1was significant (P<0.05). The correlation among other genes all showed a weak positive relation. Besides, in the tissue of duodenum in E. coli F18-resistant group a weak negative correlation was only detected in the genes of NAGA and FUT1, FUT2. And there was a strong positive relation in the genes of FUT1and FUT2, HEXB, genes among FUT2and HEXB, B3GALNT1, and genes between HEXB and B3GALNT1(P>0.05).4. The differential proteins were identified by differential proteomics technology between E.coli F18-resistant and-sensitive groups.(1) A total of20differential protein spots showed a significant change in expression among which10spots were highly expressed in the E. coli F18-resistant group and10different spots were highly expressed in the E. coli F18-susceptible group(2) ESI-MS/MS analysis was carried out for20differentially expressed proteins identified by comparison of the two groups and determinations of peptide fingerprint spectra were completed for16protein spots. According to the parameters of spectra, target proteins were identified by searching homologous proteins and peptides in the NCBInr database.A total of12proteins were identified among the16differential protein spots. Among these, two groups of spots (2287,2589,2484and2074,1967,1912) represented actin alpha2(ACTA2) and albumin (ALB) respectively. Furthermore,10significant differential proteins represented four different proteins in the E. coli F18-resistant group:upregulated transferrin (TF), similar to collapsin response mediator protein-2A (LOC100151886), ribosomal protein SA (RPSA), similar to AGAP005293-PA (LOC100153507) and six proteins in the E. coli F18-suscepitable group:upregulated vinculin (VCL), aconitase2(ACO2), actin, alpha cardiac muscle1(ACTCl), actin beta (ACTB), heat shock protein27kDa (HSP27) and smooth muscle protein22-alpha (SM22A).(3) GO analysis was used to identify genes with known functions including muscle contraction, cell surface protein localization, response to extracellular stimulus and activation of MAPKK. Pathways of differential protein-corresponding genes identified in the KEGG database included regulation of the actin cytoskeleton, adherens junction, leukocyte transendothelial migration and focal adhesion.(4) Network diagrams of interactions between differential proteins were constructed based on the KEGG database information by combining proteins in all significant pathways in order to identify the correlation of target proteins. Due to the small number of differential proteins identified in this study and limitations in the information in the database, the network diagram included only five differential proteins (others were connecting proteins):AC02, LOC100151886, ACTC1, VCL and HSP27. These five proteins were also the downstream of the network and of particular importance in this study. Combined with the function analysis of differential proteins, ACTB, VCL, Transferrin and HSP are the important protains related to E.coli F18infection.更多还原