【作者】 刘鑫；

【导师】 柳小春； 施启顺；

【作者基本信息】 湖南农业大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 仔猪腹泻是养猪生产中存在的普遍疾病,是影响养猪业经济效益的重要因素。肠毒素大肠杆菌（ETEC）是导致仔猪腹泻的最重要的病原菌,ETEC的致病性决定于它们在小肠上皮细胞的定居能力和产生肠毒素的能力,F4粘附素能否结合到猪的小肠上皮细胞,决定于猪小肠粘膜有无受体。无受体的猪在受到细菌感染时不发病,表现为抗性,因此,培育出抗性猪具有重要意义。所以,利用分子生物学手段寻找和筛选该受体的遗传标记是ETEC抗病育种的重要途径。本文采用微卫星和差异显示PCR方法技术,以沙子岭猪和大白猪为试验材料,对不同品种初生小猪的小肠和脾脏组织进行差异显示研究,筛选与ETEC F4黏附表型相关的候选基因和遗传标记,并通过通过荧光定量PCR对相关候选基因的表达进行了分析,主要研究结果如下。1、试验检测了63头初生仔猪小肠上皮细胞对ETEC F4三种抗原型（ab、ac、ad）的黏附表型。结果表明,F4ab型在两个猪种三种血清型的黏附比率最高,沙子岭猪黏附个体比率为0.727,大约克猪黏附个体比率为0.667:由此可见,初生仔猪患仔猪黄痢主要是大肠杆菌F4ab型与仔猪小肠上皮细胞刷状缘结合而引起的。F4ad型在两个猪种中的黏附比率差异显著,沙子岭猪的黏附个体比率为0.400,高于大约克的0.242（p＜0.05）。2、4个猪种在4个基因座均具有高度多态性,杂合度（H）在0.6117-0.7500之间,多态信息含量（PIC）达0.5749以上,最高为0.7247。中外猪种在各个微卫星座位上等位基因频率都存在显著差异,地方品种的基因多样性要比外来品种高。在微卫星座位SW458上,AC基因型在两个品种三种血清型黏附表型中均无黏附型,这为抗性猪的选择提供了可能,SW458座位可望作为E.coliF4抗性基因的遗传标记。3、通过DDRT-PCR技术,采用24对引物组合,对沙子岭猪和大白猪两个品种脾脏组织和小肠组织F4不同黏附表型进行了差异显示研究,共检测到57条差异片段。差异片段再扩增PCR中,有6条差异片段没能实现特异性扩增,本实验将其作为假阳性片段,没作进一步分析。反Northern点杂交验证,有5个差异片段表现为假阳性,试验共找到46个阳性差异片段,阳性率为80.7%（46/57）。4、本研究对22条差异表达片段进行了序列测定,经过BLAST同源性比对,发现有5条序列与已知功能基因同源性较高,其余17条序列同源性较低或者找到同源EST但是不知道该EST的功能,大部分为新的EST序列。本试验将22条EST登录到GenBank,登录号分别为FE597044-FE597057和FE861134-FE861141。5、在小肠组织中,ATPase,Ca²⁺ transporting,plasma membrane 1（ATP2B1）基因和Histamine receptor H1（HRH1）基因均:为93%（183/193、420/450）的同源性。ATP281即为ATP酶Ca²⁺转运质膜1,能调控肠道细胞的Na⁺和Ca²⁺平衡,其功能与腹泻紧密相关。同时,ATP281基因位于猪的13q31-q32,与猪小肠的F4ab受体基因紧密连锁。HRH1受体主要分布在外周组织,高表达于与炎症反应相关的组织和造血细胞中,HRH1被激动后使细胞内Ca²⁺增加,释放血管内皮松弛因子（EDRF）和PGI2,使小血管扩张,通透性增加。这就使肠胃的通透性增加,胃酸分泌过量,从而产生腹泻等症状。结合ATP2B、HRH1基因的同源性和功能,可分别将它们作为F4ab、F4ac受体的相候选关基因来进行下一步的研究。6、在脾脏组织中,Pre-B-cell colony enhancing factor 1（PBEF1）基因有97%（351/360）的同源性,PBEF1基因起转移糖基/烟酰胺磷酸核糖转移酶等活性作用,参与细胞信号转导、正调控细胞增殖等过程,对机体免疫、新陈代谢等有着广泛的影响,它可能是仔猪腹泻后,脾脏发生免疫应答反应而产生的产物。综合PBEF1基因的同源性和功能,可将其作为F4ad受体的相候选关基因来进行下一步的研究。7、通过荧光定量PCR技术对以上三个相关候选基因的表达分析,进一步验证了PBEF1基因可能作为仔猪腹泻的相关候选基因。ATP281基因可望作为E.coli F4ab受体的相关候选基因,而HRH1基因仍需进一步的研究。更多还原

【Abstract】 Piglets diarrhea is a widespread disease in swine industry and one of the important factors in the economic benefits of pig production. Enterotoxigenic Escherichia coli （ETEC） is the primary pathogenic bacterium leading to the diarrhea of piglet. The reason of ETEC to cause the piglet to diarrhea depend on their capability of Settlement and producing enterotoxins in the small intestine epithelial cells. F4 adhesion can combine to the piglet’s small intestine epithelial cells depends on whether there is a small intestine receptor. No receptors in the pig are not the incidence of bacterial infection, for the performance of resistance, therefore, the resistance breed pigs is of great significance. So, the use of molecular biology tools to find the receptor and screening of genetic markers ETEC is an important way for breeding disease-resistant.In this paper, we use the technique of SSRs and DDRT-PCR to do experiment in Shaziling piglets and Yorkshire piglets. Make the difference research between breeds in the small intestine and spleen organizations, screening the candidate gene and genetic markers related of ETEC F4 adhesion, analysis the relational candidacy gene’s expression by fluorescence quantitative PCR. The main conclusions of this study are as follows:1. The test detected the adhesion phenotype beween the intestinal epithelial cells and ETEC F4 three-antigen （ab, ac, ad） in 63 newborn piglets. The results showed that F4ab antigen was the highest rate of adhesion, Shaziling pig adhesion individual ratio was 0.727, Yorkshire pig adhesion individual ratio was 0.667. Thus, the F4ab antigen was the mainly reason cause newborn piglets suffering from dysentery. It was significantly in the two breeds of F4ad adhesion rate, the adhesion Shaziling pig individual ratio was 0.400, higher than the approximately Yorkshire was 0.242 （P <0.05）.2. The 4 loci in the four locas all are highly polymorphic, heterozygosity （H） ranged from 0.6117 to 0.7500. multi-information content （PIC） reached to over 0.5749, the highest was 0.7247. The allele frequencies are significant differences in various miao-satellite seats in Chinese and foreign breeds. Generally speaking, the genetic diversity of local pigs are higher than foreign. In microsatellite loci on the SW458, AC genotype in two varieties of three serum adhesion in the form of no-adhesion, which is the choice of resistance pig provides the possibility, SW458 seats is expected as the resistance gene E.coliF4 Genetic markers.3. Through DDRT-PCR technology, a 24 primer combination of primers, we studied the different spleen structures and small intestine different adhesion phonetype between the shaziling pig and the Yorkshire pig. It detected a total of 57 different segments. Among 57 differential segments, 6 segments were not re-amplified and did not get further studied. Northern hybrid-point verification, 5 different regions showed false positive, the pilot found a total of 46 positive difference in fragments, the positive rate was 80.7% （46/57）.4. This study Choose 22 different fragments to a sequencing, after BLAST homology Comparison, there were 5 sequence homology with known functional genes with high Similarity and the remaining 17 sequences, were found a lower homologous EST but did not know that the function, most of them were new EST sequence. 20.ESTs were deposited into GenBank and their accession No. were FE597044-FE597057 and FE861134-FE861141.5. In the small intestine, ATPase, Ca²⁺ transporting, plasma membrane 1 （ATP2B1） gene and Histamine receptor H1 （HRH1） gene were 93% （183/193, 420/450） homology identity. ATP2B1 is the enzyme ATP Ca2 + transit membrane 1, to control intestinal cells Na⁺ and Ca²⁺ balance, and its function is closely related to diarrhea. At the same time, ATP2B1 gene in the pig 13 q31-q32,was closely linked the F4ab receptor gene. HRH1 receptor mainly located in peripheral organizations, high expression in the inflammatory response associated with the organization and hematopoietic cells, HRH1 was so excited after Ca²⁺ cells increased, the release of vascular endothelial relaxation factor （EDRF） and PGI2, it can makes the expansion of small vessels and Permeability increase. This makes the gastrointestinal permeability increase, excessive secretion of gastric acid, resulting in diarrhea and other symptoms. With ATP2B1, HRH1 genetic identity and function, respectively them as F4ab, F4ac receptor gene is related to the candidate for the next phase of research.6.In the spleen, Pre-B-cell colony enhancing factor 1 （PBEF1） gene was 97% （351/360） homology identity. PBEF1 gene can transfer the sugar base / nicotinamide-ribose Meideng transfer enzyme, Involved the regulation and control process in cell signal transduction and cell proliferation, so it has a broad impact on the immune, metabolic and so on. May be it was the product of spleen immune reaction in response to piglets diarrhea. Comprehensive PBEF1 gene identity and function can be used as the candidate of the F4ad receptor gene related to conduct further research.7. According to the fluorescence quantitative PCR technology, we analyzed the three related gene’s expression analysis, to verify the PBEF1 gene may be related to diarrhea in pigs as a candidate gene. ATP2B1 gene is expected as E.coli F4ab receptor-related candidate genes, gene and the HRH1 need further study.更多还原