【作者】 季华员；

【导师】 黄路生； 任军；

【作者基本信息】 江西农业大学 ， 动物遗传育种与繁殖， 2011， 博士

【摘要】 产肠毒素大肠杆菌(enterotoxigenic Escherichia coli ETEC)是引发新生及断奶仔猪腹泻和水肿的主要致病菌。ETEC主要存在五种血清型,包括F4、F41、F5、F6和F18。ETEC F41作为其中的一种血清型,是引起仔猪腹泻的重要病原菌之一,其通过表面表达的黏附素与猪小肠上皮细胞的特异性受体结合,释放肠毒素进而引起大量电解质和水渗入肠腔,从而导致仔猪腹泻。本课题组前期利用183个微卫星标记在大规模白色杜洛克×二花脸资源家系群体中开展全基因组扫描,将ETECF41受体基因初步定位在猪4号染色体SW2509和S0301标记间约6.04 Mb的区域。在此基础上,本研究通过在SSC4 QTL目标区域增加7个微卫星标记,对其在同一资源群体(包括816个F2个体及所有Fo与F1个体)中开展基因分型,进一步将ETEC F41受体基因精细定位至标记SW2509至KVL2754之间约2.42 Mb的区域,为鉴别ETEC F41受体目的基因奠定了良好的工作基础。在所定位的2.42 Mb目标区域中,采用比较基因组学分析,揭示该区域目前存在21个基因,从中选取6个可能的位置候选基因(包括ST3GAL1、TMEM71、ASAP1、NDRG1、ZFAT、KCNQ3基因)通过半定量RT-PCR检测其在猪空肠组织中的表达,结果表明这6个基因在空肠中均表达。进一步结合ETEC F41受体的理化特性选择ST3p-半乳糖α-2,3唾液酸转移酶1(ST3GAL1)基因作为ETEC F41受体的重要位置候选基因加以研究。采用5’RACE和3’RACE技术,分离了ST3GAL1基因全长1637 bp的cDNA序列,其中开放阅读框(Open Reading Frame, ORF)为1032 bp,编码343个氨基酸。通过比较测序法在ST3GAL1基因内共鉴别到62个多态位点,其中2个错义突变(ST3GAL1 c.284T>A和ST3GAL1 c.661A>G),11个同义突变,49个内含子上的突变。采用PCR-RFLP和PCR-SNaPshot方法对ST3GAL1 c.284T>A(位于第1外显子)、c.477C>T(位于第2外显子)及c.661A>G(位于第3外显子)等3个多态位点在白色杜洛克×二花脸资源家系所有个体中进行多态性检测。传递不平衡检测表明除c.477C>T位点外,另两个多态位点的等位基因及所有单倍型均与ETEC F41的易感性呈极显著相关(P<0.01)。为此,本实验选择两个强关联的SNPs位点(ST3GAL1c.284T>A及c.661A>G)进一步在287头中、西方猪种远缘群体中开展遗传多态性分析,采用Case Control力法分析,结果表明ST3GAL1 c.284T>A及c.661A>G两个多态位点在中、西方猪种远缘群体中与ETEC F41黏附表型关联性均不显著。由此提示ST3GAL1基因与ETECF41黏附表型的关联性可能存在群体异质性,另外ST3GAL1基因可能不是目的基因而仅是与目的基因处于一定连锁不平衡状态的一个标记位点。为了获得更高的基因定位精度,本研究采用猪全基因组高通量60K SNP芯片对白色杜洛克×二花脸资源群体开展全基因组关联分析(Genome-wide association study, G WAS),以精细定位ETEC F41受体基因位点。GWAS结果显示,与ETEC F41黏附表型具有最显著关联的SNP位点为H3GA0011673 C>T(P=1.09E-09),与其临近的两个多态位点为ASGA0017715 T>C和ASGA0017733 C>T。进一步对这三个多态位点在287头中、西方猪种中采用PCR-SNaPshot方法进行多态性检测,结果表明仅ASGA0017733 C>T多态位点在西方猪种中与ETEC F41黏附表型显著关联。由此提示,ASGA0017733 C>T位点可能是与目的基因处于连锁不平衡的一个重要标记,这为进一步开展位置候选基因研究奠定良好的基础。ASGA0017733 C>T位点位于ZFAT基因第3内含子上,结合受体生理生化分析,本实验选择ZFAT (zinc finger and AT hook domain containing)作为ETEC F41受体位置候选基因进一步深入研究。采用RACE技术分离得到长4108 bp的猪ZFAT cDNA序列,开放阅读框(ORF)长3393 bp,编码1130个氨基酸。通过比较测序在ZFAT基因编码区鉴别到c.60C>T、c.630A>C、c.660T>C、c.1077T>C、c.1416T>C、c.2337T>C及c.2935G>C共7个多态位点,其中1个为错义突变,6个为同义突变。采用PCR-SNaPshot技术检测这7个多态位点在中、西方猪种远缘群体287个个体中的多态性。连锁不平衡(Linkage Disequilibrium, LD)分析表明大多数SNP位点均存在不同程度的连锁不平衡,但在西方猪种中的LD程度高于中国地方猪种。关联性分析表明：在中国地方猪种中仅2337T>C位点与ETEC F41黏附表型显著相关(P<0.05)；而在西方猪种中,除c.630A>C和c.660T>C位点与ETEC F41黏附表型相关性不显著外,其余5个多态位点与ETEC F41黏附表型显著相关(P<0.05),表明这些多态位点可能位于ETEC F41受体基因之中或与其处于连锁不平衡状态,这些结果为ETECF41受体基因的精细定位提供了重要的分子标记。本研究不仅证实了与ETEC F41侵染相关的主效QTL位于猪4号染色体上,而且进一步将目标区域缩小至标记SW2509-KVL2754(5.56 Mb-7.98 Mb)间,为最终确定ETEC F41受体目的基因及其因果突变奠定了重要的前期工作基础。通过对位置候选基因ST3GAL1和ZFAT基因的克隆及多态性分析,鉴别到的与ETEC F41黏附表型显著相关的位点为利用标记辅助选择(MAS)开展抗ETEC F41腹泻病分子遗传育种提供了很好的分子标记。更多还原

【Abstract】 Enterotoxigenic Eschoerchia coli (ETEC) is a main pathogen causing diarrhea and edema in neonatal and post-weaned piglets. ETEC have five main serologic variants including F4、F41、F5、F6 and F18. ETEC F41 is one of the major serologic variants causing diarrhea in piglets by adhesion with the specific receptor of epithelial cells of small intestine, releasing enterotoxin and forcing large volume of electrolyte and water entering into gut cavity, resulting in diarrhea and edema.In a previous study, we performed a whole genome scan to detect QTL for ETEC F41 receptor using a large scale White Duroc×Erhualian intercross resource population with 183 microsatellite markers covering the entire genome. A major locus for ETEC F41 receptor was mapped to a 6.04-Mb region between SW2509 and S0301 on pig chromosome 4 (SSC4). Basing on the previous study, additional 7 microsatellite markers in the QTL region on chromosome 4 were genotyped across the three-generation pedigree comprising of 19 founders,68 F1 and 816 F2 animals. The ETEC F41 receptor locus was then refined to a region of 2.42 Mb between SW2509 and KVL2754 on SSC4. This result paved an important road to identify the ETEC F41 receptor gene.According to the human-pig comparative genomic map, there are 21 annotated genes in the refined region. Six positional candidate genes including ST3GAL1. TMEM71、ASAP1、NDRG1、ZFAT、KCNQ3 were subjected to expression analysis in the small intestinal tissues with semi-quantitative RT-PCR. The results indicated that all these genes were expressed in jejunum. According to the biological characteristics of the ETEC F41 receptor, ST3GAL1 was selected as one of candidate genes for further study. The 1,637-bp full-length cDNA of porcine ST3GLAL1 was isolated by 5’RACE and 3’RACE assays, which encodes a 343-amino acids protein. A total of 62 polymorphisms including 2 missense mutations (ST3GAL1c.284T>A and ST3GALIc.661A>G),11 synonymous variants and 49 intronic polymorphisms were identified by comparative sequencing using genomic DNA of four founder animals. Of the 62 polymorphisms, ST3GAL1 c.284T>A (in exon 1), c.477C>T (in exon 2) and c.661A>G (in exon 3) were genotyped on all animals of the White Duroc×Erhualian resource population. Transmission disequilibrium test showed that all alleles and haplotypes except for c.477C>T had strong association with susceptibility to ETEC F41 (P< 0.01). Furthermore, ST3GAL1 c.284T>A and c.661A>G were genotyped in 287 purebred outbred piglets from three Western commercial breeds and Chinese indigenous breeds. Case-control analysis showed that both the two polymorphisms were not associated with ETEC F41 adhesion phenotypes. The results indicated that different association of ST3GAL1 with ETEC F41 adhesion phenotypes in deferent populations was likely caused by population heterogeneity, alternatively, ST3GAL1 was not a major gene controlling diarrhea caused by ETEC F41 in pigs, it was only a locus having linkage disequilibrium with the resposible gene.To improve the mapping resolution, the White Duroc×Erhualian intercross were genotyped with Illumina porcine 60k DNA chips. Genome-wide association (GWAS) results showed that the most significantly linked locus was H3GA0011673 C>T (P= 1.09E-09) at 6167551 bp on SSC4 followed by ASGA0017715 T>C and ASGA0017733 C＞T. The three polymorphisms were genotyped on 287 outbred pigs. The case-control analysis indicated that ASGA0017733 C＞T was associated with ETEC F41 adhesion phenotypes of Western commercial pigs. The result suggested that ASGA0017733 C>T polymorphic locus probably located in the responsible gene or is an important marker which was in high linkage disqulibrium with the causal gene. These results provided good basis for positional cloning of the gene encoding the ETEC F41 receptor.The ASGA0017733 C>T polymorphism maps to intron 3 of the ZFAT gene. According to the properties of ETEC F41 receptor, ZFAT was selected as a positional candidate gene for furtherly analysis. The 4108-bp cDNA of ZFAT was isolated by RACE assay, which contains a 3393-bp open reading frame encoding a protein of 1130 amino acids. A total of 7 SNPs including one missense mutation and six synonymous variants (c.60C>T, c.630A>C, c.660T>C, c.1077T>C, c.1416T>C, c.2337T>C and c.2935G>C) were detected by comparative sequencing using cDNA of susceptible and resistant animals. All animals of outbred populations were genotyped for the seven ZFAT polymorphisms. Linkage disequilibrium (LD) analysis indicated that strong linkage disequilibrium existed among most of ZFAT polymorphisms. Moreover, LD was higher in Western commercial pigs than that in Chinese indigenous pigs. The results of association analysis showed that only c.2337T>C had significant (P< 0.05) association with susceptibility to ETEC F41 adhesion phenotypes in Chinese native pigs. In comparison, five polymorphisms except for c.630A>C and c.660T>C had association with susceptibility to ETEC F41 in Western commercial pigs (P< 0.05). These closely associated loci (c.60C>T, c.1077T>C, c.1416T>C, c.2337T>C and c.2935G>C) could benefit fine mapping of the gene encoding the ETEC F41 receptor.This study further convinced the major QTL for susceptability to ETEC F41 on pig SSC4 and refined the critical region to the SW2509-KVL2754 interval (5.56 Mb-7.98 Mb). The results shed new insights into the final identification of the ETEC F41 receptor genes and their casuative mutation(s). The novel informatic and significant markers of ST3GAL1 and ZFAT could be used to select disease resitance to ETEC F41 in pigs by marker assisted selection (MAS) schemes.更多还原