梅山猪LBP基因的时空表达规律及其与核心启动子区甲基化水平的关系
发布时间：2018-11-09 18:22
【摘要】：产肠毒素大肠杆菌F18(E.coli F18)是引起断奶仔猪腹泻的主要病原菌,目前研究发现,E.coliF18受体合成通路和炎症免疫信号通路调控着断奶仔猪E.col F18的抗性。脂多糖结合蛋白(Lipopolysaccharide-bindingprotein,LBP)不仅能结合革兰氏阴性菌细胞壁主要成分脂多糖或内毒素(Lipopolysaccharide,LPS)激活免疫信号通路,从而杀死病原菌进而维护宿主内环境的稳态,而且血清中高浓度LBP能中和LPS,降低LPS的生物学活性,因此LBP是体内免疫防御机制中重要的组成部分。E.coliF18作为革兰氏阴性菌,LPS也是其重要的致病因子。因此,LBP基因可能在断奶仔猪E.coli F18抗性中发挥着重要的作用。本试验以梅山猪为试验材料,探究LBP基因在猪不同发育时期及不同组织中的表达规律,并探讨猪LBP基因甲基化及其对基因表达的调控机制,为了解LBP基因功能及今后更好的将其应用到猪抗病选育提供一定的试验依据和理论基础。1.本试验利用Real-time PCR检测了L P基因在35日龄梅山猪中的组织表达谱(心脏、肝脏、脾脏、肺脏、肾脏、胃、肌肉、胸腺、肠系膜淋巴结、十二指肠、空肠和回肠等12个组织);检测了其在不同发育时期(1、7、14、21、28、35和158日龄)梅山猪肝脏组织中的表达规律;利用不同浓度的LPS诱导猪肠上皮细胞(IPEC-J2),并分别在诱导后2h、4h和6h检测LBP基因在IPEC-J2细胞系中的表达。结果表明,LBP基因在肝脏组织中的表达极显著高于其他组织(P0.01),在十二指肠组织中的表达水平显著高于除肝脏组织外的其他组织(P0.05);除肝脏和十二指肠组织外,其他组织中LBP基因的表达水平较低且无显著性差异(P0.05);LB 在1和21日龄肝脏组织中的表达极显著高于7、14、28和35日龄(P0.01),158日龄肝脏组织中的LBP表达极显著高于28和35日龄(P0.01),显著高于7和14日龄(P0.05);不同浓度的LPS(0.1μg/mL、0.5μg/mL和1 μg/mL)诱导IPEC-J2细胞系后,各浓度LPS诱导后LBP的表达并未呈现一致的表达规律。本试验推测,猪LBP基因主要高度表达于肝脏组织中;肠道组织中一定程度也可合成分泌LBP;当仔猪受到外界病原菌或病原相关分子(Pathogen-associated molecular pattern,PAMP)侵染时,LBP基因的表达也会随之发生变化;肠上皮细胞可能并不是肠道组织中主要分泌和合成LBP蛋白的细胞系。2.本试验利用BDGP软件预测分析猪LBP基因核心启动子区,并基于预测结果设计缺失片段,利用双荧光素酶报告基因技术检测各片段的启动子活性。结果表明,梅山猪LBP基因启动子区段的-500～-206 bp为核心启动子区;-1500～-500 bp为调控猪LBP基因启动子活性的重要区段。3.本试验利用焦磷酸测序技术检测猪LBP基因核心启动子区的甲基化水平,其中在梅山猪不同组织中CpG2和CpG3位点的甲基化水平和LBP基因的表达呈现显著的负相关,线性相关系数分别为-0.4684和-0.3069;而在不同发育阶段的梅山猪肝组织中CpG2和CpG3位点的甲基化水平与基因表达并无显著的相关性。CpG2和CpG3位点的甲基化可能抑制转录因子YY1与启动子DNA序列的结合,从而导致猪LBP基因的组织表达差异,而外来病原菌及LPS进入仔猪体内可能是通过其他调控途径影响LBP基因的表达。
[Abstract]:Enterotoxigenic E. coli F18 (E. coli F18) is the main pathogen causing the diarrhea of weaned piglets. At present, the E. coli F18 receptor synthesis pathway and the inflammatory immune signal pathway regulate the resistance of E. col F18 in weaned pigs. Lipopolysaccharide-binding protein (LBP) can not only bind to the cell wall of gram-negative bacteria, but also activate the immune signal path with lipopolysaccharide or LPS, so as to kill the pathogenic bacteria and maintain the steady state of the environment in the host, and the high-concentration LBP in the serum can neutralize the LPS. LBP is an important part of in-vivo immune defense mechanism to reduce the biological activity of LPS. E. coliF18 is also an important pathogenic factor for Gram-negative bacteria and LPS. Therefore, the LBP gene may play an important role in the resistance of E. coli F18 in weaned pigs. The expression of the LBP gene in the different developmental stages of the pig and the different tissues was studied by using the Meishan pig as the test material, and the methylation of the LBP gene and the regulation and control mechanism of the expression of the gene were also discussed. in ord to understand that function of the LBP gene and to improve the application of the LBP gene in the breeding of the pig in the future, a certain experimental basis and a theoretical foundation are provided. The tissue expression profiles of L-P (heart, liver, spleen, lung, kidney, stomach, muscle, thymus, mesenteric lymph node, duodenum, jejunum and ileum) were detected by Real-time PCR. The expression of LBP gene in the liver of Meishan pig in different developmental stages (1, 7, 14, 21, 28, 35 and 158) was detected, and the expression of the LBP gene in the IPEC-J2 cell line was detected by LPS-induced porcine intestinal epithelial cells (IPEC-J2) at different concentrations. The results showed that the expression of the LBP gene in the liver tissue was significantly higher than that of other tissues (P0.01), and the expression level in the duodenum was significantly higher than that of other tissues except for liver tissue (P0.05); besides the liver and the duodenum, The expression of LBP in other tissues was lower and no significant difference (P0.05); the expression of LBP in the liver of 1 and 21 days was significantly higher than that of 7, 14, 28 and 35 (P0.01), and the expression of LBP in the liver of 158-day-old was significantly higher than that of 28 and 35 (P0.01). After induction of IPEC-J2 cell line by LPS (0.1. mu.g/ mL, 0.5. mu.g/ mL and 1. mu.g/ mL) at different concentrations, the expression of LBP after LPS induction did not show a consistent expression pattern. The results suggest that the expression of the LBP gene in the liver is highly expressed in the liver tissue, and the secretion of LBP may be synthesized in the intestinal tissue. The expression of the LBP gene may change when the piglets are infected by the external pathogenic bacteria or the pathogenic associated molecular pattern (PAMP). Intestinal epithelial cells may not be a cell line that mainly secretes and synthesizes the LBP protein in the intestinal tissue. The BGP software was used to predict the core promoter region of the porcine LBP gene, and the deletion fragment was designed based on the predicted results, and the promoter activity of each fragment was detected by using the double-luciferase reporter gene technique. The results showed that the-500 ~ -206 bp of the promoter region of the LBP gene in the Meishan pig was the core promoter region; -1500--500bp was the important section for regulating the promoter activity of the porcine LBP gene. The methylation level of the core promoter region of the porcine LBP gene was detected by using the pyrosequencing technique, and the methylation level of CpG2 and CpG3 sites and the expression of the LBP gene in the different tissues of Meishan pigs showed a significant negative correlation, and the linear correlation coefficients were-0.4684 and-0.3069, respectively. The methylation of CpG2 and CpG3 sites in Meishan pig liver tissue at different developmental stages was not related to gene expression. The methylation of CpG2 and CpG3 sites may inhibit the binding of the transcription factor YY1 to the promoter DNA sequence, resulting in a difference in the tissue expression of the porcine LBP gene, while the foreign pathogenic bacteria and the LPS enter the piglets may be affected by other regulatory pathways to the expression of the LBP gene.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828
本文编号：2321201
[Abstract]:Enterotoxigenic E. coli F18 (E. coli F18) is the main pathogen causing the diarrhea of weaned piglets. At present, the E. coli F18 receptor synthesis pathway and the inflammatory immune signal pathway regulate the resistance of E. col F18 in weaned pigs. Lipopolysaccharide-binding protein (LBP) can not only bind to the cell wall of gram-negative bacteria, but also activate the immune signal path with lipopolysaccharide or LPS, so as to kill the pathogenic bacteria and maintain the steady state of the environment in the host, and the high-concentration LBP in the serum can neutralize the LPS. LBP is an important part of in-vivo immune defense mechanism to reduce the biological activity of LPS. E. coliF18 is also an important pathogenic factor for Gram-negative bacteria and LPS. Therefore, the LBP gene may play an important role in the resistance of E. coli F18 in weaned pigs. The expression of the LBP gene in the different developmental stages of the pig and the different tissues was studied by using the Meishan pig as the test material, and the methylation of the LBP gene and the regulation and control mechanism of the expression of the gene were also discussed. in ord to understand that function of the LBP gene and to improve the application of the LBP gene in the breeding of the pig in the future, a certain experimental basis and a theoretical foundation are provided. The tissue expression profiles of L-P (heart, liver, spleen, lung, kidney, stomach, muscle, thymus, mesenteric lymph node, duodenum, jejunum and ileum) were detected by Real-time PCR. The expression of LBP gene in the liver of Meishan pig in different developmental stages (1, 7, 14, 21, 28, 35 and 158) was detected, and the expression of the LBP gene in the IPEC-J2 cell line was detected by LPS-induced porcine intestinal epithelial cells (IPEC-J2) at different concentrations. The results showed that the expression of the LBP gene in the liver tissue was significantly higher than that of other tissues (P0.01), and the expression level in the duodenum was significantly higher than that of other tissues except for liver tissue (P0.05); besides the liver and the duodenum, The expression of LBP in other tissues was lower and no significant difference (P0.05); the expression of LBP in the liver of 1 and 21 days was significantly higher than that of 7, 14, 28 and 35 (P0.01), and the expression of LBP in the liver of 158-day-old was significantly higher than that of 28 and 35 (P0.01). After induction of IPEC-J2 cell line by LPS (0.1. mu.g/ mL, 0.5. mu.g/ mL and 1. mu.g/ mL) at different concentrations, the expression of LBP after LPS induction did not show a consistent expression pattern. The results suggest that the expression of the LBP gene in the liver is highly expressed in the liver tissue, and the secretion of LBP may be synthesized in the intestinal tissue. The expression of the LBP gene may change when the piglets are infected by the external pathogenic bacteria or the pathogenic associated molecular pattern (PAMP). Intestinal epithelial cells may not be a cell line that mainly secretes and synthesizes the LBP protein in the intestinal tissue. The BGP software was used to predict the core promoter region of the porcine LBP gene, and the deletion fragment was designed based on the predicted results, and the promoter activity of each fragment was detected by using the double-luciferase reporter gene technique. The results showed that the-500 ~ -206 bp of the promoter region of the LBP gene in the Meishan pig was the core promoter region; -1500--500bp was the important section for regulating the promoter activity of the porcine LBP gene. The methylation level of the core promoter region of the porcine LBP gene was detected by using the pyrosequencing technique, and the methylation level of CpG2 and CpG3 sites and the expression of the LBP gene in the different tissues of Meishan pigs showed a significant negative correlation, and the linear correlation coefficients were-0.4684 and-0.3069, respectively. The methylation of CpG2 and CpG3 sites in Meishan pig liver tissue at different developmental stages was not related to gene expression. The methylation of CpG2 and CpG3 sites may inhibit the binding of the transcription factor YY1 to the promoter DNA sequence, resulting in a difference in the tissue expression of the porcine LBP gene, while the foreign pathogenic bacteria and the LPS enter the piglets may be affected by other regulatory pathways to the expression of the LBP gene.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828
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