梅山豬LBP基因的時空表達規(guī)律及其與核心啟動子區(qū)甲基化水平的關(guān)系
發(fā)布時間：2018-11-09 18:22
【摘要】：產(chǎn)腸毒素大腸桿菌F18(E.coli F18)是引起斷奶仔豬腹瀉的主要病原菌,目前研究發(fā)現(xiàn),E.coliF18受體合成通路和炎癥免疫信號通路調(diào)控著斷奶仔豬E.col F18的抗性。脂多糖結(jié)合蛋白(Lipopolysaccharide-bindingprotein,LBP)不僅能結(jié)合革蘭氏陰性菌細胞壁主要成分脂多糖或內(nèi)毒素(Lipopolysaccharide,LPS)激活免疫信號通路,從而殺死病原菌進而維護宿主內(nèi)環(huán)境的穩(wěn)態(tài),而且血清中高濃度LBP能中和LPS,降低LPS的生物學(xué)活性,因此LBP是體內(nèi)免疫防御機制中重要的組成部分。E.coliF18作為革蘭氏陰性菌,LPS也是其重要的致病因子。因此,LBP基因可能在斷奶仔豬E.coli F18抗性中發(fā)揮著重要的作用。本試驗以梅山豬為試驗材料,探究LBP基因在豬不同發(fā)育時期及不同組織中的表達規(guī)律,并探討豬LBP基因甲基化及其對基因表達的調(diào)控機制,為了解LBP基因功能及今后更好的將其應(yīng)用到豬抗病選育提供一定的試驗依據(jù)和理論基礎(chǔ)。1.本試驗利用Real-time PCR檢測了L P基因在35日齡梅山豬中的組織表達譜(心臟、肝臟、脾臟、肺臟、腎臟、胃、肌肉、胸腺、腸系膜淋巴結(jié)、十二指腸、空腸和回腸等12個組織);檢測了其在不同發(fā)育時期(1、7、14、21、28、35和158日齡)梅山豬肝臟組織中的表達規(guī)律;利用不同濃度的LPS誘導(dǎo)豬腸上皮細胞(IPEC-J2),并分別在誘導(dǎo)后2h、4h和6h檢測LBP基因在IPEC-J2細胞系中的表達。結(jié)果表明,LBP基因在肝臟組織中的表達極顯著高于其他組織(P0.01),在十二指腸組織中的表達水平顯著高于除肝臟組織外的其他組織(P0.05);除肝臟和十二指腸組織外,其他組織中LBP基因的表達水平較低且無顯著性差異(P0.05);LB 在1和21日齡肝臟組織中的表達極顯著高于7、14、28和35日齡(P0.01),158日齡肝臟組織中的LBP表達極顯著高于28和35日齡(P0.01),顯著高于7和14日齡(P0.05);不同濃度的LPS(0.1μg/mL、0.5μg/mL和1 μg/mL)誘導(dǎo)IPEC-J2細胞系后,各濃度LPS誘導(dǎo)后LBP的表達并未呈現(xiàn)一致的表達規(guī)律。本試驗推測,豬LBP基因主要高度表達于肝臟組織中;腸道組織中一定程度也可合成分泌LBP;當(dāng)仔豬受到外界病原菌或病原相關(guān)分子(Pathogen-associated molecular pattern,PAMP)侵染時,LBP基因的表達也會隨之發(fā)生變化;腸上皮細胞可能并不是腸道組織中主要分泌和合成LBP蛋白的細胞系。2.本試驗利用BDGP軟件預(yù)測分析豬LBP基因核心啟動子區(qū),并基于預(yù)測結(jié)果設(shè)計缺失片段,利用雙熒光素酶報告基因技術(shù)檢測各片段的啟動子活性。結(jié)果表明,梅山豬LBP基因啟動子區(qū)段的-500～-206 bp為核心啟動子區(qū);-1500～-500 bp為調(diào)控豬LBP基因啟動子活性的重要區(qū)段。3.本試驗利用焦磷酸測序技術(shù)檢測豬LBP基因核心啟動子區(qū)的甲基化水平,其中在梅山豬不同組織中CpG2和CpG3位點的甲基化水平和LBP基因的表達呈現(xiàn)顯著的負相關(guān),線性相關(guān)系數(shù)分別為-0.4684和-0.3069;而在不同發(fā)育階段的梅山豬肝組織中CpG2和CpG3位點的甲基化水平與基因表達并無顯著的相關(guān)性。CpG2和CpG3位點的甲基化可能抑制轉(zhuǎn)錄因子YY1與啟動子DNA序列的結(jié)合,從而導(dǎo)致豬LBP基因的組織表達差異,而外來病原菌及LPS進入仔豬體內(nèi)可能是通過其他調(diào)控途徑影響LBP基因的表達。
[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.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S828
本文編號：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.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S828
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相關(guān)期刊論文 前3條
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