球系列鞘糖脂生物合成通路关键基因启动子区甲基化对仔猪E.coli F18抗性的调控作用分析

发布时间：2018-06-17 22:33

本文选题：猪 + E.coli　；参考：《扬州大学》2015年硕士论文

【摘要】：腹泻是导致仔猪死亡的主要疾病,给养猪业带来了巨大经济损失。F18大肠杆菌(E.coli F18)菌株是引起仔猪腹泻的主要病原菌。国外研究表明,α-(1,2)岩藻糖转移酶1基因(FUT1)M307位点存在G/A的突变,该突变可以作为控制Ecoli F18粘附的遗传标记,并据此在国外猪种中实现了抗病育种,但是该位点在中国地方猪种群体中呈极端偏态分布,使得国内地方猪种抗F18大肠杆菌病育种实践陷入了困境。课题前期通过表达谱芯片技术、蛋白质组学技术,对仔猪大肠杆菌病抗性型和敏感型全同胞配对个体,进行了十二指肠组织基因表达谱差异的分析,并确定了球系列鞘糖脂生物合成通路(Glycosphinglipid biosynthesis-globo series)及通路中FUT1(α-(1,2)岩藻糖转移酶1).FUT2(α-(1,2)岩藻糖转移酶2).ST3GAL1(β-半乳糖苷a-2,3-唾液酸转移酶1)、HEXB(β-N-乙酰半乳糖胺酶B),HEXA(β-N-乙酰半乳糖胺酶A),B3GALNT1 (β-1,3- N-乙酰基半乳糖基转移酶1)和JAGA(N-乙酰基半乳糖苷酶A)等7个关键糖基转移酶基因对F18大肠杆菌病抗性具有调控作用。本研究进一步通过对35日龄仔猪肠道组织中球系列鞘糖脂生物合成通路中关键基因进行mRNA定量检测,并采用BSP(Bisulfite Sequencing PCR)+Miseq测序的方法,对十二指肠和空肠组织中这些基因启动子区的CpG岛的甲基化进行了超高精度的甲基化定量检测,同时分析了重要甲基化位点对基因mRNA表达量的影响和调控作用,进一步探讨仔猪对E.coli F18抗性的分子调控机制,为解决国内地方猪种E.coli F18抗性育种的关键科学问题提供一定的依据,并为今后利用生物工程手段调控猪E.coli F18抗性蛋白的活性提供一定的基础和依据。主要试验结果如下：1.通过荧光定量在仔猪E.coli F18抗性和敏感群体11个组织中检测球系列鞘糖脂生物合成通路中7个关键糖基化转移酶基因mRNA的表达量发现,7个基因在仔猪不同组织中的表达量具有一定的一致性,在脾、肝、肺、肾、胃及肠道组织中表达量较高,在胸腺和淋巴结等免疫组织中的表达量较低,在心和肌肉中几乎不表达；比较抗性和敏感群体肠道组织的表达量发现,FUT2基因在抗性群体十二指肠和空肠组织中的表达量极显著低于敏感性群体的表达量(P0.01),ST3GAL1基因在抗性群体十二指肠组织中的表达量显著低于敏感性群体的表达量(P0.05),其余基因在抗性和敏感群体间的表达量对比并未达到显著水平；肠道组织7个基因表达量的相关性分析表明,FUT1基因的表达量与FUT2、B3GALNT1、NAGA基因达到了显著正相关(P0.05),与ST3GAL1基因也几乎达到了显著相关(P=0.06),与HEXA基因和HEXB基因的相关性不显著；FUT2基因的表达量与FUT1、ST3GAL1、B3GALNT1基因达到了极显著正相关(P0.01),与HEXA、HEXB、NAGA基因的相关性不显著；以上结果说明,肠道组织中FUT1、FUT2基因的表达量的下调可以提高仔猪对Ecoli F18的抗性,同时,ST3GAL1基因、B3GALNT1基因、NAGA基因可能也参与到仔猪对E.coli抗性的调节过程中。2.通过比对人和猪Geneban k数据库中7个基因的序列信息发现,FUT1和B3GALNT1基因可能拥有2个可变启动子区,其余基因可能只含有1个启动子区；通过运用生物信息学技术挖掘课题组前期获得的仔猪转录组测序结果,确定了仔猪7个基因的转录起始位点和启动子区,其中FUT1基因转录起始区域具有5种可变剪切和2个启动子区域；进一步通过双荧光素酶报告基因技术检测FUTl基因2个启动子的转录活性,结果显示,FUT1基因启动子1的转录活性极显著的高于启动子2的转录活性(P0.01),启动子1在转录过程中起主导作用；通过生物信息学分析CpG岛,结果显示,仔猪FUTl启动子1以及FUT2、ST3GAL1、HEXA、HEXB和NAGA的启动子区的CpG岛的范围都在500bp以内,B3GALNT1基因启动子区没有CpG岛,且CpG位点数量也较少。3.通过BSP+Miseq的甲基化检测技术发现仔猪HEXA、HEXB、NAGA基因启动子区CpG岛未发生甲基化,而FUT1、FUT2、ST3GAL1基因启动子区CpG岛存在不同程度的甲基化；FUTl基因启动子区1CpG岛的甲基化与转录水平不存在显著的相关性,FUT2基因和ST3GAL1基因启动子区CpG岛的甲基化与转录存在一定程度的负相关；FUT2基因CpG岛上的mC-6、mC-22位点和ST3GAL1基因CpG岛上的nC-2、mC-8、mC-17位点与基因的表达量呈显著的负相关(P0.05),说明上述位点可能是调控转录的主要位点,其中FUT2基因mC-22位点和ST3GAL1基因的mC-8位点位于Spl转录因子结合位点内,推测这些位点的甲基化抑制了Spl转录因子与DNA的结合,从而影响到基因的表达,提高了仔猪对E.coli F18的抗性。
[Abstract]:Diarrhoea is the main disease causing piglet death, which has brought great economic loss to the pig industry..F18 (E.coli F18) strain is the main pathogen causing piglet diarrhea. Foreign studies have shown that the mutation of G/A in the 1 gene (FUT1) M307 loci of alpha (1,2) fucose transferase (FUT1) can be used as a genetic marker for controlling Ecoli F18 adhesion. According to this, the disease resistance breeding is realized in the foreign pig species, but the loci are extremely skewed in Chinese local pig populations, which makes the breeding practice of anti F18 colibacillosis in local local pigs is in trouble. By expressing the spectrum chip technology and proteomics technology, the resistance and sensitivity of the piglet colibacillosis in the earlier period. The gene expression profiles of duodenal tissues were analyzed, and the Glycosphinglipid biosynthesis-globo series and FUT1 (alpha (1,2) fucose transferase 1).FUT2 (alpha (1,2) fucose transferase 2).ST3GAL1 (beta galactoside a-2,3- sialic acid transfer) in the pathway was determined by the analysis of the differences in gene expression profiles in the duodenal tissue. Enzyme 1), HEXB (beta -N- acetyl galactoaminase B), HEXA (beta -N- galactoaminase A), B3GALNT1 (beta -1,3- N- acetyl galactosidase 1) and JAGA (N- acetylgalactosidase A), and other 7 key glycosyltransferase genes have a regulatory effect on the resistance to colibacillosis. This study was further studied in the intestinal tissue of 35 day old piglets. The key genes in the glycolipid biosynthesis pathway of the ball series were detected by mRNA, and the methylation of the methylation of the CpG islands in the promoter region of the duodenum and jejunum was quantified by BSP (Bisulfite Sequencing PCR) +Miseq sequencing. The important methylation site was analyzed. The effects of gene mRNA expression and regulation on the molecular regulation mechanism of the resistance of piglets to E.coli F18 provide a basis for solving the key scientific problems in the local swine E.coli F18 resistance breeding, and provide a certain basis for the future use of bioengineering methods to regulate the activity of the pig E.coli F18 resistance proteins. The main results are as follows: 1. the expression of the 7 key glycosyltransferase gene mRNA in the biosynthesis pathway of the ball series sheath glycolipid biosynthesis pathway was detected by fluorescence quantitative analysis in 11 tissues of E.coli F18 resistance and sensitive groups of piglets. The expression of 7 genes in different tissues of piglets had a certain consistency, in the spleen, liver, lung and kidney. The expression of the gastric and intestinal tissue is high, and the expression in the thymus and lymph nodes is low and almost not expressed in the heart and muscle. The expression of the FUT2 gene in the duodenum and jejunum tissues of the resistant and sensitive groups is significantly lower than that of the sensitive group. (P0.01), the expression of ST3GAL1 gene in the duodenal tissue of the resistant population was significantly lower than that of the sensitive group (P0.05). The expression of the other genes in the resistant and sensitive groups did not reach a significant level. The correlation analysis of the expression of 7 genes in the intestinal tissue showed that the expression of the FUT1 gene was associated with the FUT2, B3GALNT1, NAGA. The significant positive correlation (P0.05) was achieved (P=0.06), and the correlation between the gene and the HEXB gene was not significant. The expression of the FUT2 gene was significantly correlated with the FUT1, ST3GAL1, and B3GALNT1 genes (P0.01), and the correlation with HEXA, HEXB, and ST3GAL1 genes was not significant; the above results indicated that the intestine was in the intestines. The downregulation of the expression of FUT1 and FUT2 genes in the tract can increase the resistance to Ecoli F18 in piglets. Meanwhile, the ST3GAL1 gene, B3GALNT1 gene, and NAGA gene may also be involved in the regulation of the piglets' resistance to E.coli, and.2. can be found by comparing the sequence information of the 7 bases in the human and the Geneban K database. There are 2 variable promoter regions, and the remaining genes may contain only 1 promoter regions. By using bioinformatics technology, the transcriptional starting sites and promoter regions of the 7 genes of the piglets were determined by the sequencing of the early piglet transcriptional group obtained by the project group, of which the FUT1 gene transcriptional starting area had 5 variable shear and 2 promoters. Region; further using the double luciferase reporter gene technique to detect the transcriptional activity of the 2 promoter of the FUTl gene, the results showed that the transcriptional activity of the FUT1 gene promoter 1 was significantly higher than that of the promoter 2 (P0.01), and the promoter 1 played a leading role in the transcription process; the result of bioinformatics analysis of the island of CpG showed that the offspring could be used as a result. The range of CpG island in the promoter region of FUTl promoter 1 and FUT2, ST3GAL1, HEXA, HEXB and NAGA is within 500bp. There is no CpG island in the B3GALNT1 gene promoter region, and the number of CpG loci is less than that of the methylation detection technology. There are different degrees of methylation in the CpG island of the promoter region of the 1 gene; there is no significant correlation between the methylation and transcriptional levels of the 1CpG island in the promoter region of the FUTl gene. There is a negative correlation between the methylation and transcription of the CpG island of the promoter region of the ST3GAL1 gene and the ST3GAL1 gene, and the mC-6, mC-22, and ST3GAL1 genes on the CpG island of the FUT2 gene CpG. The nC-2, mC-8 and mC-17 loci on the island have a significant negative correlation with the gene expression (P0.05), indicating that the above locus may be the main locus for regulation of transcription, in which the mC-22 site of the FUT2 gene and the mC-8 site of the ST3GAL1 gene are located in the Spl transcription factor binding site, which suppresses the binding of these loci to the Spl transcription factor and DNA. Thus affecting the expression of genes and improving the resistance of piglets to E.coli F18.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

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