本文選題：豬 + E.coli�。� 參考：《揚(yáng)州大學(xué)》2015年碩士論文

【摘要】：腹瀉是導(dǎo)致仔豬死亡的主要疾病,給養(yǎng)豬業(yè)帶來了巨大經(jīng)濟(jì)損失。F18大腸桿菌(E.coli F18)菌株是引起仔豬腹瀉的主要病原菌。國外研究表明,α-(1,2)巖藻糖轉(zhuǎn)移酶1基因(FUT1)M307位點存在G/A的突變,該突變可以作為控制Ecoli F18粘附的遺傳標(biāo)記,并據(jù)此在國外豬種中實現(xiàn)了抗病育種,但是該位點在中國地方豬種群體中呈極端偏態(tài)分布,使得國內(nèi)地方豬種抗F18大腸桿菌病育種實踐陷入了困境。課題前期通過表達(dá)譜芯片技術(shù)、蛋白質(zhì)組學(xué)技術(shù),對仔豬大腸桿菌病抗性型和敏感型全同胞配對個體,進(jìn)行了十二指腸組織基因表達(dá)譜差異的分析,并確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)巖藻糖轉(zhuǎn)移酶1).FUT2(α-(1,2)巖藻糖轉(zhuǎn)移酶2).ST3GAL1(β-半乳糖苷a-2,3-唾液酸轉(zhuǎn)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-乙酰基半乳糖基轉(zhuǎn)移酶1)和JAGA(N-乙�；肴樘擒彰窤)等7個關(guān)鍵糖基轉(zhuǎn)移酶基因?qū)18大腸桿菌病抗性具有調(diào)控作用。本研究進(jìn)一步通過對35日齡仔豬腸道組織中球系列鞘糖脂生物合成通路中關(guān)鍵基因進(jìn)行mRNA定量檢測,并采用BSP(Bisulfite Sequencing PCR)+Miseq測序的方法,對十二指腸和空腸組織中這些基因啟動子區(qū)的CpG島的甲基化進(jìn)行了超高精度的甲基化定量檢測,同時分析了重要甲基化位點對基因mRNA表達(dá)量的影響和調(diào)控作用,進(jìn)一步探討仔豬對E.coli F18抗性的分子調(diào)控機(jī)制,為解決國內(nèi)地方豬種E.coli F18抗性育種的關(guān)鍵科學(xué)問題提供一定的依據(jù),并為今后利用生物工程手段調(diào)控豬E.coli F18抗性蛋白的活性提供一定的基礎(chǔ)和依據(jù)。主要試驗結(jié)果如下：1.通過熒光定量在仔豬E.coli F18抗性和敏感群體11個組織中檢測球系列鞘糖脂生物合成通路中7個關(guān)鍵糖基化轉(zhuǎn)移酶基因mRNA的表達(dá)量發(fā)現(xiàn),7個基因在仔豬不同組織中的表達(dá)量具有一定的一致性,在脾、肝、肺、腎、胃及腸道組織中表達(dá)量較高,在胸腺和淋巴結(jié)等免疫組織中的表達(dá)量較低,在心和肌肉中幾乎不表達(dá)；比較抗性和敏感群體腸道組織的表達(dá)量發(fā)現(xiàn),FUT2基因在抗性群體十二指腸和空腸組織中的表達(dá)量極顯著低于敏感性群體的表達(dá)量(P0.01),ST3GAL1基因在抗性群體十二指腸組織中的表達(dá)量顯著低于敏感性群體的表達(dá)量(P0.05),其余基因在抗性和敏感群體間的表達(dá)量對比并未達(dá)到顯著水平；腸道組織7個基因表達(dá)量的相關(guān)性分析表明,FUT1基因的表達(dá)量與FUT2、B3GALNT1、NAGA基因達(dá)到了顯著正相關(guān)(P0.05),與ST3GAL1基因也幾乎達(dá)到了顯著相關(guān)(P=0.06),與HEXA基因和HEXB基因的相關(guān)性不顯著；FUT2基因的表達(dá)量與FUT1、ST3GAL1、B3GALNT1基因達(dá)到了極顯著正相關(guān)(P0.01),與HEXA、HEXB、NAGA基因的相關(guān)性不顯著；以上結(jié)果說明,腸道組織中FUT1、FUT2基因的表達(dá)量的下調(diào)可以提高仔豬對Ecoli F18的抗性,同時,ST3GAL1基因、B3GALNT1基因、NAGA基因可能也參與到仔豬對E.coli抗性的調(diào)節(jié)過程中。2.通過比對人和豬Geneban k數(shù)據(jù)庫中7個基因的序列信息發(fā)現(xiàn),FUT1和B3GALNT1基因可能擁有2個可變啟動子區(qū),其余基因可能只含有1個啟動子區(qū)；通過運用生物信息學(xué)技術(shù)挖掘課題組前期獲得的仔豬轉(zhuǎn)錄組測序結(jié)果,確定了仔豬7個基因的轉(zhuǎn)錄起始位點和啟動子區(qū),其中FUT1基因轉(zhuǎn)錄起始區(qū)域具有5種可變剪切和2個啟動子區(qū)域；進(jìn)一步通過雙熒光素酶報告基因技術(shù)檢測FUTl基因2個啟動子的轉(zhuǎn)錄活性,結(jié)果顯示,FUT1基因啟動子1的轉(zhuǎn)錄活性極顯著的高于啟動子2的轉(zhuǎn)錄活性(P0.01),啟動子1在轉(zhuǎn)錄過程中起主導(dǎo)作用；通過生物信息學(xué)分析CpG島,結(jié)果顯示,仔豬FUTl啟動子1以及FUT2、ST3GAL1、HEXA、HEXB和NAGA的啟動子區(qū)的CpG島的范圍都在500bp以內(nèi),B3GALNT1基因啟動子區(qū)沒有CpG島,且CpG位點數(shù)量也較少。3.通過BSP+Miseq的甲基化檢測技術(shù)發(fā)現(xiàn)仔豬HEXA、HEXB、NAGA基因啟動子區(qū)CpG島未發(fā)生甲基化,而FUT1、FUT2、ST3GAL1基因啟動子區(qū)CpG島存在不同程度的甲基化；FUTl基因啟動子區(qū)1CpG島的甲基化與轉(zhuǎn)錄水平不存在顯著的相關(guān)性,FUT2基因和ST3GAL1基因啟動子區(qū)CpG島的甲基化與轉(zhuǎn)錄存在一定程度的負(fù)相關(guān)；FUT2基因CpG島上的mC-6、mC-22位點和ST3GAL1基因CpG島上的nC-2、mC-8、mC-17位點與基因的表達(dá)量呈顯著的負(fù)相關(guān)(P0.05),說明上述位點可能是調(diào)控轉(zhuǎn)錄的主要位點,其中FUT2基因mC-22位點和ST3GAL1基因的mC-8位點位于Spl轉(zhuǎn)錄因子結(jié)合位點內(nèi),推測這些位點的甲基化抑制了Spl轉(zhuǎn)錄因子與DNA的結(jié)合,從而影響到基因的表達(dá),提高了仔豬對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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.28

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本文編號：2032717