發(fā)布時(shí)間：2018-05-05 21:49

  本文選題：豬 + F18大腸桿菌��； 參考：《揚(yáng)州大學(xué)》2017年博士論文

【摘要】：腹瀉是導(dǎo)致斷奶仔豬死亡的重要傳染性疾病,對(duì)養(yǎng)豬業(yè)造成了巨大的經(jīng)濟(jì)損失,F18大腸桿菌(E.coliF18)菌株是引起斷奶仔豬細(xì)菌性腹瀉的主要病原菌之一。為了進(jìn)一步揭示中國(guó)地方豬品種斷奶仔豬抗E.coli F18的遺傳基礎(chǔ)和調(diào)控機(jī)制,本研究以地方品種—梅山豬為研究對(duì)象,利用不同血清型F18大腸桿菌F18ab和F18ac菌株口服攻毒試驗(yàn),并結(jié)合仔豬腸道E.coliF18細(xì)菌檢測(cè)、細(xì)菌計(jì)數(shù)以及小腸上皮細(xì)胞黏附等一系列試驗(yàn)驗(yàn)證,篩選出確證的梅山豬F18大腸桿菌抗性與敏感型斷奶仔豬個(gè)體,進(jìn)一步利用RNA-seq轉(zhuǎn)錄組和長(zhǎng)鏈非編碼RNA(lncRNA)測(cè)序?qū)γ飞截iF18大腸桿菌抗性相關(guān)調(diào)控通路、功能基因以及l(fā)ncRNA進(jìn)行了系統(tǒng)篩選,并從mRNA、蛋白質(zhì)以及細(xì)胞水平分別對(duì)重要調(diào)控通路、關(guān)鍵基因以及l(fā)ncRNA進(jìn)行系統(tǒng)的功能驗(yàn)證,以期揭示功能基因及l(fā)ncRNA在梅山斷奶仔豬F18大腸桿菌抗性過程中的調(diào)控作用及其分子機(jī)制,為解決國(guó)內(nèi)地方豬種E.coli F18抗性育種關(guān)鍵科學(xué)問題提供一定的理論參考。此外,本研究同時(shí)以培育品種—蘇太豬(杜洛克X梅山豬)為研究對(duì)象,基于課題組前期建立的蘇太豬F18大腸桿菌抗性與敏感型資源群體,利用高通量測(cè)序分析了蘇太豬F18大腸桿菌抗性相關(guān)的調(diào)控通路以及候選基因,結(jié)合關(guān)于外來豬品種F18大腸桿菌抗性基因的文獻(xiàn)挖掘結(jié)果,進(jìn)一步探討和驗(yàn)證外來豬品種F18大腸桿菌抗性相關(guān)的調(diào)控通路以及候選基因,以期揭示中外豬品種F18大腸桿菌抗性調(diào)控遺傳基礎(chǔ)的差異性。主要研究結(jié)果如下:1.梅山斷奶仔豬E..coli F18敏感和抗性型個(gè)體間十二指腸轉(zhuǎn)錄組分析(1)梅山斷奶仔豬F18大腸桿菌抗性與敏感型個(gè)體間篩選出198個(gè)差異表達(dá)基因DGEs,其中抗性組上調(diào)基因125個(gè),大部分DGEs涉及到免疫系統(tǒng)“Immune System”和感染性疾病“Infectious Diseases”通路,其中篩選出一個(gè)富集程度較高的免疫通路—Toll樣受體信號(hào)通路(Toll-likereceptor signaling pathway)以及通路中關(guān)鍵的基因CD14。(2)脂多糖(LPS)誘導(dǎo)小腸上皮細(xì)胞IPEC-J2后,qPCR和western blot檢測(cè)發(fā)現(xiàn)Toll樣受體信號(hào)通路中大部分基因(CD14、TLR4、IL-1β、ERK、IFN-α、JNK、p38、NFkB和TNFF-α)表達(dá)水平均表現(xiàn)出明顯的上調(diào),表明Toll樣受體信號(hào)通路在調(diào)控F18大腸桿菌感染過程中確實(shí)發(fā)揮重要的作用。(3)免疫組化IHC結(jié)果表明,CD14在腸道組織中廣泛分布,并且抗性組中表達(dá)水平明顯高于敏感組;利用RNAi沉默CD14基因后,F18ab菌毛與IPEC-J2黏附能力極顯著上升(P0.01),而F18ac菌毛與IPEC-J2黏附能力上升但不顯著(P0.05);通路中IL-1、IFN-α、TLR4和TNF-α基因表達(dá)水平均發(fā)生顯著下調(diào)(P0.05),而MyD88基因表達(dá)水平下調(diào)但不顯著(P0.05);白細(xì)胞介素(IL-6和IL-12)因子表達(dá)水平顯著下降(P0.05),而IL-8、MIP-1α和MIP-1p表達(dá)量降低但未達(dá)到顯著水平(P0.05)。以上結(jié)果表明CD14表達(dá)水平上調(diào)有利于提高F18大腸桿菌抗性。2.蘇太斷奶仔豬五.coli F18敏感和抗性型個(gè)體間十二指腸轉(zhuǎn)錄組分析(1)蘇太斷奶仔豬F18大腸桿菌抗性與敏感型個(gè)體間篩選出238個(gè)差異表達(dá)基因DGEs,其中抗性組上調(diào)基因112個(gè),DGEs涉及到免疫相關(guān)通路如抗原加工與呈遞(Antigen processing and presentation)、Toll 樣受體信號(hào)通路(Toll-like receptor signaling pathway),其中包括TAP2、TLR5、IL1β基因;以及鞘糖脂合成通路(Glycosphingolipidbiosynthesis-lacto andneolacto series),其中包括具有重要生物學(xué)功能的FUT2基因。(2)LPS誘導(dǎo)以及不同血清型F18大腸桿菌F18ac、F18ab分別刺激小腸上皮細(xì)胞IPEC-J2后,qPCR和Western blot檢測(cè)FUT2、TAPP2、IL-1β和TLR5表達(dá)水平均表現(xiàn)為明顯的上調(diào);組織表達(dá)譜檢測(cè)表明,FUT2基因在肝臟、脾臟、肺、腎臟、胃、淋巴結(jié)、胸腺、十二指腸和空腸等組織中均有表達(dá),其中腸道組織尤其是十二指腸表達(dá)量相對(duì)較高,qPCR和western blot檢測(cè)表明,敏感組個(gè)體十二指腸和空腸組織中FUT2基因表達(dá)水平均極顯著高于抗性組(P0.01);利用RNAi沉默F(xiàn)UT2基因后,IPEC-J2細(xì)胞對(duì)大腸桿菌F18ab和F18ac的黏附能力均顯著下降(P0.05),以上結(jié)果表明FUT2基因表達(dá)水平下調(diào)有利于提高F18大腸桿菌抗性。(3)FUT2啟動(dòng)子區(qū)甲基化分析表明,22個(gè)CpG位點(diǎn)存在不同程度的甲基化,mC-6和mC-22位點(diǎn)甲基化水平與mRNA表達(dá)存在顯著負(fù)相關(guān)(P0.05),其中mC-22位于Sp1轉(zhuǎn)錄因子結(jié)合位點(diǎn)上;凝膠遷移EMSA試驗(yàn)表明,十二指腸組織中核蛋白與FUT2野生型非甲基化探針結(jié)合,加入Sp1抗體后,非甲基化野生型探針出現(xiàn)了超遷移(supershift)現(xiàn)象,表明核蛋白中Sp1轉(zhuǎn)錄因子可以特異性結(jié)合FUT2野生型非甲基化探針,以上結(jié)果表明,FUT2啟動(dòng)子區(qū)mC-22位點(diǎn)甲基化修飾抑制了 Sp1轉(zhuǎn)錄因子與啟動(dòng)子DNA結(jié)合,降低了FUT2基因表達(dá)水平,進(jìn)而提升了 F18大腸桿菌抗性。3.梅山與蘇太斷奶仔豬E.coli F18敏感和抗性型個(gè)體間十二指腸長(zhǎng)鏈非編碼RNA分析(1)lncRNA測(cè)序結(jié)合生物信息學(xué)分析在梅山豬和蘇太豬中共獲得2056個(gè)候選lncRNA分子,根據(jù)p-value0.05原則,梅山斷奶仔豬F18大腸桿菌抗性型與敏感型個(gè)體之間存在24個(gè)差異表達(dá)lncRNA,其中抗性組中上調(diào)21個(gè);蘇太豬存在23個(gè)差異表達(dá)lncRNA,其中抗性組中上調(diào)7個(gè)。基于cis和trans機(jī)制進(jìn)行靶基因預(yù)測(cè)發(fā)現(xiàn),cis機(jī)制預(yù)測(cè)出梅山豬所有差異表達(dá)lncRNA上下游共存在59個(gè)可能靶基因,而蘇太豬所有差異表達(dá)lncRNA上下游存在67個(gè)可能靶基因;trans機(jī)制預(yù)測(cè)出梅山豬RNA-seq中存在517個(gè)基因與差異lncRNA存在顯著的表達(dá)相關(guān)性,而蘇太豬RNA-seq中存在96個(gè)基因與差異lncRNA存在顯著的表達(dá)相關(guān)性。靶基因參與GO功能以及KEGG通路分析表明,靶基因涉及到信號(hào)傳導(dǎo)通路(如 NF-kappaB signaling pathway)、免疫相關(guān)通路(如 Toll-like receptor signaling pathway)、疾病感染通路(如Salmonella infection)、糖脂類合成相關(guān)通路(如Glycosaminoglycanbiosynthesis-KS)等。(2)梅山豬與蘇太豬抗性與敏感型個(gè)體十二指腸間存在3個(gè)共同差異表達(dá)lncRNA:TCONS_0183659、TCONS_00352975、TCONS_00053650;結(jié)合靶基因預(yù)測(cè)及其功能分析,篩選出一個(gè)與F18大腸桿菌抗性相關(guān)的重要lncRNA—TCONS_00183659,其序列100 kb范圍內(nèi)發(fā)現(xiàn)一個(gè)可能靶基因FUT3。TCONS_00183659位于豬2號(hào)染色體,長(zhǎng)度為5831 bp,具有兩個(gè)exon(5746 bp和85 bp),是不連續(xù)的,屬于基因間的lncRNA。(3)qPCR檢測(cè)結(jié)果表明,TCONS_00183659在梅山豬和蘇太豬F18抗性組個(gè)體十二指腸組織中表達(dá)水平極顯著高于敏感組個(gè)體(P0.01);熒光原位雜交技術(shù)FISH分析表明,TCONS_00183659在細(xì)胞核和細(xì)胞質(zhì)中均有分布;成功建立沉默TCONS_00183659豬小腸上皮細(xì)胞IPEC-J2系,干擾效率達(dá)到69.58%;RNA pull down結(jié)合western blot驗(yàn)證表明,TCONS_00183659 與 Histone H4 存在相互作用;TCONS_00183659 干擾前后 IPEC-J2蛋白組學(xué)結(jié)合western blot驗(yàn)證分析表明,干擾TCONS_00053650后Mx1、Mx2、IFIT2蛋白表達(dá)升高。以上結(jié)果表明,TCONS_00183659可能通過與組蛋白Histone H4結(jié)合,從而調(diào)控Mx1、Mx2、IFIT2蛋白表達(dá)。結(jié)合前期國(guó)內(nèi)外相關(guān)研究結(jié)果,本研究進(jìn)一步揭示了中外豬品種F18大腸桿菌抗性調(diào)控的遺傳基礎(chǔ)確實(shí)存在差異,Toll樣受體信號(hào)通路及其CD14基因在梅山豬抵抗F18大腸桿菌感染過程中發(fā)揮著免疫調(diào)控作用,其中CD14基因表達(dá)上調(diào)有利于提高仔豬對(duì)E.coli F18感染抗性;而鞘糖脂合成通路及其FUT2基因可能在外來豬品種F18大腸桿菌受體形成過程中起關(guān)鍵作用,其中FUT2基因表達(dá)下調(diào)有利于提高仔豬對(duì)E.colE.F18感染抗性。此外,本研究系統(tǒng)揭示了長(zhǎng)鏈非編碼RNA在斷奶仔豬F18大腸桿菌抗性過程中的調(diào)控機(jī)制,篩選出1個(gè)關(guān)鍵lncRNA:TCONS_00183659,并且推測(cè)該lncRNA可能通過與組蛋白Histone H4結(jié)合,從而調(diào)控Mx1、Mx2、IFIT2蛋白表達(dá),今后需要進(jìn)一步驗(yàn)證TCONS_00183659調(diào)控作用是否與Histone H4表觀遺傳修飾有關(guān)。
[Abstract]:Diarrhoea is an important infectious disease that leads to the death of weanling piglets, causing huge economic losses to the pig industry. F18 Escherichia coli (E.coliF18) strain is one of the main pathogens causing bacterial diarrhea in weanling piglets. In order to further reveal the genetic basis and regulation mechanism of the Chinese local pig breaned piglets against E.coli F18, the research on the genetic basis and regulation mechanism of the Chinese local pig breeds piglets has been further revealed. Taking the local breed - Meishan pig as the research object, using different serotype F18 Escherichia coli F18ab and F18ac strains oral attack test, combined with a series of tests of intestinal E.coliF18 bacteria detection, bacterial count and intestinal epithelial cell adhesion, screening out the corroborated resistance and sensitive weanling of F18 Escherichia coli in Meishan pigs. Pig individuals, further using RNA-seq transcriptome and long chain non coded RNA (lncRNA) sequencing, systematically screened the regulation pathway of resistance to Escherichia coli in Meishan pigs, functional genes and lncRNA, and verified the important regulatory pathways, key genes and lncRNA from mRNA, protein and cell levels, respectively. The regulatory role of functional genes and lncRNA in the resistance process of F18 colibacilli in Meishan weaned piglets and its molecular mechanism provide a certain theoretical reference for solving the key scientific problems of E.coli F18 resistance breeding in domestic pigs. In addition, this study is based on the cultivation of Sutai pig (Durok X Meishan pig) as the research object and based on the study. The group of Escherichia coli resistance and sensitive type of Sutai pig F18 was established by the project group. The regulation pathway and candidate genes related to the resistance of F18 Escherichia coli in Sutai pig were analyzed by high throughput sequencing, and the results of literature mining on the resistance genes of foreign pig breeds F18 Escherichia coli were combined to further explore and verify the foreign pig breeds, F18 The regulation pathway and candidate genes related to the resistance of Escherichia coli to reveal the differences in the genetic basis of resistance regulation of F18 Escherichia coli in Chinese and foreign pigs. The main results are as follows: 1. E..Coli F18 sensitive and resistant interindividual duodenal transcriptome analysis in Meishan weaned piglets (1) resistance and sensitivity of F18 colibacilli in weanling piglets 198 differentially expressed genes DGEs were screened among the individuals, of which 125 were up-regulated in the resistant group, and most of the DGEs involved the immune system "Immune System" and the infectious disease "Infectious Diseases" pathway, in which a high enrichment of the immune pathway, Toll like receptor signaling pathway (Toll-likereceptor signaling P) was screened. Athway) and the key gene CD14. (2) LPS (LPS) induced IPEC-J2 in the small intestinal epithelial cells. QPCR and Western blot detected that most of the genes in the Toll like receptor signaling pathway (CD14, TLR4, IL-1 beta, ERK, alpha, IPEC-J2, and alpha) showed obvious up-regulation, indicating that the receptor signaling pathway is in the modulation. In the process of controlling F18 Escherichia coli infection, it did play an important role. (3) immuno histochemical IHC results showed that CD14 was widely distributed in the intestinal tissue, and the expression level in the resistant group was significantly higher than that in the sensitive group. The adhesion ability of F18ab pilus to IPEC-J2 was significantly increased (P0.01) after the RNAi silencing of CD14 gene, and F18ac pilus and IPEC-J2 adhesion energy were found. The expression level of IL-1, IFN-, TLR4 and TNF- in the pathway decreased significantly (P0.05), but the expression level of MyD88 gene was down but not significantly (P0.05), and the level of the expression of interleukin (IL-6 and IL-12) factor decreased significantly (P0.05), while IL-8, IFN- and TNF- were decreased but not significant. The above results showed that the up regulation of CD14 expression was beneficial to the analysis of the five.Coli F18 sensitive and resistant interindividual duodenal transcriptome analysis of F18 Escherichia coli resistance of.2. Suzhou weanling piglets. (1) 238 differentially expressed DGEs were screened from F18 Escherichia coli resistance and susceptible individuals in Sutai weanling piglets, and 112 of the genes were up regulated in the resistant group. DGEs involves immune related pathways such as antigen processing and presentation (Antigen processing and presentation), Toll like receptor signaling pathway (Toll-like receptor signaling pathway), including TAP2, TLR5, beta gene, and sheath glycolipid pathway. The FUT2 gene of biological function. (2) LPS induction and different serotype F18 Escherichia coli F18ac, F18ab stimulated small intestinal epithelial cells IPEC-J2, qPCR and Western blot detected FUT2, TAPP2, IL-1 beta and expression levels were all obviously up-regulated; tissue expression profiles showed that the gene was in the liver, spleen, lung, kidneys, stomach, lymph, and lymph QPCR and Western blot detection showed that the expression level of FUT2 gene in the duodenum and jejunum tissues of the sensitive group was significantly higher than that in the resistant group (P0.01), and IPEC-J2 cells were used to silence the FUT2 gene by RNAi. The adhesion ability of Escherichia coli F18ab and F18ac decreased significantly (P0.05). The above results showed that the downregulation of FUT2 gene expression level was beneficial to the resistance to F18 Escherichia coli. (3) methylation analysis of FUT2 promoter region showed that there were different degrees of methylation in the 22 CpG loci, and there was a significant negative correlation between the level of mC-6 and mC-22 locus methylation and mRNA expression. (P0.05), in which mC-22 was located on the Sp1 transcription factor binding site, and the gel migration EMSA test showed that the nuclear protein in the duodenum was combined with the FUT2 wild type non methylation probe and added to the Sp1 antibody, and the non methylation wild type probe appeared hyper migration (supershift) image, indicating that the Sp1 transcription factor in the nuclear protein could be specifically combined with FUT2. The results of the wild type non methylation probe showed that the methylation modification at the mC-22 site of FUT2 promoter inhibited the binding of Sp1 transcription factors to promoter DNA, reduced the expression level of FUT2 gene, and then enhanced the F18 sensitivity and the non coding of the long chain duodenal long chain between F18 Escherichia coli resistance.3. Meishan and Sutai weanling piglets. RNA analysis (1) lncRNA sequencing combined with bioinformatics analysis of 2056 candidate lncRNA molecules in Meishan pigs and Sutai pigs. According to the p-value0.05 principle, there were 24 differential expressions of lncRNA between the F18 Escherichia coli resistant and sensitive individuals in Meishan weaned piglets, of which 21 were up-regulated in the resistance group, and 23 different lncR expressed lncR in Sutai pigs. NA was up to 7 in the resistance group. The target gene prediction based on the CIS and trans mechanism found that the CIS mechanism predicted that there were 59 possible target genes in the upper and lower reaches of the Meishan pig, while all the differences expressed in the Sutai pigs had 67 possible target genes in the upper and lower reaches of lncRNA, and the trans mechanism predicted that there were 517 bases in the RNA-seq of Meishan pigs. There is a significant correlation with the difference lncRNA, and there is a significant correlation between the 96 genes in Sutai pig RNA-seq and the difference lncRNA. The target genes involved in the GO function and the KEGG pathway analysis show that the target genes are involved in the signal transduction pathway (such as NF-kappaB signaling pathway) and the immune related pathway (Toll-like receptor s). Ignaling pathway), disease infection pathways (such as Salmonella infection), glycolipid synthesis related pathways (such as Glycosaminoglycanbiosynthesis-KS), etc. (2) there are 3 common differences in the expression of lncRNA: TCONS_0183659, TCONS_00352975, and TCONS_00053650 in the duodenum of the resistant and sensitive individuals of Meishan and Sutai pigs. Functional analysis, screening an important lncRNA - TCONS_00183659 related to F18 resistance to Escherichia coli. The sequence of 100 kb in the range of 100 kb was found to be located on the porcine chromosome 2, the length of 5831 BP, with two exon (5746 BP and 85 BP), which was discontinuous, and belonged to the lncRNA. (3) qPCR detection result table between genes. The expression level of TCONS_00183659 in the F18 resistance group of Meishan pig and Sutai pig was significantly higher than that of the sensitive group (P0.01). The fluorescence in situ hybridization technique FISH analysis showed that TCONS_00183659 was distributed in the nucleus and cytoplasm, and the IPEC-J2 system of the small intestinal epithelial cells of the silent TCONS_00183659 pig was successfully established. The rate reached 69.58%; RNA pull down combined with Western blot showed that TCONS_00183659 had interaction with Histone H4; IPEC-J2 proteomics before and after TCONS_00183659 interference combined Western blot validation analysis showed that the protein expression increased after interference. Protein Histone H4 binding, which regulates the expression of Mx1, Mx2, and IFIT2 protein. Combined with the previous research results at home and abroad, this study further reveals that the genetic basis of the resistance regulation of F18 Escherichia coli in Chinese and foreign pig breeds is indeed different. The Toll like receptor signaling pathway and its CD14 gene play a role in the resistance of Meishan pigs to the infection of F18 Escherichia coli. The up regulation of CD14 gene expression is beneficial to improve the resistance to E.coli F18 infection in piglets, and the sheath glycolipid synthesis pathway and its FUT2 gene may play a key role in the formation of F18 Escherichia coli in foreign pig breeds, and the down regulation of FUT2 gene expression is beneficial to improve the resistance to E.colE.F18 infection in piglets. The research system reveals the regulation mechanism of long chain non coding RNA in the process of F18 colibacilli resistance in weanling piglets, screening 1 key lncRNA:TCONS_00183659, and speculates that the lncRNA may be combined with histone Histone H4 to regulate the expression of Mx1, Mx2, and IFIT2 protein. The role of TCONS_00183659 regulation is further verified in the future. It is not related to epigenetic modification of Histone H4.

【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.28

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