断奶仔猪F18大肠杆菌抗性基因和lncRNA的筛选与调控机制分析

发布时间：2018-05-05 21:49

本文选题：猪 + F18大肠杆菌　；参考：《扬州大学》2017年博士论文

【摘要】：腹泻是导致断奶仔猪死亡的重要传染性疾病,对养猪业造成了巨大的经济损失,F18大肠杆菌(E.coliF18)菌株是引起断奶仔猪细菌性腹泻的主要病原菌之一。为了进一步揭示中国地方猪品种断奶仔猪抗E.coli F18的遗传基础和调控机制,本研究以地方品种—梅山猪为研究对象,利用不同血清型F18大肠杆菌F18ab和F18ac菌株口服攻毒试验,并结合仔猪肠道E.coliF18细菌检测、细菌计数以及小肠上皮细胞黏附等一系列试验验证,筛选出确证的梅山猪F18大肠杆菌抗性与敏感型断奶仔猪个体,进一步利用RNA-seq转录组和长链非编码RNA(lncRNA)测序对梅山猪F18大肠杆菌抗性相关调控通路、功能基因以及lncRNA进行了系统筛选,并从mRNA、蛋白质以及细胞水平分别对重要调控通路、关键基因以及lncRNA进行系统的功能验证,以期揭示功能基因及lncRNA在梅山断奶仔猪F18大肠杆菌抗性过程中的调控作用及其分子机制,为解决国内地方猪种E.coli F18抗性育种关键科学问题提供一定的理论参考。此外,本研究同时以培育品种—苏太猪(杜洛克X梅山猪)为研究对象,基于课题组前期建立的苏太猪F18大肠杆菌抗性与敏感型资源群体,利用高通量测序分析了苏太猪F18大肠杆菌抗性相关的调控通路以及候选基因,结合关于外来猪品种F18大肠杆菌抗性基因的文献挖掘结果,进一步探讨和验证外来猪品种F18大肠杆菌抗性相关的调控通路以及候选基因,以期揭示中外猪品种F18大肠杆菌抗性调控遗传基础的差异性。主要研究结果如下:1.梅山断奶仔猪E..coli F18敏感和抗性型个体间十二指肠转录组分析(1)梅山断奶仔猪F18大肠杆菌抗性与敏感型个体间筛选出198个差异表达基因DGEs,其中抗性组上调基因125个,大部分DGEs涉及到免疫系统“Immune System”和感染性疾病“Infectious Diseases”通路,其中筛选出一个富集程度较高的免疫通路—Toll样受体信号通路(Toll-likereceptor signaling pathway)以及通路中关键的基因CD14。(2)脂多糖(LPS)诱导小肠上皮细胞IPEC-J2后,qPCR和western blot检测发现Toll样受体信号通路中大部分基因(CD14、TLR4、IL-1β、ERK、IFN-α、JNK、p38、NFkB和TNFF-α)表达水平均表现出明显的上调,表明Toll样受体信号通路在调控F18大肠杆菌感染过程中确实发挥重要的作用。(3)免疫组化IHC结果表明,CD14在肠道组织中广泛分布,并且抗性组中表达水平明显高于敏感组;利用RNAi沉默CD14基因后,F18ab菌毛与IPEC-J2黏附能力极显著上升(P0.01),而F18ac菌毛与IPEC-J2黏附能力上升但不显著(P0.05);通路中IL-1、IFN-α、TLR4和TNF-α基因表达水平均发生显著下调(P0.05),而MyD88基因表达水平下调但不显著(P0.05);白细胞介素(IL-6和IL-12)因子表达水平显著下降(P0.05),而IL-8、MIP-1α和MIP-1p表达量降低但未达到显著水平(P0.05)。以上结果表明CD14表达水平上调有利于提高F18大肠杆菌抗性。2.苏太断奶仔猪五.coli F18敏感和抗性型个体间十二指肠转录组分析(1)苏太断奶仔猪F18大肠杆菌抗性与敏感型个体间筛选出238个差异表达基因DGEs,其中抗性组上调基因112个,DGEs涉及到免疫相关通路如抗原加工与呈递(Antigen processing and presentation)、Toll 样受体信号通路(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学功能的FUT2基因。(2)LPS诱导以及不同血清型F18大肠杆菌F18ac、F18ab分别刺激小肠上皮细胞IPEC-J2后,qPCR和Western blot检测FUT2、TAPP2、IL-1β和TLR5表达水平均表现为明显的上调;组织表达谱检测表明,FUT2基因在肝脏、脾脏、肺、肾脏、胃、淋巴结、胸腺、十二指肠和空肠等组织中均有表达,其中肠道组织尤其是十二指肠表达量相对较高,qPCR和western blot检测表明,敏感组个体十二指肠和空肠组织中FUT2基因表达水平均极显著高于抗性组(P0.01);利用RNAi沉默FUT2基因后,IPEC-J2细胞对大肠杆菌F18ab和F18ac的黏附能力均显著下降(P0.05),以上结果表明FUT2基因表达水平下调有利于提高F18大肠杆菌抗性。(3)FUT2启动子区甲基化分析表明,22个CpG位点存在不同程度的甲基化,mC-6和mC-22位点甲基化水平与mRNA表达存在显著负相关(P0.05),其中mC-22位于Sp1转录因子结合位点上;凝胶迁移EMSA试验表明,十二指肠组织中核蛋白与FUT2野生型非甲基化探针结合,加入Sp1抗体后,非甲基化野生型探针出现了超迁移(supershift)现象,表明核蛋白中Sp1转录因子可以特异性结合FUT2野生型非甲基化探针,以上结果表明,FUT2启动子区mC-22位点甲基化修饰抑制了 Sp1转录因子与启动子DNA结合,降低了FUT2基因表达水平,进而提升了 F18大肠杆菌抗性。3.梅山与苏太断奶仔猪E.coli F18敏感和抗性型个体间十二指肠长链非编码RNA分析(1)lncRNA测序结合生物信息学分析在梅山猪和苏太猪中共获得2056个候选lncRNA分子,根据p-value0.05原则,梅山断奶仔猪F18大肠杆菌抗性型与敏感型个体之间存在24个差异表达lncRNA,其中抗性组中上调21个;苏太猪存在23个差异表达lncRNA,其中抗性组中上调7个。基于cis和trans机制进行靶基因预测发现,cis机制预测出梅山猪所有差异表达lncRNA上下游共存在59个可能靶基因,而苏太猪所有差异表达lncRNA上下游存在67个可能靶基因;trans机制预测出梅山猪RNA-seq中存在517个基因与差异lncRNA存在显著的表达相关性,而苏太猪RNA-seq中存在96个基因与差异lncRNA存在显著的表达相关性。靶基因参与GO功能以及KEGG通路分析表明,靶基因涉及到信号传导通路(如 NF-kappaB signaling pathway)、免疫相关通路(如 Toll-like receptor signaling pathway)、疾病感染通路(如Salmonella infection)、糖脂类合成相关通路(如Glycosaminoglycanbiosynthesis-KS)等。(2)梅山猪与苏太猪抗性与敏感型个体十二指肠间存在3个共同差异表达lncRNA:TCONS_0183659、TCONS_00352975、TCONS_00053650;结合靶基因预测及其功能分析,筛选出一个与F18大肠杆菌抗性相关的重要lncRNA—TCONS_00183659,其序列100 kb范围内发现一个可能靶基因FUT3。TCONS_00183659位于猪2号染色体,长度为5831 bp,具有两个exon(5746 bp和85 bp),是不连续的,属于基因间的lncRNA。(3)qPCR检测结果表明,TCONS_00183659在梅山猪和苏太猪F18抗性组个体十二指肠组织中表达水平极显著高于敏感组个体(P0.01);荧光原位杂交技术FISH分析表明,TCONS_00183659在细胞核和细胞质中均有分布;成功建立沉默TCONS_00183659猪小肠上皮细胞IPEC-J2系,干扰效率达到69.58%;RNA pull down结合western blot验证表明,TCONS_00183659 与 Histone H4 存在相互作用;TCONS_00183659 干扰前后 IPEC-J2蛋白组学结合western blot验证分析表明,干扰TCONS_00053650后Mx1、Mx2、IFIT2蛋白表达升高。以上结果表明,TCONS_00183659可能通过与组蛋白Histone H4结合,从而调控Mx1、Mx2、IFIT2蛋白表达。结合前期国内外相关研究结果,本研究进一步揭示了中外猪品种F18大肠杆菌抗性调控的遗传基础确实存在差异,Toll样受体信号通路及其CD14基因在梅山猪抵抗F18大肠杆菌感染过程中发挥着免疫调控作用,其中CD14基因表达上调有利于提高仔猪对E.coli F18感染抗性;而鞘糖脂合成通路及其FUT2基因可能在外来猪品种F18大肠杆菌受体形成过程中起关键作用,其中FUT2基因表达下调有利于提高仔猪对E.colE.F18感染抗性。此外,本研究系统揭示了长链非编码RNA在断奶仔猪F18大肠杆菌抗性过程中的调控机制,筛选出1个关键lncRNA:TCONS_00183659,并且推测该lncRNA可能通过与组蛋白Histone H4结合,从而调控Mx1、Mx2、IFIT2蛋白表达,今后需要进一步验证TCONS_00183659调控作用是否与Histone H4表观遗传修饰有关。
[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.

【学位授予单位】：扬州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S858.28

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