E.tenella感染雏鸡脾脏差异表达基因的cDNA-SRAP分析

发布时间：2018-04-14 12:17

本文选题：柔嫩艾美耳球虫 + cDNA-SRAP　；参考：《西南大学》2016年硕士论文

【摘要】：球虫病是集约化养鸡业最为高发且危害严重的一类寄生虫病,在我国发病率较高,经济损失巨大,其中柔嫩艾美耳球虫为危害最强最严重的虫种。尽管近年来对球虫免疫学研究越来越深入,但由于球虫复杂的生活史,庞大的基因组等问题,导致现阶段对球虫入侵和宿主防御的分子机制还不明确,感染引起宿主基因的表达变化也不清楚。本研究通过c DNA-SRAP技术筛选和分析与E.tenella感染相关基因,加强对E.tenella和宿主之间相互作用的研究,为从转录组水平上揭示E.tenella与宿主间的相互作用规律奠定基础。主要研究结果如下:(1)雏鸡脾脏RNA样本的c DNA-SRAP体系的建立。用Trizol法提取14日龄雏鸡脾脏总RNA,采用SMART Scribe Reverse Transcriptase试剂盒进行反转录,以脾脏c DNA为模板,以单因素试验设计和正交试验设计对c DNA-SRAP反应体系进行优化,获得最佳反应体系和扩增程序。反应体系为:d NTPs:0.25m M;Mg2+:2.0m M;Taq聚合酶:2.25U;引物:0.55μM;模板c DNA:20ng。扩增程序为:94℃预变性5min,94℃变性1min,36℃退火1min,72℃延伸1min,共5个循环,94℃变性1min,55℃退火1min,72℃延伸1min,共35个循环,72℃延伸10min。利用优化好的程序和体系进行c DNA-SRAP扩增,能够获得条带清晰、多态性好的扩增条带。(2)E.tenella感染雏鸡脾脏转录组c DNA-SRAP分析。以雏鸡脾脏c DNA为模板,采用最优扩增体系及程序,通过256对c DNA-SRAP任意引物对感染组和对照组进行扩增,经1.6%琼脂糖凝胶电泳和紫外分析,对差异显著的条带进行回收。初次筛选后感染组共获得12个上调表达基因,通过动物实验和c DNA-SRAP对该12个基因进行验证后,经过二次PCR、克隆测序后共获得9个基因序列。在NCBI中通过BLAST-N工具进行在线比对,结果显示9个基因均有高度同源性序列,其中6个为已知功能基因,分别为MLL5蛋白基因(XM_003640341.2)、嘌呤受体P2RY8基因(XM_011588440.1)、磷脂酶PLCD1基因(XM_004939119.2)、亮氨酸氨基肽酶LAP3基因(XM_010220558.1)、IL-17 RA基因(XM_013172986.1)、Wolf-Hirschhom畸变综合征的候选相关基因WHSC1L1基因(XM_015297415.1)。(3)E.tenella感染雏鸡脾脏差异表达基因定量分析。对6个功能基因设计特异性引物,采用SYBR Green I法进行Real time RT-PCR定量分析。结果表明感染E.tenenlla后,MLL5基因上调表达1.52倍、P2RY8基因上调表达2.33倍、PLCD1基因上调表达1.48倍、LAP3基因上调表达1.57倍、IL-17RA基因上调表达1.41倍、WHSC1L1基因上调表达1.73倍。对数据进行独立样本T检验显著性分析,其中P2RY8、WHSC1L1基因为极显著差异(P0.01),MLL5、PLCD1、LAP3、IL-17 RA为显著差异(P0.05)。E.tenella感染雏鸡能够引起脾脏中MLL5、P2RY8、PLCD1、LAP3、IL-17RA和WHSC1L1基因的上调,表明在E.tenella感染宿主早期,上述基因发生表达变化可能与E.tenella入侵宿主以及宿主的防御机制相关。PLCD1能够放大Ca2+信号调节途径,参与固有免疫反应,LAP3帮助抗原肽在膜表面形成MHCI类分子复合物,IL-17RA作为细胞因子受体参与炎症反应过程,三个基因具有的免疫学功能预示其表达变化可能与球虫感染引起的免疫反应相关;MLL5基因具有促进细胞增殖的作用,WHSC1L1基因多与癌症发展过程相关,P2RY8基因具体功能还不太明确,三者同球虫感染相关作用机制仍然需要进一步研究。
[Abstract]:Coccidiosis is the most frequent and serious harm parasitic diseases in intensive poultry industry, in China's high incidence of huge economic losses, the Eimeria species is the most serious harm to the strongest. Although in recent years, more and more in-depth research on coccidia immunology, but due to the complex life history of coccidia, genome such a large problem, leading to the molecular mechanism at the present stage of invasion and host defense is not clear, infection caused by expression of host genes is not clear. This study by C DNA-SRAP and E.tenella infection screening and analysis of related genes, strengthen the research on the interaction between E.tenella and host, lay a foundation for revealing each other interaction between E.tenella and the host from the transcriptome level. The main results are as follows: (1) the establishment of RNA C DNA-SRAP chicken spleen samples from 14 days of age. The system with Trizol method Chicken spleen total RNA, reverse transcription using SMART Scribe Reverse Transcriptase kit, C DNA in spleen of C as template, DNA-SRAP reaction system was optimized by single factor test and orthogonal test, the best reaction system and PCR procedure. The reaction system is: D NTPs:0.25m M; Mg2+: 2.0m M; Taq polymerase primer: 2.25U; 0.55 M; C template DNA:20ng. amplification procedure: 94 C predegeneration 5min 94 C 1min 1min degeneration, annealed at 36 C, 72 C extension 1min, a total of 5 cycles, 94 degrees degeneration 1min, annealed at 55 C 1min, 72 C extension 1min, a total of 35 cycles, 72 degrees extension 10min. C DNA-SRAP by using the optimization procedure and the system can get clear bands, amplified bands with high polymorphism. (2) E.tenella chickens infected C DNA-SRAP spleen transcriptome analysis. In chicken spleen C DNA as template, using the optimal amplification system and procedures, by 256 to C DNA- SRAP arbitrary primers of infection group and control group were analyzed by 1.6% agarose gel electrophoresis and UV, the difference bands were recovered. There were 12 up-regulated genes were infected after screening for the first time, 12 genes were verified by animal experiment and C DNA-SRAP, after two times of PCR, 9 gene sequences were cloned and sequenced. The BLAST-N tool for online comparison in NCBI, the results showed that 9 genes were highly homologous sequences, including 6 known genes, including MLL5 protein gene (XM_003640341.2), purine receptor P2RY8 gene (XM_011588440.1), phospholipase PLCD1 gene (XM_004939119.2), leucine aminopeptidase LAP3 gene (XM_010220558.1), IL-17 RA (XM_013172986.1) gene, a candidate gene for WHSC1L1 Wolf-Hirschhom gene aberration syndrome (XM_015297415.1). (3) E.tenella infected chicken spleen Quantitative analysis of gene expression difference. Dirty on the 6 functional gene specific primers were designed for Real time RT-PCR SYBR Green I by quantitative analysis method. The results showed that after E.tenenlla infection, MLL5 gene expression up-regulated P2RY8 gene expression by 1.52 times, 2.33 times, 1.48 times up-regulated PLCD1 gene expression, LAP3 gene expression in 1.57 times, IL-17RA the gene expression of 1.41 times, 1.73 times. The WHSC1L1 gene expression was analyzed by independent sample T test were analyzed, the data including P2RY8, WHSC1L1 gene was significant difference (P0.01), MLL5, PLCD1, LAP3, IL-17, RA were significant difference (P0.05).E.tenella infection in chickens can cause spleen MLL5, P2RY8, PLCD1. LAP3, IL-17RA and WHSC1L1 genes up-regulated in E.tenella showed that the early infection of the host, the gene expression changes of E.tenella and invasion of host and host defense mechanisms related to amplify Ca2+.PLCD1 Signal pathway, involved in innate immune responses, LAP3 help MHCI antigen peptide to form molecular complexes on the membrane surface, IL-17RA as a cytokine receptor involved in inflammatory reaction, immunological function of three genes has indicated its expression may be associated with the immune response induced by Eimeria infection; MLL5 gene can promote cell proliferation, WHSC1L1 genes associated with cancer progression, P2RY8 gene specific function is not clear, the three with the coccidial infection mechanism still needs further study.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.31

【相似文献】