转录组分析杂色鲍在高温和缺氧应激下免疫相关基因及通路

发布时间：2018-05-26 15:52

本文选题：杂色鲍 + 缺氧　；参考：《集美大学》2017年硕士论文

【摘要】：杂色鲍(Haliotis diversicolor),隶属软体动物门(Mollusca)、腹足纲(Gastropoda)、前鳃亚纲(Prosobranchia)、原始腹足目(Archaeogastropoda)、鲍科(Haliotidae)、鲍属(Haliotis)。杂色鲍自然分布于我国浙江以南沿海以及日本、越南等地,是我国南方沿海一种重要的经济养殖贝类。当前随着养殖业集约化的发展导致生产中面临着应激性疾病频发的严峻挑战,尤其在夏季水体的高温缺氧影响杂色鲍正常的生理状态,降低其应对外界病原侵染的能力,对鲍养殖业的健康发展产生较大的影响。本研究通过二代高通量测序平台IIlumina HiseqTM2000对杂色鲍缺氧应激下不同时相的血细胞进行了大规模测序,比对了不同转录样品之间的差异,筛选出差异表达和特异表达的unigenes。在转录组测序结果的基础上,筛选免疫相关的重要基因及免疫相关通路多个,重点研究了作为先天免疫调节过程中的重要信号通路PI3K-AKT信号通路在杂色鲍应对外界环境刺激的调节机制。主要研究结果如下:1、对杂色鲍缺氧应激下的14个血细胞RNA样品进行了转录组测序共获得307395572reads,所有reads数经拼接组装后共产生99774条unigenes,其中有47154条unigenes获得注释,为后续挖掘与免疫调节机制相关的基因和通路提供了大量基础数据。对已拼接完成的unigene进行筛选分析,共得到免疫通路相关基因242条。这些基因涉及PI3K-AKT信号通路(PI3K-AKT signaling pathway)、MAPK信号通路(MAPK signaling pathway)、P53信号通路(P53 signaling pathway)、NF-κB信号通路(NF-κB signaling pathway)、HIF信号通路(HIF-1 signaling pathway)等免疫、凋亡主要相关通路。对获得的多个转录组的样本进行了生物信息学比较分析,得到多条差异表达unigenes,其中缺氧应激实验组0h样本和对照组0h样本之间具有最多的差异表达unigenes,共24189条,注释率为59%;实时荧光定量PCR实验验证了41条筛选后的差异表达基因,其中27条的表达趋势与转录组分析结果一致。2、首次克隆获得杂色鲍PI3K-AKT信号通路关键基因:磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)、蛋白质丝氨酸/苏氨酸激酶(protein-serine-threonine kinase,AKT),并分别命名为HdAKT和HdPI3K。HdAKT cDNA总长2126 bp,可编码479个氨基酸,其中包含1440 bp的开放阅读框(ORF),198bp的5’非翻译区(UTR)和488bp的3’UTR。HdPI3K cDNA全长序列为6052 bp(GenBank登录号:KX245017),包括5’UTR 496 bp,3’UTR 2262 bp和3294 bp的ORF,可编码1097个氨基酸。3、选择了PI3K-AKT信号通路中的共14条相关基因进行了实时荧光定量PCR分析。结果表明这14条基因在检测的杂色鲍7个组织中均有广泛表达,且HdAKT基因在血细胞中表达量最高(p0.05),其次是肝胰腺(p0.05),HdPI3K在血细胞的表达水平高于其他组织。在正常的温度和溶氧条件下,副溶血弧菌感染后HdAKT,HdPI3K和其他PI3KAKT信号通路成员基因表达水平显著上调,表明PI3K-AKT信号通路在杂色鲍先天免疫系统中发挥重要的作用。但是当处于环境应激(高温、缺氧和高温缺氧联合)条件下,再次受到弧菌感染时,这些基因的表达水平在鳃、血细胞和肝胰腺组织中被显著抑制,表明PI3K-AKT信号通路可能参与杂色鲍在环境压力下诱导产生免疫抑制的过程,导致杂色鲍的免疫系统处于抑制状态。4、对杂色鲍血细胞中HdAKT基因进行dsRNA干扰实验,结果表明:在dsRNA干扰血细胞后的各时相(4h、12h、24h),HdAKT基因的表达水平均出现显著下调(P0.05)。同时,PI3K-AKT信号通路相关基因的表达情况与绿色荧光蛋白组以及空白对照组相比也均有不同程度的显著变化(P0.05)。同时利用Cytoscape构建了HdAKT RNA干扰后PI3KAKT信号通路相关基因各时相的相互作用网络。随着干扰时间的加长,HdAKT的干扰效果加深,PI3K-AKT信号通路相关基因也逐渐显现出被抑制的趋势,这一结果也说明了HdAKT在对上述基因的调控过程中起到了关键性的作用,并对PI3K-AKT信号通路相关基因具有正向调控作用。
[Abstract]:Haliotis diversicolor, belonging to the mollusk gate (Mollusca), the gastropods (Gastropoda), the pre branchial subclass (Prosobranchia), the primitive gastropods (Archaeogastropoda), Boko (Haliotidae) and the abalone (Haliotis). The abalone is naturally distributed in the south coast of Zhejiang and in Japan and Vietnam, and is an important channel in the coastal areas of the south of our country. With the intensive development of the aquaculture industry, the intensive development of the aquaculture industry has led to the severe challenge of frequent stress diseases. Especially in summer, the high temperature anoxia affects the normal physiological state of the abalone, reduces the ability to deal with the external pathogen infection, and has a great influence on the healthy development of the abalone breeding industry. The two generation high throughput sequencing platform IIlumina HiseqTM2000 sequenced the blood cells of the different simultaneous phases under the anoxic stress of abalone, compared the difference between different transcriptional samples and screened out the differential expression and specific expression of unigenes. on the basis of the sequencing results of the transcriptional group, screening important immune related genes and immune correlation. The main research results are as follows: 1, the main results are as follows: 1, a total of 14 blood cell RNA samples under anoxic stress of abalone were sequenced and 307395572reads was sequenced. All reads numbers were obtained. After the assembly, 99774 unigenes were produced, of which 47154 unigenes were annotated. A large number of basic data were provided for subsequent mining of genes and pathways related to immunomodulatory mechanisms. A total of 242 immune pathway related genes were screened and analyzed for the spliced UniGene. These genes involved the PI3K-AKT signaling pathway (PI3K-A KT signaling pathway), the MAPK signaling pathway (MAPK signaling pathway), the P53 signaling pathway (P53 signaling pathway), the NF- kappa signaling pathway, the main pathway of apoptosis, and the bioinformatics analysis of the samples of the multiple transcriptional groups obtained. Multiple differential expression of unigenes, among which 0h samples from the experimental group of hypoxia stress and the 0h samples from the control group had the most differential expression of unigenes, a total of 24189, and the annotation rate was 59%. The real time fluorescence quantitative PCR test verified the 41 differentially expressed genes after the screening, and 27 of them were consistent with the results of the transcriptional analysis and were first cloned for the first time. The key genes of PI3K-AKT signaling pathway in abalone are obtained: phosphatidyl inositol 3- kinase (phosphatidylinositol 3-kinase, PI3K), protein serine / threonine kinase (Protein-Serine-Threonine kinase, AKT), and respectively named HdAKT and HdPI3K.HdAKT cDNA 2126 BP, which can encode 479 amino acids, including 1440 open reading frames (ORF), the 3 'UTR.HdPI3K cDNA full length sequence of the 5' untranslated region (UTR) and 488bp of the 198bp is 6052 BP (GenBank login number: KX245017), including 5 'UTR 496 BP, 3' UTR 2262 and 3294 amino acids, which can encode 1097 amino acids, and select a total of 14 related genes in the signaling pathway to carry out real-time quantitative quantitative analysis. These 14 genes are widely expressed in 7 tissues of abalone, and the expression of HdAKT in blood cells is the highest (P0.05), followed by hepatopancreas (P0.05), and the expression level of HdPI3K in blood cells is higher than that of other tissues. Under normal temperature and dissolved oxygen conditions, Vibrio parahaemolyticus is infected with HdAKT, HdPI3K and other PI3KAKT signals. The gene expression level of the members of the road is significantly up-regulated, indicating that the PI3K-AKT signaling pathway plays an important role in the innate immune system of abalone. However, the expression levels of these genes are significantly suppressed in the gills, blood cells and hepatopancreas when environmental stress (high temperature, hypoxia and high temperature anoxia) is associated with Vibrio infection again. The result shows that the PI3K-AKT signaling pathway may participate in the process of immune suppression induced by abalone under environmental pressure. The immune system of abalone is in the inhibitory state of.4, and the dsRNA interference experiment on HdAKT gene in the blood cells of abalone shows that the expression of HdAKT gene expression after dsRNA interferes with the blood cells (4h, 12h, 24h) and the expression of the HdAKT gene. At the same time, the expression of PI3K-AKT signaling pathway related genes was significantly different from that of the green fluorescent protein group and the blank control group (P0.05). At the same time, the interaction network of the PI3KAKT signaling pathway related genes after HdAKT RNA interference was constructed with Cytoscape. With the lengthening of the interference time, the interference effect of HdAKT is deepened, and the genes related to the PI3K-AKT signaling pathway also gradually appear to be suppressed. This result also shows that HdAKT plays a key role in the regulation of the above genes and has a positive regulation on the genes related to the PI3K-AKT signaling pathway.
【学位授予单位】：集美大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

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