牙鲆免疫相关基因及热休克蛋白基因的转录表达

发布时间：2018-08-02 09:50

【摘要】：牙鲆(Paralichthys olivaceus),是我国最有经济价值的海水养殖种类之一。牙鲆病害问题日益突出,缺乏优良抗病品种,制约了产业的可持续发展。牙鲆免疫系统及病原防御机制研究缺乏,从转录组水平了解牙鲆免疫相关基因及信号通路,有助于深入了解鱼类的免疫系统和免疫防御机制。本研究进行了牙鲆脾脏转录组测序,发现了免疫相关基因及其信号通路,进一步研究了牙鲆热休克蛋白在温度刺激、病原感染和疫苗免疫下的表达,对深入理解鱼类的免疫系统和牙鲆的病害防治有很重要的意义。实验主要结果如下:1、研究了牙鲆感染鳗弧菌前后脾脏的转录组:取牙鲆感染鳗弧菌前后的脾脏制备文库,经Illumina Miseq测序后,共得到314,377个转录本。经Trinity拼接组装后得到96,627个非冗余Unigenes,在Nr数据库同源比对后得到21,392个Unigenes。注释后的Unigenes通过与GO、COG和KEGG数据库进行Blast X比对获得基因注释和功能分类信息,有12,503个Unigenes注释到GO的52个功能条目中;有19,547个预测蛋白注释到COG26个分子家族中,有10,649 unigene参与到KEGG六大类生化代谢途径中。对拼接后得到的96,627个Unigenes和斑马鱼的Unigenes以及牙鲆EST进行了同源性比较分析。有11,799的Unigenes和牙鲆EST序列有同源性,有4,442条Unigenes和斑马鱼的Unigenes同源。对注释后得到的21,392个unigenes进行了分类统计分析,有18,627个Unigenes来自鱼类,占整个注释Unigenes的87.1%;有778个来自其他脊椎动物(哺乳动物、鸟类、两栖类、爬行动物),占3.6%,有1.987个来自于植物和微生物,占9.3%。通过KEGG功能分类发现1,563个免疫相关基因序列,定位到15条免疫相关通路,首次发现TOLLIP、IRAK1/4、TRAF3/6、C2、C6、C8g、C5AR1等免疫基因在牙鲆的表达,为深入鱼类分子生物学和免疫防御机制研究奠定基础。从314,377个转录本中预测出47,362个SNP位点,其中转换位点为28,142,颠换位点有19,220个。在21,392个Unigenes中发现了40,928个SSR位点;在1.563个免疫通路相关基因中发现有1,579个SNP位点,分布在339个Unigenes上。比较感染鳗弧菌和未感染鳗弧菌的转录组,发现有189个差异表达显著的unigene,其中上调表达的38个,下调表达的151个。2、牙鲆热休克蛋白基因的转录表达通过荧光定量PCR技术检测了温度刺激、鳗弧菌感染、疫苗免疫后牙鲆热休克蛋白基因Hsp10、Hsp40A4、Hsp60、Hsp70和Hsp110的表达。比较了热休克蛋白家族基因在健康牙鲆脾脏中的表达变化。Hsp90β的表达量最高,其次是Hsp90α和Hsp70,而Hsp10和Hsp110的表达量最低。热休克蛋白基因在牙鲆的肝、脾、头肾、后肾,心脏,腮,脑、胃、中肠、血、腹肌,背鳍、背皮12个组织中都有不同程度的表达,不同的热休克蛋白基因在不同组织间表达的差异较大。以中肠、肝脏和腹肌等组织中的表达量最高。牙鲆在5℃、10℃、16℃、22℃、28℃和32℃不同温度处理1h后,不同的热休克蛋白基因间的表达差异较大。Hsp40和Hsp70对冷胁迫不敏感,对热激敏感,在28℃和32℃热激时,大量表达;Hsp10在冷胁迫和32℃热激时表达下调,28℃热激时表达量增加;Hsp60对低温和高温都不敏感;Hsp110对低温敏感,在28℃热激时表达量显著增加。热休克蛋白基因在冷胁迫(10℃)和热激(28℃)1h、3h、5h、8h后,表达量发生不同程度的变化。Hsp10和Hsp70在冷胁迫后表达量下调;Hsp40、Hsp60和Hsp110表达类似,表达量上调,在5h时表达量最高。热休克蛋白基因对热激敏感,除了Hsp60表达量没有显著变化,其余热休克蛋白基因表达量显著增加,3h达到峰值,特别是Hsp70和Hsp40在28℃热激后,表达量迅速增加。研究了热休克蛋白基因在鳗弧菌M3感染和灭活鳗弧菌免疫的牙鲆脾脏中的转录表达。鳗弧菌感染后,不同的热休克蛋白基因在6h、12h、24h和72h表达变化不同。大部分热休克蛋白基因在细菌侵染后,表达是下调的,而Hsp60表达上调。灭活鳗弧菌免疫1d、3d、7d、14d后,热休克蛋白基因在免疫初期1d时,表达都是上调的,3d时除了Hsp60表达上调外,其余都是表达下调,7d和14d时大多回到正常水平,和对照组没有显著差异。
[Abstract]:Paralichthys olivaceus is one of the most valuable marine culture species in China. The disease problem of flounder is becoming more and more serious. The lack of good disease resistant varieties has restricted the sustainable development of the industry. The research on immune system and pathogenic mechanism of flounder is lacking. It is helpful to understand the immune related genes and signal pathways of flounder flounder from the transcriptional group. In order to understand the immune system and immune defense mechanism of fish, the splenic transcriptional group of flounder was sequenced, the immune related genes and their signaling pathways were found, and the expression of heat shock proteins in temperature stimulation, pathogen infection and vaccine immunization were further studied, and the immune system and disease of flounder were deeply understood. The main results of the prevention and control are as follows: 1, the splenic transcriptional group before and after the infection of Vibrio eel in Paralichthys olivaceus: the spleen preparation library before and after infection of Vibrio eel in Paralichthys olivaceus, 314377 transcripts were obtained after Illumina Miseq sequencing. 96627 non redundant Unigenes were obtained after the splicing of Trinity, and the homologous ratio of the Nr database was found. The Unigenes after the 21392 Unigenes. annotations is obtained by Blast X alignment with GO, COG, and KEGG databases to obtain gene annotation and functional classification information, with 12503 Unigenes annotations to 52 functional items of GO; 19547 predictive proteins are annotated to COG26 family, and 10649 UniGene participates in the biochemistry of KEGG six. 96627 Unigenes and Unigenes of zebrafish and EST of Paralichthys olivaceus were compared in metabolic pathways. 11799 of Unigenes and EST sequences of Paralichthys olivaceus were homologous, 4442 Unigenes and zebrafish were homologous to Unigenes. The 21392 unigenes obtained after the annotation were classified and analyzed, 18627 Unigenes comes from fish, accounting for 87.1% of the whole annotation Unigenes; 778 from other vertebrates (mammals, birds, amphibians, reptiles), 3.6%, 1.987 from plants and microbes, accounting for 1563 immunological sequences of 9.3%. through KEGG functional classification, located to 15 immune related pathways, and for the first time the discovery of TOLL The expression of IP, IRAK1/4, TRAF3/6, C2, C6, C8g, C5AR1 and other immune genes in Paralichthys olivaceus lay the foundation for the study of molecular biology and immune defense mechanism in fish. 47362 SNP loci were predicted from 314377 transcripts, of which the conversion site was 28142, the change site was 19220. 40928 SSR loci were found in 21392 Unigenes; 1 in 1. 1579 SNP loci were found in.563 immuno pathway related genes and distributed on 339 Unigenes. Compared with the transcriptional group infected with Vibrio Anguilla and Vibrio Anguilla, there were 189 distinct UniGene expressions, of which 38 were up, 151.2 was downregulated, and the transcriptional expression of the heat shock protein gene of Paralichthys olivaceus was quantified by fluorescence quantitative P The expression of heat shock protein gene Hsp10, Hsp40A4, Hsp60, Hsp70 and Hsp110 in Paralichthys olivaceus after immunization was detected by CR technique, and the expression of the expression of heat shock protein family gene in the spleen of healthy flounder was the highest, followed by Hsp90 A and Hsp70, while the expression of Hsp10 and Hsp110 was the lowest. The shock protein gene was expressed in 12 tissues of the liver, spleen, head kidney, back kidney, heart, gills, brain, stomach, midgut, blood, abdominal muscle, dorsal fin and dorsal skin, and different expression of heat shock protein genes in different tissues. The highest expression in tissues such as midgut, liver and abdominal muscles was 5, 10, 16, 22. After treating for 1H at 28 degrees C and 32 c, the expression of different heat shock protein genes was significantly different,.Hsp40 and Hsp70 were not sensitive to cold stress, and the heat shock sensitivity was greatly expressed at 28 and 32 c. The expression of Hsp10 was downregulated at cold and 32 c, and the expression increased at 28 centigrade heat shock, and Hsp60 was not sensitive to low temperature and high temperature. Hsp110 was sensitive to low temperature and increased significantly at 28 centigrade heat shock. After cold stress (10 C) and heat shock (28) 1H, 3h, 5h, 8h, the expression of.Hsp10 and Hsp70 decreased after cold stress; Hsp40, Hsp60 and Hsp110 expressions were similar, the expression amount was up up, and the expression amount was the highest at 5h. Heat shock eggs The white gene was sensitive to heat shock, except that the expression of Hsp60 was not significantly changed. The expression of other heat shock proteins increased significantly, and the 3H reached its peak value. The expression of Hsp70 and Hsp40 increased rapidly after heat shock at 28 C. The transcriptional expression of heat shock protein gene in M3 infection of Vibrio Anguilla and inactivated Vibrio eel's spleen was studied. After infection of Vibrio anguinae, the expression of different heat shock protein genes in 6h, 12h, 24h and 72h was different. Most of the heat shock protein gene was down regulated after bacterial infection, and the expression of Hsp60 was up regulated. After immunization of Vibrio anguinae, 1D, 3D, 7d, 14d, the expression of heat shock protein gene was up-regulated at the 1D of early immunization, and 3D was expressed in addition to Hsp60 expression. Up regulation, the rest were downregulated, while 7d and 14d returned to normal levels, and there was no significant difference between the two groups.
【学位授予单位】：中国科学院研究生院(海洋研究所)
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S917.4

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