牙鲆免疫相关基因及热休克蛋白基因的转录表达

IOCAS-IR > 实验海洋生物学重点实验室

	牙鲆免疫相关基因及热休克蛋白基因的转录表达
	黄琳
学位类型	博士
导师	莫照兰
	2015-05-22
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	海洋生物学
关键词	牙鲆转录组免疫相关基因热休克蛋白
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BlastX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。
其他摘要	Japanese flounder (Paralichthys olivaceus) is one of economically important marine fish in China. However, diseases of Japanese flounder have become increasingly prominent, and there were few disease-resistant species, which restricted sustainable development of Japanese flounder aquiculture. However, immune system and defend mechanism to pathogen were scarce, the comprehension of immune-related genes and signaling pathways of Japanese flounder from the point of view of the transcriptome level will contribute to deeply understand immune system and defend mechanism of fish. In this study, we conducted transcriptome sequencing of Japanese flounder spleen and discovered immune-related genes and signaling pathways. Moreover, we further studied the gene expression changes of heat shock proteins of Japanese flounder after temperature stimulation, pathogen infection and vaccine immunity. This study will have a very important significance to deeply understand of fish immune system and disease control of Japanese flounder. Main results of the study were as follows: 1. We studied the spleen transcriptome of Japanese flounder after infected with Vibrio anguillarum. Two cDNA libraries (the spleen of Japanese flounder before and after infected with V. anguillarum) were successfully constructed. 314,377 transcripts were obtained from Illumina Mi-seq sequencing. Using Trinity software, a total of 96,627 unigenes were assembled, which were annotated using the Nr databases. There were a total of 21,392 (22.14%) unigenes with significant blast hits to known proteins. Approximately 12,503 (58.45%) unigenes were categorized into three Gene Ontology groups, 19,547 (91.38 %) were classified into 26 Cluster of Orthologous Groups, and 10,649 (49.78%) were assigned to six Kyoto Encyclopedia of Genes and Genomes pathways. The Japanese flounder assembled unigene sequences were compared to unigenes from zebrafish (Danio rerio) and known flounder EST and nucleotide sequences in the NCBI database. 11,799 of the unigenes could be matched in the EST sequences, 4,442 sequences were shared between the flounder unigenes and zebrafish unigenes. Among the species distribution of BLAST hits, 18,627 (87.1%) originated from fish species, 778 (3.6%) from other vertebrates, including mammals, birds, amphibians, and reptiles, and 1,987 (9.3%) from other species, mainly plant and microorganisms. We identified 1,563 putative immune-associated unigenes that mapped to 15 immune signaling pathways. Many genes, such as TOLLIP, IRAK1/4, TRAF3/6, C2, C6, C8g, C5AR1 were even first found to express in Japanese flounder. A total of 47,362 putative SNPs were identified from the 314,377 transcripts, including 28,142 transitions and 19,220 transversions. Moreover, a total of 40,928 putative SSR loci were discovered in 21,392 unigenes; Among the 1,563 immune unigenes, a total of putative 1,579 SNPs were detected, which were distributed in 339 unigene sequences. A total of 189 differentially expressed genes (DEGs) were detected between the spleen transcriptomes of the infected and control group. 38 genes were up-regulated while 151 were down-regulated in the two transcriptomes. 2. Using qRT-PCR detection, we initially constructed an expression pattern of Hsp10, Hsp40A4, Hsp60, Hsp70 and Hsp110 of Japanese flounder after temperature stimulation, pathogen infection and vaccine immunity. The expression pattern of HSP genes of healthy Japanese flounder in spleen was compared. The highest expression quantities was Hsp90β, followed by Hsp90α和Hsp70, and the lowest expression was Hsp10 and Hsp110. HSP genes of healthy Japanese flounder were expressed in different degrees in 12 different tissues. The expression difference of HSP genes between 12 tissues was large. The expression quantity in the midintestine, liver and muscle were the highest. The expression changes of HSP genes were great after 1 hour at 5℃, 10℃, 16℃, 22℃, 28℃ and 32℃. Hsp40 and Hsp70 were not sensitive to cold stress, but sensitive to heat shock, the expression quantitiy of the two genes increased abundantly. The expression level of Hsp10 genes was lower under cold stress and 32℃ heat shock than that at normal conditions. And the expression level of Hsp10 increased abundantly at 28℃ heat shock. Hsp60 was not sensitive to cold stress and heat shock. Hsp110 was sensitive to cold stress, and the expression level increased abundantly at 28℃ heat shock. QRT-PCR was performed to detect the expression pattern of HSP genes of Japanese flounder spleen after cold stress (10℃) and heat shock (28℃) 1h, 3h, 5h, 8h. The mRNA expression level of Hsp10 and Hsp70 decreased after cold stress. Otherwise, the expression level of Hsp40, Hsp60 and Hsp110 were similar, expression quantity increased over time and were the highest at 5 hour after cold stress. Heat shock protein genes were sensitive to heat shock. The mRNA expression level of HSP increased significantly and was the highest at 3 hour after heat shock, in addition to the expression amount of Hsp60 did not change significantly. Especially the expression quantity of Hsp70 and Hsp40 increased rapidly after heat shock (28℃). QRT-PCR was performed to detect the expression pattern of HSP genes in the spleen of Japanese flounder after infected with V. anguillarum and immuned by inactvated V. anguillarum. Different heat shock protein genes seemed to show different expression changes at 6 h, 12 h, 24 h and 72h after infected with V. anguillarum. The expression of most of HSP genes was down-regulated, but the expression of Hsp60 was up-regulated. By contrast, the expression of most of HSP genes was up-regulated at 1 day after vaccine immunity, subsequently down-regulated at 3 day. Hsp60 was up-regulated at 3 day after vaccine immunity. The expression level of HSP genes were back to normal levels at 7 and 14 day, which were not differ with the control group significantly.
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/23257
专题	实验海洋生物学重点实验室
作者单位	中科院海洋所
第一作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	黄琳. 牙鲆免疫相关基因及热休克蛋白基因的转录表达[D]. 北京. 中国科学院大学,2015.

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