牙鲆免疫相關(guān)基因及熱休克蛋白基因的轉(zhuǎn)錄表達(dá)
發(fā)布時(shí)間：2018-08-02 09:50
【摘要】：牙鲆(Paralichthys olivaceus),是我國(guó)最有經(jīng)濟(jì)價(jià)值的海水養(yǎng)殖種類之一。牙鲆病害問(wèn)題日益突出,缺乏優(yōu)良抗病品種,制約了產(chǎn)業(yè)的可持續(xù)發(fā)展。牙鲆免疫系統(tǒng)及病原防御機(jī)制研究缺乏,從轉(zhuǎn)錄組水平了解牙鲆免疫相關(guān)基因及信號(hào)通路,有助于深入了解魚(yú)類的免疫系統(tǒng)和免疫防御機(jī)制。本研究進(jìn)行了牙鲆脾臟轉(zhuǎn)錄組測(cè)序,發(fā)現(xiàn)了免疫相關(guān)基因及其信號(hào)通路,進(jìn)一步研究了牙鲆熱休克蛋白在溫度刺激、病原感染和疫苗免疫下的表達(dá),對(duì)深入理解魚(yú)類的免疫系統(tǒng)和牙鲆的病害防治有很重要的意義。實(shí)驗(yàn)主要結(jié)果如下:1、研究了牙鲆感染鰻弧菌前后脾臟的轉(zhuǎn)錄組:取牙鲆感染鰻弧菌前后的脾臟制備文庫(kù),經(jīng)Illumina Miseq測(cè)序后,共得到314,377個(gè)轉(zhuǎn)錄本。經(jīng)Trinity拼接組裝后得到96,627個(gè)非冗余Unigenes,在Nr數(shù)據(jù)庫(kù)同源比對(duì)后得到21,392個(gè)Unigenes。注釋后的Unigenes通過(guò)與GO、COG和KEGG數(shù)據(jù)庫(kù)進(jìn)行Blast X比對(duì)獲得基因注釋和功能分類信息,有12,503個(gè)Unigenes注釋到GO的52個(gè)功能條目中;有19,547個(gè)預(yù)測(cè)蛋白注釋到COG26個(gè)分子家族中,有10,649 unigene參與到KEGG六大類生化代謝途徑中。對(duì)拼接后得到的96,627個(gè)Unigenes和斑馬魚(yú)的Unigenes以及牙鲆EST進(jìn)行了同源性比較分析。有11,799的Unigenes和牙鲆EST序列有同源性,有4,442條Unigenes和斑馬魚(yú)的Unigenes同源。對(duì)注釋后得到的21,392個(gè)unigenes進(jìn)行了分類統(tǒng)計(jì)分析,有18,627個(gè)Unigenes來(lái)自魚(yú)類,占整個(gè)注釋Unigenes的87.1%;有778個(gè)來(lái)自其他脊椎動(dòng)物(哺乳動(dòng)物、鳥(niǎo)類、兩棲類、爬行動(dòng)物),占3.6%,有1.987個(gè)來(lái)自于植物和微生物,占9.3%。通過(guò)KEGG功能分類發(fā)現(xiàn)1,563個(gè)免疫相關(guān)基因序列,定位到15條免疫相關(guān)通路,首次發(fā)現(xiàn)TOLLIP、IRAK1/4、TRAF3/6、C2、C6、C8g、C5AR1等免疫基因在牙鲆的表達(dá),為深入魚(yú)類分子生物學(xué)和免疫防御機(jī)制研究奠定基礎(chǔ)。從314,377個(gè)轉(zhuǎn)錄本中預(yù)測(cè)出47,362個(gè)SNP位點(diǎn),其中轉(zhuǎn)換位點(diǎn)為28,142,顛換位點(diǎn)有19,220個(gè)。在21,392個(gè)Unigenes中發(fā)現(xiàn)了40,928個(gè)SSR位點(diǎn);在1.563個(gè)免疫通路相關(guān)基因中發(fā)現(xiàn)有1,579個(gè)SNP位點(diǎn),分布在339個(gè)Unigenes上。比較感染鰻弧菌和未感染鰻弧菌的轉(zhuǎn)錄組,發(fā)現(xiàn)有189個(gè)差異表達(dá)顯著的unigene,其中上調(diào)表達(dá)的38個(gè),下調(diào)表達(dá)的151個(gè)。2、牙鲆熱休克蛋白基因的轉(zhuǎn)錄表達(dá)通過(guò)熒光定量PCR技術(shù)檢測(cè)了溫度刺激、鰻弧菌感染、疫苗免疫后牙鲆熱休克蛋白基因Hsp10、Hsp40A4、Hsp60、Hsp70和Hsp110的表達(dá)。比較了熱休克蛋白家族基因在健康牙鲆脾臟中的表達(dá)變化。Hsp90β的表達(dá)量最高,其次是Hsp90α和Hsp70,而Hsp10和Hsp110的表達(dá)量最低。熱休克蛋白基因在牙鲆的肝、脾、頭腎、后腎,心臟,腮,腦、胃、中腸、血、腹肌,背鰭、背皮12個(gè)組織中都有不同程度的表達(dá),不同的熱休克蛋白基因在不同組織間表達(dá)的差異較大。以中腸、肝臟和腹肌等組織中的表達(dá)量最高。牙鲆在5℃、10℃、16℃、22℃、28℃和32℃不同溫度處理1h后,不同的熱休克蛋白基因間的表達(dá)差異較大。Hsp40和Hsp70對(duì)冷脅迫不敏感,對(duì)熱激敏感,在28℃和32℃熱激時(shí),大量表達(dá);Hsp10在冷脅迫和32℃熱激時(shí)表達(dá)下調(diào),28℃熱激時(shí)表達(dá)量增加;Hsp60對(duì)低溫和高溫都不敏感;Hsp110對(duì)低溫敏感,在28℃熱激時(shí)表達(dá)量顯著增加。熱休克蛋白基因在冷脅迫(10℃)和熱激(28℃)1h、3h、5h、8h后,表達(dá)量發(fā)生不同程度的變化。Hsp10和Hsp70在冷脅迫后表達(dá)量下調(diào);Hsp40、Hsp60和Hsp110表達(dá)類似,表達(dá)量上調(diào),在5h時(shí)表達(dá)量最高。熱休克蛋白基因?qū)峒っ舾?除了Hsp60表達(dá)量沒(méi)有顯著變化,其余熱休克蛋白基因表達(dá)量顯著增加,3h達(dá)到峰值,特別是Hsp70和Hsp40在28℃熱激后,表達(dá)量迅速增加。研究了熱休克蛋白基因在鰻弧菌M3感染和滅活鰻弧菌免疫的牙鲆脾臟中的轉(zhuǎn)錄表達(dá)。鰻弧菌感染后,不同的熱休克蛋白基因在6h、12h、24h和72h表達(dá)變化不同。大部分熱休克蛋白基因在細(xì)菌侵染后,表達(dá)是下調(diào)的,而Hsp60表達(dá)上調(diào)。滅活鰻弧菌免疫1d、3d、7d、14d后,熱休克蛋白基因在免疫初期1d時(shí),表達(dá)都是上調(diào)的,3d時(shí)除了Hsp60表達(dá)上調(diào)外,其余都是表達(dá)下調(diào),7d和14d時(shí)大多回到正常水平,和對(duì)照組沒(méi)有顯著差異。
[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.
【學(xué)位授予單位】：中國(guó)科學(xué)院研究生院(海洋研究所)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：S917.4
本文編號(hào)：2159049
[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.
【學(xué)位授予單位】：中國(guó)科學(xué)院研究生院(海洋研究所)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：S917.4
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