發(fā)布時間：2018-05-26 15:52

  本文選題：雜色鮑 + 缺氧��； 參考：《集美大學(xué)》2017年碩士論文

【摘要】：雜色鮑(Haliotis diversicolor),隸屬軟體動物門(Mollusca)、腹足綱(Gastropoda)、前鰓亞綱(Prosobranchia)、原始腹足目(Archaeogastropoda)、鮑科(Haliotidae)、鮑屬(Haliotis)。雜色鮑自然分布于我國浙江以南沿海以及日本、越南等地,是我國南方沿海一種重要的經(jīng)濟養(yǎng)殖貝類。當(dāng)前隨著養(yǎng)殖業(yè)集約化的發(fā)展導(dǎo)致生產(chǎn)中面臨著應(yīng)激性疾病頻發(fā)的嚴(yán)峻挑戰(zhàn),尤其在夏季水體的高溫缺氧影響雜色鮑正常的生理狀態(tài),降低其應(yīng)對外界病原侵染的能力,對鮑養(yǎng)殖業(yè)的健康發(fā)展產(chǎn)生較大的影響。本研究通過二代高通量測序平臺IIlumina HiseqTM2000對雜色鮑缺氧應(yīng)激下不同時相的血細胞進行了大規(guī)模測序,比對了不同轉(zhuǎn)錄樣品之間的差異,篩選出差異表達和特異表達的unigenes。在轉(zhuǎn)錄組測序結(jié)果的基礎(chǔ)上,篩選免疫相關(guān)的重要基因及免疫相關(guān)通路多個,重點研究了作為先天免疫調(diào)節(jié)過程中的重要信號通路PI3K-AKT信號通路在雜色鮑應(yīng)對外界環(huán)境刺激的調(diào)節(jié)機制。主要研究結(jié)果如下:1、對雜色鮑缺氧應(yīng)激下的14個血細胞RNA樣品進行了轉(zhuǎn)錄組測序共獲得307395572reads,所有reads數(shù)經(jīng)拼接組裝后共產(chǎn)生99774條unigenes,其中有47154條unigenes獲得注釋,為后續(xù)挖掘與免疫調(diào)節(jié)機制相關(guān)的基因和通路提供了大量基礎(chǔ)數(shù)據(jù)。對已拼接完成的unigene進行篩選分析,共得到免疫通路相關(guān)基因242條。這些基因涉及PI3K-AKT信號通路(PI3K-AKT signaling pathway)、MAPK信號通路(MAPK signaling pathway)、P53信號通路(P53 signaling pathway)、NF-κB信號通路(NF-κB signaling pathway)、HIF信號通路(HIF-1 signaling pathway)等免疫、凋亡主要相關(guān)通路。對獲得的多個轉(zhuǎn)錄組的樣本進行了生物信息學(xué)比較分析,得到多條差異表達unigenes,其中缺氧應(yīng)激實驗組0h樣本和對照組0h樣本之間具有最多的差異表達unigenes,共24189條,注釋率為59%;實時熒光定量PCR實驗驗證了41條篩選后的差異表達基因,其中27條的表達趨勢與轉(zhuǎn)錄組分析結(jié)果一致。2、首次克隆獲得雜色鮑PI3K-AKT信號通路關(guān)鍵基因:磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)、蛋白質(zhì)絲氨酸/蘇氨酸激酶(protein-serine-threonine kinase,AKT),并分別命名為HdAKT和HdPI3K。HdAKT cDNA總長2126 bp,可編碼479個氨基酸,其中包含1440 bp的開放閱讀框(ORF),198bp的5’非翻譯區(qū)(UTR)和488bp的3’UTR。HdPI3K cDNA全長序列為6052 bp(GenBank登錄號:KX245017),包括5’UTR 496 bp,3’UTR 2262 bp和3294 bp的ORF,可編碼1097個氨基酸。3、選擇了PI3K-AKT信號通路中的共14條相關(guān)基因進行了實時熒光定量PCR分析。結(jié)果表明這14條基因在檢測的雜色鮑7個組織中均有廣泛表達,且HdAKT基因在血細胞中表達量最高(p0.05),其次是肝胰腺(p0.05),HdPI3K在血細胞的表達水平高于其他組織。在正常的溫度和溶氧條件下,副溶血弧菌感染后HdAKT,HdPI3K和其他PI3KAKT信號通路成員基因表達水平顯著上調(diào),表明PI3K-AKT信號通路在雜色鮑先天免疫系統(tǒng)中發(fā)揮重要的作用。但是當(dāng)處于環(huán)境應(yīng)激(高溫、缺氧和高溫缺氧聯(lián)合)條件下,再次受到弧菌感染時,這些基因的表達水平在鰓、血細胞和肝胰腺組織中被顯著抑制,表明PI3K-AKT信號通路可能參與雜色鮑在環(huán)境壓力下誘導(dǎo)產(chǎn)生免疫抑制的過程,導(dǎo)致雜色鮑的免疫系統(tǒng)處于抑制狀態(tài)。4、對雜色鮑血細胞中HdAKT基因進行dsRNA干擾實驗,結(jié)果表明:在dsRNA干擾血細胞后的各時相(4h、12h、24h),HdAKT基因的表達水平均出現(xiàn)顯著下調(diào)(P0.05)。同時,PI3K-AKT信號通路相關(guān)基因的表達情況與綠色熒光蛋白組以及空白對照組相比也均有不同程度的顯著變化(P0.05)。同時利用Cytoscape構(gòu)建了HdAKT RNA干擾后PI3KAKT信號通路相關(guān)基因各時相的相互作用網(wǎng)絡(luò)。隨著干擾時間的加長,HdAKT的干擾效果加深,PI3K-AKT信號通路相關(guān)基因也逐漸顯現(xiàn)出被抑制的趨勢,這一結(jié)果也說明了HdAKT在對上述基因的調(diào)控過程中起到了關(guān)鍵性的作用,并對PI3K-AKT信號通路相關(guān)基因具有正向調(diào)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.
【學(xué)位授予單位】：集美大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S917.4

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