本文選題：雜色鮑　切入點(diǎn)：血藍(lán)蛋白　出處：《上海海洋大學(xué)》2016年碩士論文

【摘要】：血藍(lán)蛋白(hemocyanin)又稱血藍(lán)素,與蚯蚓血紅蛋白(hemerythrin)、血紅蛋白(hemoglobin)并稱自然界三大呼吸蛋白。血藍(lán)蛋白主要分布在節(jié)肢動物和軟體動物血淋巴中,含量非常豐富,具有運(yùn)輸氧氣、調(diào)節(jié)滲透壓、表皮固化、調(diào)節(jié)蛻皮過程、轉(zhuǎn)運(yùn)金屬離子、儲存蛋白質(zhì)、抗菌、抗病毒和抗腫瘤等多種功能。然而,近年來對于血藍(lán)蛋白的研究多集中在其蛋白結(jié)構(gòu)和功能方面,而對于血藍(lán)蛋白的基因結(jié)構(gòu)尤其是軟體動物血藍(lán)蛋白基因多亞型和其多功能之間的關(guān)系方面研究較少。本文運(yùn)用基因序列分析、熒光定量PCR(qPCR)和熒光原位雜交技術(shù)(FISH)對雜色鮑(Haliotis diversicolor)血藍(lán)蛋白的基因結(jié)構(gòu)及其對病原刺激的響應(yīng)等進(jìn)行了研究,對深入了解軟體動物血藍(lán)蛋白這種多序列、多功能、高分子量免疫分子的基因演化和免疫響應(yīng)機(jī)理具有重要的意義。首先,運(yùn)用生物信息學(xué)工具軟件對課題組克隆得到的雜色鮑血藍(lán)蛋白基因Hd Hc1、Hd Hc2-1和Hd Hc2-2序列進(jìn)行分析。結(jié)果顯示,Hd Hc與歐洲鮑(H.tuberculata)、鑰孔戚(M.Crenulata)等腹足綱動物的Hc聚為一支,遺傳距離較近;Hd Hc基因結(jié)構(gòu)與Ht Hc的基因結(jié)構(gòu)相似,也有8個功能單元(FU-a~FU-h),FU-a、FU-f、FU-g分別由4、3、2個外顯子編碼,其余5個功能單元則是連續(xù)的。和歐洲鮑相同的是,Hd Hc也含有0、1、2三種內(nèi)含子相位,不同的是Hd Hc1和Hd Hc2-1的7個連接內(nèi)含子均是1相位,6個內(nèi)部內(nèi)含子中有3個0相位,1個1相位,2個2相位,而Hd Hc2-2的7個連接內(nèi)含子都是0相位,6個內(nèi)部內(nèi)含子有1個0相位,2個1相位,3個2相位。其次,人工注射病原哈維氏弧菌(Vibrio harveyi)、鮑類皰疹病毒(Abalone herpesvirus,Ab HV)刺激健康雜色鮑,檢測血淋巴中可溶性總蛋白的濃度、超氧化物歧化酶(Superoxide Dismutase,SOD)、酸性磷酸酶(Acid Phosphatase,ACP)、堿性磷酸酶(Alkaline Phosphatase,AKP)活性的變化以及無細(xì)胞血漿的抑菌性,結(jié)果表明,(1)哈維弧菌和Ab HV刺激后,雜色鮑血淋巴中可溶性總蛋白濃度先顯著升高,后顯著下降。(2)哈維弧菌和Ab HV刺激后,雜色鮑血淋巴中ACP和AKP活性顯著升高,哈維弧菌刺激組的SOD先顯著升高,后顯著下降,Ab HV刺激組的SOD顯著下降。(3)雜色鮑無細(xì)胞血淋巴對哈維氏弧菌、創(chuàng)傷弧菌(Vibrio vulnificus)和溶珊瑚弧菌(Vibrio coralliiluyitcus)這三種病原均表現(xiàn)出了不同程度的抑菌性增強(qiáng)的作用。這些結(jié)果表明病原刺激引起了雜色鮑免疫相關(guān)因子的變化,使得血淋巴的抑菌性增強(qiáng)。然而,作為血淋巴中含量最高的一種免疫分子,血藍(lán)蛋白的表達(dá)可能也受到了調(diào)控。運(yùn)用熒光定量PCR(qPCR)技術(shù)檢測鮑胚胎發(fā)育各時期以及哈維氏弧菌,溶珊瑚弧菌,Ab HV,Poly(I:C)刺激后雜色鮑腹足、外套膜、鰓、肝胰腺中血藍(lán)蛋白基因Hd Hc1、Hd Hc2-1和Hd Hc2-2相對表達(dá)量的變化,從而分析其與血淋巴免疫活性變化的關(guān)系。結(jié)果顯示:(1)血藍(lán)蛋白兩個基因Hd Hc1、Hd Hc2-1在雜色鮑的發(fā)育初期即有表達(dá),Hd Hc2-1的相對表達(dá)量高于Hd Hc1的表達(dá)量,未見Hd Hc2-2的表達(dá)。(2)哈維氏弧菌、溶珊瑚弧菌、Ab HV、Poly(I:C)刺激雜色鮑后,腹足、肝胰腺、鰓、外套膜中的血藍(lán)蛋白基因Hd Hc1、Hd Hc2-1的相對表達(dá)量均有變化,但是不同組織、不同刺激組的變化規(guī)律不同。與0 h的空白對照組相比,哈維氏弧菌刺激組Hd Hc1表達(dá)量先升高后降低,Hd Hc2-1表達(dá)量先降低后升高再降低;溶珊瑚弧菌刺激組Hd Hc1表達(dá)量呈降低趨勢,Hd Hc2-1表達(dá)量升高降低再升高;Ab HV刺激組Hd Hc1表達(dá)量先降低后升高再降低,Hd Hc2-1表達(dá)量先降低后升高再降低;Poly(I:C)刺激組Hd Hc1表達(dá)量先降低,在48 h突然升高,然后再降低,Hd Hc2-1表達(dá)量先降低在48 h突然升高,然后再降低;注射PBS的對照組:Hd Hc1和Hd Hc2-1表達(dá)量變化趨勢相同,均是先升高后降低;注射PBS的對照組以及四個刺激組的雜色鮑的4種組織中均未檢測到Hd Hc2-2的表達(dá)。根據(jù)qPCR結(jié)果,進(jìn)一步運(yùn)用FISH技術(shù)對外界刺激后血藍(lán)蛋白基因在組織中的具體表達(dá)部位進(jìn)行了研究。Poly(I:C)刺激48 h后腹足中的Hd Hc的FISH結(jié)果表明,Hd Hc1陽性信號主要集中在腹足的邊緣處,即接觸環(huán)境的那一側(cè),而腹足肌肉的內(nèi)部Hd Hc1陽性信號較少;腹足邊緣Hd Hc2-1的陽性信號明顯少于Hd Hc1,腹足肌肉內(nèi)部的Hd Hc2-1的陽性信號也成零星分布。綜上所述,雜色鮑血藍(lán)蛋白與歐洲鮑血藍(lán)蛋白親緣關(guān)系最近,軟體動物血藍(lán)蛋白不同亞基的進(jìn)化早于種屬的進(jìn)化,Hd Hc2-2可能是假基因。雜色鮑血藍(lán)蛋白基因Hd Hc1和Hd Hc2-1為誘導(dǎo)型表達(dá)基因,參與了幼體發(fā)育、抗細(xì)菌及抗病毒的免疫過程;細(xì)菌和病毒刺激后,雜色鮑無細(xì)胞血漿的抑菌性顯著增強(qiáng)可能與血藍(lán)蛋白的表達(dá)調(diào)控有關(guān)。
[Abstract]:Hemocyanin (hemocyanin) also called hemocyanin, and earthworm hemoglobin (hemerythrin), hemoglobin (hemoglobin) and three respiratory proteins. The nature of hemocyanin mainly distributed in arthropod animal and animal blood with Bazhong software, very rich in content, with the transport of oxygen, regulating the osmotic pressure, skin curing, regulating molting the process of transport, metal ions, storage protein, antibacterial, antiviral and anti-tumor function. However, in recent years the study of hemocyanin mainly focused on the structure and function of the protein, and the relationship between the gene structure of blue blood protein especially soft animal hemocyanin gene subtype and its function the research of gene sequence analysis. In this paper, fluorescence quantitative PCR (qPCR) and fluorescence in situ hybridization (FISH) (Haliotis diversicolor) of Haliotis diversicolor hemocyanin gene structure of pathogenic thorn Shock response and so on, in-depth understanding of software animal hemocyanin this sequence, multi function, gene evolution and the immune response mechanism of the high molecular weight of immune molecules is of great significance. Firstly, using biological information on research group clone tool abalone hemocyanin gene Hc2-1 and Hd Hd Hc1. Hd Hc2-2 sequence analysis. The results showed that Hd Hc and the European Abalone (H.tuberculata), keyhole limpet (M.Crenulata) and other gastropod animal Hc were clustered into one clade, genetic distance; gene structure of Hd Hc and Ht Hc gene structure is similar, there are 8 functional units (FU-a~FU-h, FU-a). FU-f, FU-g respectively by 4,3,2 exons encoding, the other 5 functional units is continuous. The same and the European abalone, Hd Hc also contains 0,1,2 three intron phase, the difference is that Hd Hc1 and Hd Hc2-1 7 are connected within intron 1 phase, 6 internal There are 3 phase 0 introns, 1 phase 1, 2 phase 2, and the 7 intron Hd Hc2-2 connection is 0 phase, 6 internal intron 1 phase 0, 2 phase 1, 3 phase 2. Secondly, artificial injection of pathogenic Vibrio harveyi (Vibrio harveyi abalone), herpes simplex virus (Abalone herpesvirus, Ab HV) to stimulate healthy H.diversicolor, concentration of total soluble protein was detected in the haemolymph, superoxide dismutase (Superoxide Dismutase, SOD), acid phosphatase (Acid Phosphatase, ACP), alkaline phosphatase (Alkaline Phosphatase, AKP) activity and cell free plasma the results showed that the antibacterial activity, (1) v. Harvey and Ab after HV stimulation, soluble total protein concentration in the hemolymph of abalone first increased significantly, decreased significantly. (2) v. Harvey and Ab after HV stimulation, abalone hemolymph of ACP and the activity of AKP increased significantly, SOD stimulation group v. Harvey increased significantly Ab, decreased significantly after HV stimulation group, SOD decreased significantly. (3) abalone cell free haemolymph of Vibrio harveyi, Vibrio vulnificus (Vibrio vulnificus) and Vibrio coralliilyticus (Vibrio coralliiluyitcus) the three pathogens showed different degrees of inhibitory effects. These results suggest that the pathogen stimulates the change of immune related factors in abalone hemolymph, making antibacterial activity enhanced. However, as a kind of immune molecule content in hemolymph of the highest, the expression of hemocyanin may be regulated. By using fluorescence quantitative PCR (qPCR) of each period as well as the development of abalone embryo detection of Vibrio harveyi, Vibrio coralliilyticus, Ab HV Poly, (I:C) after stimulation inthe foot mantle, gill, hepatopancreas, hemocyanin gene in Hd Hc1, the relative expression of Hd Hc2-1 and Hd Hc2-2, to analyze its relationship with hemolymph immune activity changes. The results showed that: (1) blue blood protein two gene Hd Hc1, Hd Hc2-1 in the early development of Haliotis diversicolor was expressed relative to the expression of Hd the expression of Hc2-1 was higher than that of Hd Hc1, there was no expression of Hd Hc2-2. (2) of Vibrio harveyi, Vibrio coralliilyticus, Ab HV, Poly (I:C) complex stimulus the color of abalone, foot, hepatopancreas, gill, mantle of the hemocyanin gene Hd Hc1 and Hd Hc2-1 relative expression quantity changes, but changes in different tissues, different stimulation groups. 0 h compared with the blank control group, Vibrio harveyi stimulated Hd Hc1 expression increased first and then decreased Hd, the expression of Hc2-1 decreased after the first increased and then decreased; Vibrio coralliilyticus stimulated Hd Hc1 expression decreased, Hd expression increased Hc2-1 reduced and then increased; Ab HV stimulated Hd Hc1 expression decreased after the first increased and then decreased, the Hd expression of Hc2-1 decreased first and then increased and then decreased; Poly (I:C Hd Hc1 stimulation group) The expression decreased at 48 h, suddenly increased, then decreased, the Hd expression of Hc2-1 decreased after a sudden increase in 48 h, then decreased; the control group injected with PBS: Hd Hc1 and Hd Hc2-1 expression in the same trend was first increased and then decreased; the control group was injected with PBS and four stimulus the 4 group of Haliotis diversicolor tissue was not detected the expression of Hd Hc2-2. According to the results of qPCR, further use of FISH technology for the research of.Poly specific expression of hemocyanin gene in the tissues of external stimuli (I:C) FISH results after 48 h stimulation were Hd Hc showed positive signals Hd Hc1 mainly concentrated at the edge of the contact side of the foot, the environment, and the internal Hd muscle gastropod Hc1 positive signal is less; the positive signal of Hd was less than Hc2-1 from the edge of the Hd Hc1, the positive signals of Hd Hc2-1 gastropod inside the muscle is fully scattered. The recent relationship between hemocyanin and the European abalone Haliotis diversicolor hemocyanin genetic, animal software hemocyanin subunits evolved early in the evolution of species, Hd Hc2-2 may be a pseudogene. Abalone hemocyanin gene Hc1 and Hd Hd Hc2-1 for inducible gene expression in larval development, immune the process of anti bacteria and anti virus; bacterial and viral stimulation, inhibition of Haliotis diversicolor cell-free plasma significantly enhanced expression may be related with the regulation of hemocyanin.

【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S917.4

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