杂色鲍血蓝蛋白基因结构和表达规律研究

发布时间：2018-04-08 18:16

本文选题：杂色鲍　切入点：血蓝蛋白　出处：《上海海洋大学》2016年硕士论文

【摘要】：血蓝蛋白(hemocyanin)又称血蓝素,与蚯蚓血红蛋白(hemerythrin)、血红蛋白(hemoglobin)并称自然界三大呼吸蛋白。血蓝蛋白主要分布在节肢动物和软体动物血淋巴中,含量非常丰富,具有运输氧气、调节渗透压、表皮固化、调节蜕皮过程、转运金属离子、储存蛋白质、抗菌、抗病毒和抗肿瘤等多种功能。然而,近年来对于血蓝蛋白的研究多集中在其蛋白结构和功能方面,而对于血蓝蛋白的基因结构尤其是软体动物血蓝蛋白基因多亚型和其多功能之间的关系方面研究较少。本文运用基因序列分析、荧光定量PCR(qPCR)和荧光原位杂交技术(FISH)对杂色鲍(Haliotis diversicolor)血蓝蛋白的基因结构及其对病原刺激的响应等进行了研究,对深入了解软体动物血蓝蛋白这种多序列、多功能、高分子量免疫分子的基因演化和免疫响应机理具有重要的意义。首先,运用生物信息学工具软件对课题组克隆得到的杂色鲍血蓝蛋白基因Hd Hc1、Hd Hc2-1和Hd Hc2-2序列进行分析。结果显示,Hd Hc与欧洲鲍(H.tuberculata)、钥孔戚(M.Crenulata)等腹足纲动物的Hc聚为一支,遗传距离较近;Hd Hc基因结构与Ht Hc的基因结构相似,也有8个功能单元(FU-a~FU-h),FU-a、FU-f、FU-g分别由4、3、2个外显子编码,其余5个功能单元则是连续的。和欧洲鲍相同的是,Hd Hc也含有0、1、2三种内含子相位,不同的是Hd Hc1和Hd Hc2-1的7个连接内含子均是1相位,6个内部内含子中有3个0相位,1个1相位,2个2相位,而Hd Hc2-2的7个连接内含子都是0相位,6个内部内含子有1个0相位,2个1相位,3个2相位。其次,人工注射病原哈维氏弧菌(Vibrio harveyi)、鲍类疱疹病毒(Abalone herpesvirus,Ab HV)刺激健康杂色鲍,检测血淋巴中可溶性总蛋白的浓度、超氧化物歧化酶(Superoxide Dismutase,SOD)、酸性磷酸酶(Acid Phosphatase,ACP)、碱性磷酸酶(Alkaline Phosphatase,AKP)活性的变化以及无细胞血浆的抑菌性,结果表明,(1)哈维弧菌和Ab HV刺激后,杂色鲍血淋巴中可溶性总蛋白浓度先显著升高,后显著下降。(2)哈维弧菌和Ab HV刺激后,杂色鲍血淋巴中ACP和AKP活性显著升高,哈维弧菌刺激组的SOD先显著升高,后显著下降,Ab HV刺激组的SOD显著下降。(3)杂色鲍无细胞血淋巴对哈维氏弧菌、创伤弧菌(Vibrio vulnificus)和溶珊瑚弧菌(Vibrio coralliiluyitcus)这三种病原均表现出了不同程度的抑菌性增强的作用。这些结果表明病原刺激引起了杂色鲍免疫相关因子的变化,使得血淋巴的抑菌性增强。然而,作为血淋巴中含量最高的一种免疫分子,血蓝蛋白的表达可能也受到了调控。运用荧光定量PCR(qPCR)技术检测鲍胚胎发育各时期以及哈维氏弧菌,溶珊瑚弧菌,Ab HV,Poly(I:C)刺激后杂色鲍腹足、外套膜、鳃、肝胰腺中血蓝蛋白基因Hd Hc1、Hd Hc2-1和Hd Hc2-2相对表达量的变化,从而分析其与血淋巴免疫活性变化的关系。结果显示:(1)血蓝蛋白两个基因Hd Hc1、Hd Hc2-1在杂色鲍的发育初期即有表达,Hd Hc2-1的相对表达量高于Hd Hc1的表达量,未见Hd Hc2-2的表达。(2)哈维氏弧菌、溶珊瑚弧菌、Ab HV、Poly(I:C)刺激杂色鲍后,腹足、肝胰腺、鳃、外套膜中的血蓝蛋白基因Hd Hc1、Hd Hc2-1的相对表达量均有变化,但是不同组织、不同刺激组的变化规律不同。与0 h的空白对照组相比,哈维氏弧菌刺激组Hd Hc1表达量先升高后降低,Hd Hc2-1表达量先降低后升高再降低;溶珊瑚弧菌刺激组Hd Hc1表达量呈降低趋势,Hd Hc2-1表达量升高降低再升高;Ab HV刺激组Hd Hc1表达量先降低后升高再降低,Hd Hc2-1表达量先降低后升高再降低;Poly(I:C)刺激组Hd Hc1表达量先降低,在48 h突然升高,然后再降低,Hd Hc2-1表达量先降低在48 h突然升高,然后再降低;注射PBS的对照组:Hd Hc1和Hd Hc2-1表达量变化趋势相同,均是先升高后降低;注射PBS的对照组以及四个刺激组的杂色鲍的4种组织中均未检测到Hd Hc2-2的表达。根据qPCR结果,进一步运用FISH技术对外界刺激后血蓝蛋白基因在组织中的具体表达部位进行了研究。Poly(I:C)刺激48 h后腹足中的Hd Hc的FISH结果表明,Hd Hc1阳性信号主要集中在腹足的边缘处,即接触环境的那一侧,而腹足肌肉的内部Hd Hc1阳性信号较少;腹足边缘Hd Hc2-1的阳性信号明显少于Hd Hc1,腹足肌肉内部的Hd Hc2-1的阳性信号也成零星分布。综上所述,杂色鲍血蓝蛋白与欧洲鲍血蓝蛋白亲缘关系最近,软体动物血蓝蛋白不同亚基的进化早于种属的进化,Hd Hc2-2可能是假基因。杂色鲍血蓝蛋白基因Hd Hc1和Hd Hc2-1为诱导型表达基因,参与了幼体发育、抗细菌及抗病毒的免疫过程;细菌和病毒刺激后,杂色鲍无细胞血浆的抑菌性显著增强可能与血蓝蛋白的表达调控有关。
[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.

【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S917.4

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