栉孔扇贝与海湾扇贝免疫学比较研究

IOCAS-IR > 海洋环流与波动重点实验室

	栉孔扇贝与海湾扇贝免疫学比较研究
其他题名	The comparative immunological study on two scallops Chlamys farreri and Argopecten irradians
	高强
学位类型	博士
	2007-06-08
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	海湾扇贝栉孔扇贝热休克蛋白 Mrna表达免疫防御重金属
摘要	扇贝是我国重要的海水养殖品种，但自1997年以来，养殖扇贝陆续爆发的大规模死亡，不但造成了巨大的经济损失，而且直接威胁到现有产业的生存和发展。引起扇贝大规模死亡原因是多方面的，其主要原因是病原滋生、养殖环境恶化和种质衰退。因此，深入研究扇贝免疫防御机制，探讨提高机体抗病力的有效途径和方法，改良种质和培育抗病品系，促进我国扇贝养殖业的健康、可持续发展。本文通过比较栉孔扇贝和海湾扇贝正常状态和重金属污染胁迫下相关免疫指标的变化及HSP70和HSP90在重金属胁迫后表达规律，以期更好的了解贝类的防御机制，为扇贝病害防治提供资料。本研究采用流式细胞仪技术对栉孔扇贝和海湾扇贝血细胞死亡率、细胞吞噬率和呼吸爆发进行了测定，发现正常状态下两种扇贝的细胞死亡率相差不大，呼吸爆发的基础值相差也不大，而海湾扇贝细胞吞噬率显著高于栉孔扇贝。在血淋巴和肝胰腺中，海湾扇贝SOD、ACP酶活性均显著高于栉孔扇贝；海湾扇贝的MDA含量也高于栉孔扇贝，但差异不显著。鳗弧菌感染实验发现，海湾扇贝累积死亡率远低于栉孔扇贝。上述结果表明，海湾扇贝对细菌等异物的吞噬和杀灭能力以及机体自身的抗氧化能力高于栉孔扇贝，这为海湾扇贝比栉孔扇贝具有更高的抗逆性提供了证据。不同浓度Pb2+刺激后，海湾扇贝血细胞的死亡率明显低于栉孔扇贝。海湾扇贝细胞吞噬率和呼吸爆发均高于栉孔扇贝。对两种扇贝体液免疫指标的测定发现，海湾扇贝的SOD、ACP活性和MDA含量均高于栉孔扇贝。结果表明，在Pb2+刺激下两种扇贝都处于重金属氧化胁迫下，其免疫系统均受到了影响，但相同剂量的Pb2+对两种扇贝的毒害程度不同，海湾扇贝受到的影响要低于栉孔扇贝。采用同源克隆和cDNA 末端快速扩增（RACE）技术，从海湾扇贝血淋巴中克隆到了热休克蛋白90（AiHSP90）基因。AiHSP90的cDNA序列全长为2669bp，其中5' 非编码区（UTR）为90bp，3' UTR为44bp，开放阅读框（Open Reading Frame, ORF）包括2175bp，2524-2528位AATAA为推测的AiHSP90基因的 mRNA 多聚腺苷酸加尾信号（Polyadenylation Signal）。开放阅读框编码724个氨基酸残基，推测的理论分子量为83.08 kDa，等电点为4.81。利用荧光实时定量PCR 技术检测了AiHSP90基因在革兰氏阴性菌鳗弧菌和革兰氏阳性菌藤黄微球菌感染后不同时间点相对于β-actin基因的表达。在鳗弧菌和藤黄微球菌刺激后，除72h外AiHSP90均上调表达，在9h其mRNA表达达到最大值，分别约为空白组的6.8倍和3倍；随后随着时间的推移，其表达下降，在48h恢复到接近空白组的水平；到72h时降到最低点。鳗弧菌对AiHSP90的诱导比藤黄微球菌要高，这也反映出鳗弧菌对海湾扇贝有更强的毒力，诱导的HSP90表达也更强。本研究首次揭示出在软体动物中HSP90在细菌感染条件下的表达情况，结果表明海湾扇贝AiHSP90参与了机体对细菌感染的应答。采用荧光实时定量PCR技术，对栉孔扇贝和海湾扇贝在不同浓度、不同种类重金属胁迫后HSP70和HSP90基因表达规律进行了比较。结果显示，镉和铅对栉孔扇贝处理10天后即诱导HSP70以较高水平表达，20天后镉处理组HSP70恢复到与对照接近的水平，而在铅处理组也呈现出下降的趋势。而海湾扇贝HSP70基因表达与栉孔扇贝恰好相反，处理20天后其HSP70表达才出现较高水平。铜离子处理对诱导两种扇贝HSP70的影响相似，对栉孔扇贝HSP70的诱导强度更大。对于同一种重金属，不同的浓度诱导的HSP70表达也各不相同。而对于HSP90，海湾扇贝在受到三种重金属刺激后HSP90的表达量均高于栉孔扇贝，而且长时间的刺激（20天）诱导了更高的HSP90基因表达。不同的浓度的同一种重金属离子诱导的HSP90表达也各不相同。这些结果表明，在重金属胁迫下HSP70以及HSP90在栉孔扇贝和海湾扇贝中均表现出不同的应答方式。结合前面海湾扇贝抗逆性较强的综合结果，两种扇贝热休克蛋白基因表达的比较提示我们栉孔扇贝HSP70的激烈反应预示着抗逆能力的不足，而海湾扇贝HSP90的高表达却预示着更强的抗逆能力，有待于更多应激实验如热刺激、病原刺激等的进一步验证。
其他摘要	Scallop aquaculture is a big industry and contributes enormously to the economic development of coastal provinces in China. Since the summer of 1997, large-scale mortality of cultured scallop has caused catastrophic losses to scallop aquaculture, which resulted in the production decreasing drastically. There are a range of factors causing the mortality, which could be divided into three subgroups as pathogens, breed degeneration and deteriorative environment. Intensive investigation on scallop immune defense mechanism, looking for ways to enhance resistance to diseases and improving breed quality would be the final solution to the problems of scallop aquaculture and would be of great importance to sustainable development of aquaculture in China. In this paper, comparative study on immune parameters of Zhikong scallop and bay scallop was executed under both stress and stressless condition. The comparison of HSP70 and HSP90 gene expression between the two scallops under heavy metal stress was made. This will help to better understanding the internal defense mechanisms of scallop immunity, to select disease-resistant strains, and provide basic data for diseases prevention in shellfish aquaculture. Flow-cytometric method was adopted to determine hemocyte mortality, phagocytosis capacity and the respiratory burst of Zhikong scallop and bay scallop. There were slightly differences in hemocyte mortality and respiratory burst between the two scallops. But the phagocytosis rate in bay scallop was significantly higher than that of Zhikong scallp. In haemolymph and hepatopancreas, bay scallop had a significantly higher SOD and ACP activities than Zhikong scallop. MDA content in bay scallop was also higher, but there was no significant difference between the two scallops. In the Vibrio anguilarum infection experiment, Zhikong scallop displayed a higher accumulated mortality rate. It indicated that bay scallop was more capable of destroying invading pathogens and protect organism from oxidative damage than Zhikong scallop. Under the stress of different Pb2+ stress, the haemocyte mortality rate was lower in bay scallop than that of Zhikong scallop. Bay scallop also had a higher phagocytosis rate and respiratory burst. Measurement of humoral immunity parameter showed that SOD, ACP activities and MDA content was higher in bay scallop than in Zhikong scallop. These parameters demonstrated both scallops encountered oxidative stress exposed to Pb2+ but the immunity of bay scallop was less affected. The full length cDNA of bay scallop Argopecten irradians HSP90 (designated AiHSP90) was cloned by the techniques of homology cloning and rapid amplification of cDNA ends (RACE). It was of 2669 bp, including a 5’-terminal untranslated region (UTR) of 90 bp, a 3’-terminal UTR of 404 bp with a canonical polyadenylation signal sequence AATAAA, a poly (A) tail, and an open reading frame (ORF) of 2175 bp encoding a polypeptide of 724 amino acids, with a predicted molecular weight of 83.08 kDa and theoretical isoelectric point of 4.81. Fluorescent real-time quantitative RT-PCR was used to examine the expression pattern of AiHSP90 mRNA in haemocytes of scallops challenged by Gram-negative bacteria Vibrio anguillarum and Gram-positive bacteria Micrococcus luteus. In both bacterial challenged groups, AiHSP90 mRNA level increased and reached maximal level at 9h after injection and then dropped progressively. The increase of AiHSP90 transcript was higher in V. anguillarum challenged groups than that in M. luteus challenged groups, which suggested that V. anguillarum was a stronger inducer of AiHSP90. This is the first time such a response of HSP90 has been observed in a molluscan species, which indicated that AiHSP90 was involved in scallop’s response to bacteria infection. Such data could help to understand the significance of HSP expression to scallop immune defence. Fluorescent real-time quantitative RT-PCR was used to examine the mRNA expression pattern of HSP70 and HSP90 from Zhikong scallop (designated CfHSP70 and CfHSP90, respectively) and bay scallop (designated AiHSP70 and AiHSP90, respectively) in haemocytes of scallops under the stress of different concentrations of Cd2+, Pb2+ and Cu2+. CfHSP70 mRNA expression was at high level after 10 days of Cd2+ or Pb2+ exposure. After 20 days of exposure, CfHSP70 in Cd2+ group recovered to nearly the same level of control group and in Pb2+ group CfHSP70 also displayed a trend to decline. On the contrary, AiHSP70 mRNA expression reached high level only after 20 days of Cd2+ or Pb2+ exposure. Cu2+ treatment showed similar effects on both HSP70s of the two scallops. But a higher induction of HSP70 was observed in bay scallop. For the same heavy metal, different concentrations led to different induction of HSP70. For HSP90, AiHSP90 showed a higher expression than CfHSP90 in all the three heavy metal treated groups and a longer exposure induced higher expression of AiHSP90. For the same heavy metal, different concentrations led to different induction of HSP90. The results indicated that gene expression manner of HSP70 and HSP90 was different under heavy metal stress. Considering bay scallop living better under stress, which was demonstrated in the former experiments, the comparison of HSPs gene expression between the two scallops under heavy metal stress indicated that higher expression of Zhikong scallop HSP70 implied weaker resistance to stress while higher expression of bay scallop HSP90 implied stronger resistance to stress. Further investigations on more stress experiments such as heat, pathogen challenges shoud be carried out to testify the deduction.
页数	129
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/453
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	高强. 栉孔扇贝与海湾扇贝免疫学比较研究[D]. 海洋研究所. 中国科学院海洋研究所,2007.

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