【作者】 吴任；

【导师】 谢数涛；

【作者基本信息】 暨南大学 ， 水生生物学， 2006， 硕士

【摘要】 热休克蛋白(HSPs)也称为应激蛋白，是一个高度保守的蛋白质家族，广泛存在于所有的细菌、酵母、植物、无脊椎动物和脊椎动物的细胞内。细胞在正常生理情况下构成性地表达该应激蛋白(constitutive HSPs)，也可由一系列应激因素包括细胞缺氧、能量衰竭、组织创伤、高温、重金属、病菌感染等应激产生诱导型的应激蛋白(inducible HSPs)。真核细胞内的热休克蛋白分子量从7 kDa到105 kDa大小不等，其中以热休克蛋白70(HSP70)在热应激中升高最为显著，作用最具代表性，而被广泛关注。非应激状态下，热休克蛋白70与错误折叠或未折叠的新生蛋白质相互作用，参与其细胞内的折叠、装配和转位，因而被称为“分子伴侣”蛋白(chaperones)，应激状态下，细胞的HSP70大量表达，并且对应激胁迫的耐受能力得到提高，避免受到进一步的损伤。因此，研究水生动物HSP70s与环境耐受性的相互关系对于改善水生动物的分布、生存和繁育有着极大推动作用。 本文首先通过热应激实验，确立凡纳滨对虾(Litopenaeus vannamei)的亚致死温度和致死温度，进而设计10℃温差来热休克凡纳滨对虾，继而再用致死高温胁迫，与未经预热休克的对照组相比，对虾的存活率达到50％以上；以同样条件热处理对虾，然后注射入致死量白斑综合症病毒(white spot syndromvirus，WSSV)，与对照组相比(46.89h)，对虾存活时间得到显著的提高(63.28-101.43h)；热休克后，在2-24h的恢复期中，对虾的存活时间在2小时恢复表现最高，恢复6小时后，对虾存活时间表现出明显下降。这表明热应激可诱导凡纳滨对虾对高温和病毒的耐受性。 利用RACE(rapid amplification of cDNA ends)方法，从凡纳滨对虾的鳃组织克隆到一种组成型的HSC70基因，该基因长2223 bp，含有一个长1959 bp的开放阅读框，29 bp的5’端非编码区和237 bp的3’端非编码区，3’端末端有一加尾信号(AATAAA)；利用特异引物从对虾基因组DNA中扩增HSC70的全长编码区，发现与HSC70 cDNA序列相一致，说明这种HSC70基因结构不包含有内含子；HSC70的推导氨基酸序列含有652个氨基酸，为一种胞质HSC70蛋白。更多还原

【Abstract】 Heat shock proteins (HSPs), also known as stress proteins, belong to one of highly conserved protein families. It is widly found in most cells or organs, from bacteria, yeasts, plants and animls. Under normal conditions, certain HSPs constitutively express in cells, and the expression of those inducible HSPs can be induced by a number of physiological signals, such as anoxia, energy depletion, injury, hyperthermia, heavy metals and pathogens. Eukaryotic HSPs are composed of HSP105/HSP100, HSP90, HSP70, HSP60 and low-molecular weight HSPs. The heat-depandent HSP70s is one of most representative families and undertakes fundamental functions in cells. Therefore, HSP70s have intrigued researchers’ great interest in its actions and mechanisms. To date, we know that HSP70s play an essential role in protein metabolism in both stressed and unstressed cells. HSP70s are implicated in nascent protein folding, membrane translocation, formation and degradation of denatured proteins. In addition, HSP70s demonstrate its potential protection against environmental stressors and participate in immune intermediation. Because of speciality of HSP70s, further studies on interaction between HSP70s and environmental adaptability in aquatic animals would greatly ameliorate aquatic animals’ distribution, survival and reproduction.We determined the sublethal and lethal temperatures for Litopenaeous vannamei via heat-shocked experiments. L. vannamei was treated by a jumped temperature (from 25 ℃ to 35 ℃) for 2 hours and then exposed to the lethal temperature (40 ℃) water, the survival rate increased up to 54.2 %. After exposed to the sublethal temperature for 2 hours, four divided groups of L. vannamei were maintained at room temperature (25±0.5℃) for 2, 6, 12 and 24 hours, respectively. Subsequently, the shrimps were injected with white spot syndrome virus (WSSV). The result showed that the mean survival time of the infected shrimps with recovery times of 2, 6,12 and 24 hours was 101.43,98.07,76.92 and 63.28 hours, respectively. The mean survival time of the control group, which was directly injected with WSSV without treatment of sublethal temperature, was 46.89 hours. The mean survival times of the更多还原