【作者】 王丽燕；

【导师】 樊廷俊；

【作者基本信息】 中国海洋大学 ， 细胞生物学， 2010， 博士

【摘要】 卤虫又名盐水丰年虫,隶属于节肢动物门,甲壳纲,具有广温、耐高盐等特性,因其营养价值极高,且其休眠卵便于保存,运输等优点成为海水养殖业中非常重要的饵料生物。近年来,在甲壳动物疾病感染机制、生物体抗性反应机制和饵料营养价值分析等方面被作为一种极好的模式生物进行了的相关研究。与其他无脊椎动物类似,卤虫缺乏特异性免疫系统,没有免疫记忆能力,只能依靠非特异性免疫反应来抵御病原菌侵染,其中,由酚氧化酶原激活系统、溶酶体酶(含溶菌酶)和抗氧化酶类(含超氧化物歧化酶)等构成的体液免疫反应,在其免疫防御中发挥着十分重要的作用,其中,酚氧化酶、溶菌酶和超氧化物歧化酶已被用作评价甲壳动物免疫力的指标之一近年来,甲壳动物如虾、蟹等的养殖病害的发生日趋严重,其中,细菌类疾病中由弧菌引起的养殖动物的致死率最高,使养殖业遭受了巨大的经济损失,已成为阻碍水产养殖业健康发展的限制性因素。使用化学药物进行防治的方法,因易造成环境污染、药物残留、危害人体健康等问题,其应用已受到了严格的限制。开拓新的有效的病害防治方法,变得尤为迫切。通过研究甲壳动物的免疫机制,提高甲壳动物免疫力,从而增强其抗病力的途径被认为是解决养殖甲壳动物疾病爆发的关键。本文比较研究了野生型(WT)面包酵母菌(SC)和其细胞壁同源突变型(mnn9)两种SC酵母菌株作为卤虫饵料,对卤虫抗弧菌VC感染的免疫机制的影响作用。结果发现,分别投喂卤虫WT和mnn9,于培养两天后,两组卤虫的存活率出现显著性差异,同时,对其进行VC侵染实验发现,于侵染24h时,两组之间也出现显著性差异,而且均是投喂mnn9的卤虫明显高于投喂WT的卤虫,随着培养时间的延长,这种差异越来越大。对培养至第三天时投喂WT或mnn9的卤虫体长分别进行了测定发现,投喂mnn9的卤虫体长明显大于投喂WT的卤虫。另外,又对投喂WT和mnn9的卤虫的PO、SOD和LSZ活性变化进行了检测,发现于侵染后不同时间,投喂mnn9的卤虫的三种免疫酶活性均明显高于投喂WT的卤虫。在无弧菌侵染的情况下,得到了与此相一致的结论。分析认为,mnn9相对于WT,对卤虫具有更高的营养价值,并且其细胞壁突变使其成分发生变化,β-葡聚糖及几丁质含量均高于WT酵母菌,因此推测,mnn9首先可以通过营养强化增强卤虫体质,又可以通过细胞壁免疫刺激成份如β-葡聚糖和几丁质形成对卤虫非特异性免疫系统持续有益的刺激,从而提高卤虫的免疫酶活性表达水平,增强其抗VC感染的免疫力,进而提高了存活率。弧菌VC对卤虫进行侵染后发现,于侵染后24h时,投喂WT的卤虫的存活率明显降低,并随着侵染时间的延长,急剧下降。对其PO、SOD和LSZ活性的检测同样发现,三种免疫酶活性也均于侵染后不同时间出现了显著性下降。说明弧菌VC侵染导致了卤虫先天性免疫系统受损,从而降低了相关的免疫酶活性,引起卤虫存活率降低。WT或mnn9经热激诱导后,蛋白免疫印迹实验显示其热激蛋白Hsp70的表达量增多,投喂卤虫热激WT后发现,于侵染后48 h-72 h时,其存活率比投喂正常培养WT的卤虫的明显更高。对其PO、SOD和LSZ三种免疫相关酶活性的检测同样发现,投喂热激WT的卤虫具有更高的酶活性。因此分析认为,酵母菌热激后热激蛋白的增多可能是导致卤虫免疫系统增强的主要因素,通过某种仍然未知的机制,刺激了卤虫的非特异性免疫系统,使其免疫酶活性水平增高,最终提高了卤虫抗感染的能力,使存活率升高。本文利用已知美国卤虫的酚氧化酶原cDNA序列全长设计引物,利用反转录-聚合酶链式反应(RT-PCR)方法,克隆并拼接获得了中国卤虫proPO的基因序列全长共2125bp,编码699个氨基酸残基,分子量为80.37kDa,等电点为6.48。与美国卤虫的相似性达到99%。对其序列的保守功能结构域进行分析,发现proPO含有一个蛋白酶切位点推测位于Arg75-Gly76,2个铜离子结合位点和一个1个硫羟酸酯结构域。2个铜离子结合位点(CuA和CuB)内含有六个高度保守的组氨酸残基(位于氨基酸225,229,251和379,383,419处)。1个硫羟酸酯结构域(GCGWPEHM)位于氨基酸592-599处,也高度保守。通过分析得知中国卤虫proPO无信号肽序列。对其二级及三级结构的预测发现其铜离子保守结构区为α-螺旋结构。系统进化分析发现,卤虫远离其他十足目甲壳动物,与水蚤形成甲壳动物的另一进化分支。以卤虫β-actin基因为内参,以获得的proPO基因序列设计特异性引物,利用半定量RT-PCR方法对中国卤虫5个不同发育时期(0h,6h, E (E), InstarⅠ和InstarⅡ期)proPO基因的转录表达水平进行了检测,发现卤虫的proPO基因从E期开始出现转录表达,从E期到InstarⅡ期的表达水平没有明显差异。同时对这5个不同发育时期卤虫的PO活性进行了检测,发现只有InstarⅡ期幼虫才有明显的PO活性。说明卤虫proPO转录本与PO酶活性的表达变化在胚胎发育过程中受到了调控。本文比较研究了弧菌侵染、饵料营养与免疫成分刺激及热激蛋白对卤虫免疫机制的影响作用,对进一步了解目前仍不详细的甲壳动物的免疫机制奠定了一定的理论基础,并为开拓新的增强甲壳动物免疫力的方法如利用免疫刺激物或Hsp的应用等提供了重要的理论依据。并首次对PO在卤虫早期发育中的表达进行研究,为深入了解proPO在体内的表达调控机制及其免疫功能等打下基础,并为免疫防病的研究提供理论基础。更多还原

【Abstract】 The brine shrimp, Artemia, belong to Phylum Arthropoda, Class Crustacea, is one of the most important live feeds for commercial production of fish and shellfish larvae. This organism is unique in the animal kingdom, because it is extremely euryhaline and can produce cysts (encapsulated embryos), which can be easily stored and transported. Recently, it has been used as an excellent model organism to study the biology of infections in Crustaceans, stress response and feed quality analysis and so on. Similar to the other invertebrates, Artemia can only rely on their non-specific immunology system and have no immunological memory. So the immune effectors in haemolymph such as prophenoloxidase-activating system(proPO-AS), superoxide dismutase (SOD) and lysozyme (LSZ) play a critical role for the immune reaction. At the present time, PO, SOD and LSZ have been used as indicator of health status and pathogen infection in crustacean. Recently, crustacean such as shrimp and crab culture around the world has suffered problems linked to deteriorating environments due to development of intensification, subsequently resulting in stress-induced disease incidence. Bacteria such as vibrio are by far the most serious, causing massive mortalities of cultured organisms worldwild. Diseases have become the major inhibitor of aquaculture production, and lead to the massive financial losses. The use of chemotherapeutic drug have sever negative impact on living organism and the environment, such as inducing the development of resistant pathogens, and moreover, contaminating the environment and accumulating in tissues of seafood, then could be harmful to people’s health So its use has been restricted. In order to solve this problems, some new bio-control strategies and environmently friendly prophylactic alternatives were required urgently, and the health of organisms and enhancement of their immunity are of primary concern.In this study, wild type (WT) of Saccharomyces cerevisiae (SC) and its cell wall mutant mnn9 were chose as Artemia larvae feed, effects of WT and mnn9 on protecting Artemia to resist Vibrio campebllii were studied. The survival of Artemia fed with mnn9 was significantly higher than that of Artemia fed with WT after two days incubation with the feed schedule, at the mean time, the same significant difference occured after 24 h of vibrio infection, and this significance between them became bigger in the following incubation.What’s more, the average length of Artemia larvae was measured, and the results showed that Artemia larvae fed with mnn9 had longer length of the whole body. Regardless of larval survival and larval length, PO, SOD and LSZ activity of Artemia fed with WT or mnn9 were checked following the Vibrio challenge or non-challenge test, then the similar results were found that these three enzyme activity showed higher in Artemia fed with mnn9 in different incubation time whatever under Vibrio challenge or not. Therefore, mnn9 was deduced to maily have two effects on Artemia larvae. Firstly, mnn9 has higher nutritional quality for Artemia than WT, so it can make Artemia larvae to be more healthier and stronger. Secondly, mnn9 for its mutation in cell wall has moreβ-glucan and chitin which are immunostimulants as known, then these immunostimulants could stimulant the innate immune system of Artemia larvae, and enhance the immune reaction by improving the immune-related enzyme activity such as PO, SOD and LSZ, and improve the survival of Artemia larvae in the end.Larval surval of Artemia fed with WT significantly decreased after 24h challenging with Vibrio campbellii, and decreased quikly in the following incubation. In addition, PO, SOD and LSZ activity of Artemia larvae were checked after Vibrio challenge and also showed significantly decrease in different challenging time. what means that Vibrio campbellii could damage the host immune system, then lead to increasing mortality of Artemia larvae by inhibiting the immune-related enzyme activity.Artemia were fed with heat shocked WT and mnn9 respectively which were induced to produce more Hsp70, then Vibrio challenge test was arranged. The results showed larval survival of Artemia fed with WT significantly increase in 48-72h challenging time. In addition, PO, SOD and LSZ activity of Artemia larvae fed with heat shocked WT showed significantly increased in different vibrio challenge time. So the yeast of Hsps may stimulate Artemia immune response to resist the Vibrio infection by one kind of still unclear mechnism.A full length of 2125 bp AsproPO cDNA was cloned by RT-PCR using oligonucleotide primers based on the proPO sequence of Artemia franciscana. It contains an open reading frame of 2100 bp, and 25 bp of the 3’-untranslated region, and encodes protein of 699 amino acids with a predicted molecular weight of 80.37 kDa and with an estimated pI of 6.48. Analysis of the deduced amino acid sequces of AsproPO shows that The AsproPO has a proteolytic activation site (Arg75-Gly76), two putative copper-binding sites with six conserved histidine residues (CuA:225, 229,251and CuB:379,383,419) and a thiol-ester-like motif (GCGWPEHM) which are highly conserved inarthropods. However, no signal peptide sequence was detected. Analysis of sequence similarity in a range of arthropod taxa suggested that the proPO gene in Artemia was as dissimilar to other crustaceans as it was to insects.Semi-quantification RT-PCR was used to study expression of proPO in Artemia at five different developmental stages (0h,6h, Emergence(E), Instar I and Instar II), while the expression ofβ-actin gene as inner control. And PO enzymatic activity was also checked.The results showed that proPO expression was expressed at E stage, InstarⅠand InstarⅡstages, while significant PO activity was only found in larvae at InstarⅡstage. The expression analysis indicates that proPO system were modulated in Artemia early developing stages.In conclusion, effects of nutritional quality of feed and immunostimulation or vibrio infection and Hsps on Artemia immune response were studied,which suplies the academic groundwork for the still unclear crustacean immune mechanism. And some methods such as immunostimulation or Hsps to enhance Crustacean immune response were explored. For the first time, the expression of proPO and PO was studied and analyzed, which could help to deeply understand the immune fuction and modulation mechanism of proPO system, and make the academic support for disease control in Crustacean.更多还原