论文标题:腰带长体茧蜂抵御其寄主亚洲玉米螟细胞免疫反应的机理研究
Mechanisms That Polyembryonic Parasitoids Microcentrus Cingulum Resist the Immune Reaction of Host Ostrinia Furnacalis
论文作者 胡建
论文导师 符文俊,论文学位 博士,论文专业 动物学
论文单位 中国科学院研究生院（上海生命科学研究院）,点击次数 139,论文页数 128页File Size1535k
2003-06-01论文网 http://www.lw23.com/lunwen_26643767/ 腰带长体茧蜂;亚洲玉米螟;血细胞;包囊;黑化;多克隆抗体;cDNA 文库;基因克隆
Macrocentrus cingulum;Ostrinia furnacalis;hemocytes;encapsulation;melanization;polyclonal antibody;cDNA libaray;gene clone
多胚发育寄生蜂腰带长体茧蜂(Microcentrus cingulum)可以成功的寄生亚洲玉米螟(Ostrinia furnacalis)幼虫。作为寄生蜂防御寄主免疫反应的重要因子之一，蜂卵表面成分在保护寄生蜂逃避寄主的免疫反应过程中起重要作用。本文主要从亚洲玉米螟幼虫的细胞免疫反应、腰带长体茧蜂寄生对寄主幼虫细胞免疫反应的影响和寄生蜂逃避寄主免疫反应的机制进行了生理、生化和分子生物学研究。通过这些研究为利用寄生蜂防治重要农业害虫提供一些理论依据。本文主要研究内容如下：1．根据光镜和电子显微镜的观察结果将亚洲玉米螟幼虫血细胞分为五类：粒细胞、浆细胞、类绛色细胞、原血细胞和球形血细胞。调查了血细胞总数（THC）和不同类型血细胞数量（DHC）的变化。从三龄末期到五龄第五天期间，亚洲玉米螟幼虫的THC在蜕皮前后会下降，蜕皮后12小时左右降到最低点然后慢慢回升。在五龄蜕皮后12小时至五龄第五天期间，浆血细胞在五龄第三天前呈增加趋势，之后开始下降，而粒细胞则呈相反趋势。浆细胞和粒细胞具有附着延展性，可以附着在载玻片表面，但浆细胞的延展能力要明显强于粒细胞，而其它三种血细胞没有这个性质。血细胞可以包囊Sephadex A-25凝胶珠，在注射后2小时内就有79%的珠子被包囊，到注射后24小时已有98%的珠子被包囊，包囊程度随时间的增加而增强。部分被包囊的珠子会出现黑化现象。2．通过对寄生与未寄生的亚洲玉米螟幼虫血细胞在血细胞总数（THC）、不同类型血细胞数量（DHC）、形态、血细胞的延展性、和对Sephadex A-25葡聚糖凝胶珠的包囊以及包囊的黑化几个方面进行了比较，发现寄生后的幼虫与未寄生幼虫的血细胞在以上几个方面都没有明显差异。说明腰带长体茧蜂的寄生对寄主幼虫血细胞的免疫反应并没有产生明显的影响。3．位于腰带长体茧蜂雌蜂卵小管中的未成熟的卵母细胞表面有一层滤泡细胞，而位于侧输卵管中的成熟卵表面则没有。在注入寄主幼虫体内后，卵母细胞和崩溃酶处理后的成熟的卵可以被寄主血细胞包囊而侧输卵管中的成熟的卵则不能被包囊。电镜结果表明成熟卵表面覆盖有一层纤维层，崩溃酶处理后，成熟卵表面的纤维层被除掉。表明卵表面纤维层在保护寄生蜂逃避寄主免疫反应过程中起一定的作用。寄生蜂卵、胚胎和幼虫都可以被FITC-lectin标记，但卵和胚胎的标记程度较深。卵和胚胎中的一种97kDa的蛋白可以被明显的与FITC-lectin结合，推测这种97kDa 的蛋白在寄生蜂抵抗寄主免疫反应过程中起重要作用。4．构建了腰带长体茧蜂胚胎的cDNA文库。首先从寄生到寄主幼虫体内６d的寄生蜂胚胎中提取总RNA和mRNA，使用分离到的微量mRNA合成cDNA第1链。然后通过长距离 PCR扩增得到足够量的双链cDNA。将Sfi I酶切后并纯化的cDNA片段连接到经Sfi I酶切的噬菌体λTripIEX2上, 并对噬菌体进行包装建成cDNA的全长库。经大肠杆菌XL1- Blue平板检测未扩增文库的滴度为0.75(106 pfu?ml-1, 克隆重组百分率为96.77%, 扩增后文库的滴度为0.5(1010 pfu?ml-1。5．通过制备胶电泳分离所需的目标蛋白，割下目标蛋白条带直接免疫BALB/c小鼠，制备了97kDa蛋白的多克隆抗体，ELISA检测和点杂交的方法确定抗血清的效价。使用多克隆抗体作为免疫学探针在腰带长体茧蜂cDNA文库中筛选到阳性克隆，然后分别进行转化、质粒提取、酶切鉴定和测序。同时使用PCR方法从cDNA文库中得到一1000kb左右的基因片段。
Polyembryonic wasp, Microcentrus cigululm may parasitize the larvae of Ostrinia furnacalis successfully. As one of the important factors that parasitoids avoid the immune system of host, materials in wasps egg surface play an important role in protecting parasitoids from host"s immune reaction. This paper includes researches on physiology, biochemistry and molecular biology in the fields of hemocyte immune reactions of Ostrinia furnacalis larvae, effects of parasitization by Microcentrus cigulum on the immune reaction of host"s hemocytes and strategies that wasps avoid host"s immune system. The results are as follows: 1. Using phase contrast microscopy and electronic transmission microscopy, five types of hemocytes were determined in the hemolymph of Ostrinia furnacalis larvae, namely Granular hemocytes, Plasmatocytes, Oenocytoids, Prohemocytes and Spherule cells. Total hemocyte counts (THC) and differential hemocyte counts (DHC) were compared in the hemolymph of the larvae. From the late stage of third instar till fifth day of fifth instar, it is interesting to note that THC increased shortly before and after ecdysis and thereafter decreased to a basal level. During the first day to the fifth day of the fifth instar, the amount of plasmatocytes increased before the third day and decreased after that but granular hemocytes showed a reversed profile. In vitro test both plastomatocytes and granularcytes showed spreading and adhesive behaviour but their spreading abilities were different. When Sephadex A-25 beads were injected into the fifth-instarlarvae they were soon encapsulated and some of them were melanized. Hemocytes were also able to encapsulate the latex beads in vitro test. No difference of the capsule structure could be seen.2. Comparing hemocyte counts ( THC and DHC), morphologies, spreading behavior, encapsulation on Sephadex A-25 and melanization of capsules of hemocytes in parasitized host larvae and normal larvae, no distinct differences were observed. Therefore, parasitization of Macrocentrus cingulum has no significant effect on the immune reaction of the hemcoytes of host, Ostrinia furnacalis larvae.3. Immature egg excised from ovariole of the female wasp has a sheath of follicular epithelium but mature egg from lateral oviducts does not have it. Injected M. cingulum mature eggs were not encapsulated, while immature eggs and Driselase treated eggs provoked an encapsulation response. Inspection of eggs by transmission electron microscopy revealed that there is a fibrous layer on the surface of mature eggs and Driselase collapsed the surface fibrous layer of the eggs, indicating that surface fibrous layer may play a role in protecting eggs from host"s immune attack. Egg, embryonic and larvae of M. cingulum can be labelled by FITC-lectin, but labelling on larvae was weak. A 97kDa protein in eggs and embryonics can be conjugated with FITC-lectin, speculating this 97kDa protein play an important role in wasp resisting host"s immune reaction.4. A cDNA library of Macrocentrus cingulum embryos, excised from their host Ostrinia furnacalis 6 days after parasitism has been constructed. After synthesizing the first-strand cDNA using limited amount of mRNA isolated from total RNA, the double-strand cDNA was amplified with long-distance PCR method. Then the ligation of the Sfi I digested cDNA to the Sfi I digested vector λTripIEX2 was perpormed and the phage was packaged to construct the full longth cDNA library. It was determined that the titer of unamplified library was 0.75(106 pfu?ml-1 and thepercentage of recombinant clones was 96.77% and the titer of amplified library was 0.5(1010 pfu?ml-1 by plating E.coli XL1- Blue. 5. 97 kDa protein combined with other proteins extracted from embryo was run SDS-polyacrylamide electrophoresis and 97kDa protein was cut from gel to immunize BALB/c mice directly. ELISA and Dot-blotting were used to determine the titer of the above prepared antisera. Using polyclonal antibody as immune probe to screen cDNA libaray, a positive plaque was pi
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