【作者】 刘青娥；

【导师】 徐均焕；

【作者基本信息】 浙江大学 ， 微生物学， 2003， 硕士

【摘要】 酚氧化酶原激活系统（ProPO-AS）是一个有效的非自我识别系统，在昆虫的防御反应中起着重要的作用，它参与超氧化物的产生、黑色素合成以及抵御外源物质入侵等一系列反应。虫霉是一种重要的昆虫病原真菌，关于该菌诱导昆虫免疫方面的研究少见报道，在国内尚属空白。本研究以根虫瘟霉（Zoopthora radicans）-小菜蛾（Plutella xylostella）为免疫互作研究模型，通过不同寄主来源的根虫瘟霉菌株对小菜蛾的毒力测定、入侵过程中引起的酚氧化酶（PO）活力变化分析、小菜蛾血淋巴酚氧化酶原（ProPO）的存在部位与免疫学特性、β-1，3-葡聚糖结合蛋白（βGRP）的分离纯化与功能分析、根虫瘟霉-ProPO-βGRP三者间免疫互作过程的研究，初步探明了ProPO-AS的作用及有效防御机理。 不同根虫瘟霉菌株对小菜蛾的毒力 在不同寄主来源的4株根虫瘟霉对小菜蛾二龄幼虫的生物测定中，发现源于小菜蛾的菌株ARSEF1100毒力最强，在0.5～319个孢子／mm²的剂量下，接种后第8d累计死亡率为2.38％～97.44％，虫尸全部表现典型的虫瘟霉症状；源于叶蝉的ARSEF2699和F99101菌株的同日累计死亡率分别为2.4％～50％（剂量为1.6～315个孢子／mm²）和2.4～60.5％（剂量为1.8～484个孢子／mm²），而源于菜粉蝶的ARSEF1342菌株在3.5～633个孢子／mm²的剂量下只引发6.5％～13.6％的累计死亡率，后3个菌株致死的小菜蛾幼虫仅部分表现典型症状。所获数据经时间—剂量—死亡率模型模拟分析，剂量效应参数依次为ARSEF1100（1.89）＞F99101（1.48）＞ARSEF2699（1.23）＞ARSEF1342（0.37），相互间差异均达极显著水平。接种后4～8d内，ARSEF1100的LD₅₀值分别为232、113、71、41和35个孢子／mm²，其毒力远高于其余3个菌株；ARSEF2699的相应LD₅₀值为1344、922、556、410和397个孢子／mm²；F99101的LD₅₀为666、452、414、351和333个孢子／mm²，而ARSEF1342的毒力太弱难以估计。这些结果表明，源于原始寄主的ARSEF1100菌株对小菜蛾的毒力最强，较容易克服寄主的免疫防御系统。 入侵过程中寄主血淋巴PO的变化 经不同菌株感染后，小菜蛾血淋巴PO活性均高于未接种的寄主，且不同菌株引起的寄主PO活性变化也不同。源于同翅目寄主的两个菌株所引起的PO活性较高，尤其是菌株ARSEF2699，所引起的寄主PO活性是未接种的7-16倍；而源于鳞翅目寄主的两菌株所引起的PO活性则较低。另外，各菌株感染的小菜蛾血淋巴PO活性都有一个先升高后降低的过程，菌株F99101、ARSEF1100、ARSEF2699和ARSEF1342接种后PO活性水平最高峰出现时间分别为1d、2d、3d和4d，这或许与它们的入侵速度有关。菌株F99101的入侵速度最快，其次为ARSEF1100，ARSEF2699，而ARSEF1342的入侵速度最慢。随后寄主血淋巴PO活性逐渐受到抑制并开始下降，如ARSEF99101和ARSEFll00菌株引起的PO活性接种后第4d还显著低于对照。研究发现，不同寄主来源的根虫瘟霉菌株对小菜蛾毒力强弱与其所引起的PO活性成负相关，这符合原始寄主亲缘关系较近的菌株较易克服并逃避原始寄主的免疫识别的假设。但是，源于菜粉蝶的ARSEF 1 342菌株引起PO活性显著低于其它三个菌株，而且毒力也最低，这可能与该菌抱子极少入侵小菜蛾或是入侵速度非常缓慢有关。 ProPO的存在部位和免疫学特性分析在昆布多糖存在下测定小菜蛾血浆、血细胞裂解液和血细胞碎片的PO活性，发现血细胞碎片中的PO活性为最高，达26.80U;其次为血细胞裂解液，达16.68U。血浆中最低，仅为2.53U。该结果表明ProPO主要位于血细胞膜和血细胞中，而血浆中很少含有该酶。有活性的PO对昆布多糖和根虫瘟霉菌丝裂解液具有一定的吸附能力，且对不同寄主来源菌株的吸附能力也不同。PO对ARSEF1342菌株的吸附能力最强，对其余三菌株的吸附能力较弱，尤其是对ARSEFlloo菌株的吸附能力最弱。进一步研究发现，昆布多糖等微生物多糖和根虫瘟霉菌株裂解液能够激活小菜蛾血淋巴的ProPO。此外，血浆能够增强由昆布多糖和根虫瘟霉菌丝裂解液所引起的PO活性，但它本身并不能激活ProPO，这或许表明小菜蛾血浆中存在着一些识别蛋白，当真菌等异物入侵时能促使血细胞的ProPO快速转化为PO。 pGRP的分离及免疫活性分析用p一1，3一葡聚糖亲和沉淀法，从小菜蛾血淋巴中分离到两种pGR卫，分子量分别为75.9 kDa和83.2 kDa。它们是单链蛋白，主要位于小菜蛾的血浆中，能特异性地结合p一1，3一葡聚糖，如昆布多糖和酵母聚糖等。但用纤维素（p一1，4-葡聚糖）、右旋糖普（a一葡聚糖）及甘露聚糖亲和沉淀法极少分离到相应的结合蛋白。研究发现，pGRP和昆布多糖共同存在下对血细胞裂解液中ProPO的激活作用最强，显著高于其它各处理。昆布多糖单独存在时也能激活血细胞裂解液中ProPO，显著高于只加p一1，3一葡聚糖结合蛋白及对照处理，而后两者无显著性差异。在pGRP存在的情况下，由原生质体裂解液引起的PO活性极显著地低于由菌丝裂解液引起的酶活。并且，不同寄主来源的菌株，其原生质体裂解液所引发的PO活性也不同，原始寄主来源的ARSEFll00菌株引起的PO活性与对照相似，极显著地低于其余三菌株引?更多还原

【Abstract】 The prophenoloxidase-activating system (ProPO-AS) is an efficient non-self recognition system which involves in the generation of superoxide, melanin synthesis, and subsequent sequestration of foreign matter entering the hemocoel of the insect. The entomophthorales is one of the most promising entomopathogenic fungi, while few studies have been made on insect immunity which was activated by entomopathogenic fungi and there is no report in China. This thesis is a preliminary study on immune interaction between Zoophthora radicans and Plutella xylostella. It presents the results on the virulence of Z. radicans against P. xylostella, changes of insect phenoloxidase (PO) during invasion by the Z. radicans, location and characteristics of ProPO, purification and immunological characteristics of ji-l,3-glucan binding protein (PGRP), and effect of Z. radicans and (5GRP on PO activities.Virulence of original-host-different Z. radicans isolates against P. xylostella larvae Four Z. radicans isolates from different original hosts were bioassayed against the second instar larvae of the diamondback moth, P. xylostella in laboratory. Each isolate included eight dosages resulting from time-varying spore shower. On day 8 after exposure to spore shower for inoculation, a P. xylostella-derived isolate, ARSEF 1100, caused a cumulative mortality of 2.4-97.4% at 0.5-319 conidia/mm2 with all cadavers exhibiting typical Zoophthora syndrome. Two isolates derived from Empoascaflavescens, ARSEF 2699 and F99101, killed 2.4-50% and 2.4-60.5% at 1.6-315 and 1.8-484 conidia/mm2, respectively, whereas the isolate ARSEF 1342 from Pieris brassicae killed only 6.52-13.63% at 3.5-633 conidia/mm2. Typical Zoophthora syndrome was observed only in approximately one third of cadavers killed by the latter three isolates. Fitting the data to a time-dose-mortality model generated the estimates of parameters for time and dose effects of each isolate. The parameter estimate for dose effect (B) was 1.89 for ARSEF 1100, 1.48 for F99101, 1.23 for ARSEF 2699, and 0.37 for ARSEF 1342, being significantly different from one to another. The LD50 values estimated for ARSEF 1100 were 232, 113, 71, 41 and 35 conidia/mm2 on days 4-8 after exposure, respectively. The corresponding estimates were 1344, 922, 556, 410 and 397 conidia/mm2 for ARSEF 2699, and 666, 452, 414, 351 and 333 conidia/mm2 for F99101. The virulence for ARSEF 1342 was too weak for its LD50 to be estimated. Conclusively, the isolate ARSEF 1100 was most virulent to P. xylostella, and can escape from immune system of the host.Changes of PO activity in host hemolymph during Z. radicans invasion Afterinfection by different Z. radicans isolates, the PO activities in the hemolymph of the P. xylostella larvae infected was significantly higher than that of the uncontaminated hosts. Different original-host isolates induced different changes of PO activities. PO activities activated by two isolates from Homoptera is much higher than thoes by two isolates from Lepidoptera, the highest PO activity was happened at ARSEF2699 isolate, which was 7-16 folds higher than that of uncontaminated hosts. In addition, The PO activities induced Z. radicans was changed from low to high and then to low. The peaks of PO activitise for F99101, ARSEF1100, ARSEF2699 and ARSEF 1342 was happened on the dl, d2, d3, and d4 respectively, Which may be corresponded to their aggressive speed for each isolate. Then, PO activities began to decrease, especially those induced by ARSEF99101 and ARSEF 1100 on the forth day after inoculation, they are even lower than that of the uncontaminated hosts. The results suggested that the PO activity in hemolymph of host was controlled at later periods due to the fungus infection. Meanwhile, there is the negative relation between virulence of original-host-different Z. radicans isolates against P. xylostella and PO activity induced by them. This maybe indicates that the isolates from similar original-hosts will easily conquer and escape from immune system. But, there is an exception. PO更多还原