【作者】 张丽红；

【导师】 彭建新；

【作者基本信息】 华中师范大学 ， 微生物学， 2004， 硕士

【摘要】 光增白剂对杆状病毒具有增效和保护作用，能提高虫体对杆状病毒敏感性，这对于扩大杆状病毒杀虫剂的应用具有重要的意义。光增白剂对杆状病毒的增效作用的机理存在两种推测一种观点认为光增白剂是通过破坏围食膜结构的完整性，促使更多的病毒粒子穿越围食膜而发动感染的；另一种意见认为光增白剂能延迟中肠上皮细胞的脱落，促进病毒的复制繁殖。本文利用以β—半乳糖苷酶基因为标志基因的重组苜蓿银纹夜蛾核型多角体病毒（AcMNPV-hsp70／lacZ）在中肠组织水平上研究荧光增白剂对该病毒的增效作用及其增效作用方式。 三龄甜菜夜蛾经口服感染病毒的生物测定表明：1％光增白剂FB-28对该重组病毒具有明显的增效作用。添加荧光增白剂FB-28的病毒感染3龄末甜菜夜蛾幼虫其LC₅₀从2.31×10⁷ PIBs／ml降低到5.18×10⁴ PIBs／ml，增效比值达到448倍。当病毒浓度为1.55×10⁷ PIB／m1时，加有FB-28的LT₅₀为4.6d（r=0.9437），单剂量的病毒液感染3龄幼虫的LT₅₀为7.0d（r=0.9798），杀虫速度提高了34.3％。 此外，对于处于不同虫龄的幼虫进行病毒感染时发现：对3¹⁶幼虫毒力生物测定时，荧光增白剂的增效作用达到50％，但对4龄初的供试幼虫进行测定，荧光增白剂的增效作用却不很明显，仅为10％。此结果表明荧光增白剂对不同虫龄阶段的幼虫增效能力存在较大的差异。 选用2.25×10⁷ PIB／ml的病毒悬液感染3¹⁶、4⁰甜菜夜蛾幼虫。病毒感染后的8h，12h，16h，20h提取中肠组织，按常规石蜡包埋切片进行洗涤、透明、浸蜡、包埋、切片、粘片等操作程序，制成连续切片，在倒置显微镜下逐一进行观察计数。结果如下： 幼虫中肠的报告基因表达率（病毒在中肠部位形成的病灶）数字显示：荧光增白剂确实可以提高幼虫对病毒的敏感性。实验组幼虫中肠的报告基因表达率（病毒在中肠部位形成的病灶）要明显高于对照组中幼虫中肠的报告基因表达率。此外，实验组中供试幼虫中肠的报告基因表达率均随感染时间的延长而增高，但在感染后20h左右，供试幼虫中肠的报告基因表达息硕士学泣论文、!、从卜又’荞!乡;三、卜率却有所降低，对此，我们分析其原因可能是由于病毒的感染部位发生了转移。 从病毒在中肠中形成的病灶数月可以看出:316，40接种的甜菜夜蛾幼虫中肠组织中病灶随着感染时间的延长逐渐增多。在相同时间内实验组中的病灶数目明显多于对照组中的病灶数目。可见，荧光增白剂的添加不仅可以加快病毒在中肠组织的感染，还能增强病毒在中肠组织的感染能力。可见，对于处于不同时龄的供试幼虫接种病毒时，添加荧光增白剂均可提高病毒在中肠组织中的感染能力。 从病毒在中肠中形成的病灶部位可以看出，实验组与对照组的病灶均靠近中肠的基底膜，荧光增白剂的加入并不能改变病毒在中肠组织的感染部位。实验结果表明，在幼虫的病毒接种物中添加合适浓度的荧光增白剂（0 .01 g.mL一’）不会改变病毒在幼虫中肠组织中的感染部位，却可以提高幼虫对病毒的敏感性，增加了病毒毒力。此外，血淋巴细胞感染观察表明:在幼虫的病毒接种物中添加合适浓度的荧光增白剂可以提高幼虫对病毒的敏感性，产生更多的病灶。 此外，采用分子探针R18（荧光标记物）标记病毒囊膜，体外分离中肠上皮细胞，将标记的病毒粒子与离体中肠上皮细胞混合后保温，病毒吸附Zh后，通过检测荧光的变化来监测病毒粒子与上皮细胞的融合。从而了解荧光增白剂是否在膜的融合过程中存在影响。实验结果表明荧光增白剂影响了病毒与中肠上皮细胞的融合，荧光增白剂促进了病毒与中肠上皮细胞的融合。更多还原

【Abstract】 It has been demonstrated that optical brightener (or fluorescent brightener) can enhance viral activity, provide UV protection for nucleopolyhedrovirus (NPV) and increase host susceptibility. The mechanism enhancement of the optical brightener is not known. Shapiro et al. postulated that selected brightener including M2R inhibit or alter the chitinous peritrophic membrane (PM), creating gaps in the membrane or gut lining and perhaps allowing more virions to pass from the gut lumen into the hemocoel. We used a reporter gene recombinant of Autographa californica, MNPV(AcMNPV-hsp70/lacZ) to investigate the enhancing effects of FB-28 on pathogenesis in midgut, mortality and time to death in larvae of Spodoptera exiguaStudies were designed to determine the effect of the selected optical brighter FB-28 on the infectivity of the AcMNPV-hsp70/lacZ.the viral preparation were diluted in water (l%)and applied to the surface of artificial diet, the addition of the FB-28 to the virus in the bioassay greatly enhanced infectivity of the AcMNPV-hsp70/lacZ of FB-28 to the AcMNPV-hsp70/lacZ, reduced the LC50 values from 2.31 X 107 PIB per ml to 5.18 X 104 PIBs/ml and at the highest virus concentration 1.55 X 107PIBs/ ml, the LT50 values was reduced from 7.0d to 4.6d. suggesting that the larvae of Spodoptera exigua died more quickly when FB-28 were added to the virus .The larvae of Spodoptera exigua inoculated as newly molted fourth instars (40s) and at various hours after molting (316s), we sacrificed larvae at various time intervals after inoculation to process the larvae for lacZ expression in midgut by using tissue section techniques on early phase infection. Optical brighteners can’t change the part of infection, but it improves viral enhancement on pathogenesis of midgut. We detect the differentiation of the control and the test to study synergism of optical brighteners to viral enhancement and its mechanisms. Fixed with gluteraldehyde, Stained with pH 7.4 phosphate buffered X-gal Dye Solution,observed by reverse microscope, the results are as follow: the percent of larvae expressing lacZ in midgut showed that FB-28 improved the sensitivity of larvae to virus. The percent of larvae expressing lacZ gradually increased for various time points after inoculation, but the percent of larvae expressing lacZ reduced after 20 hours post inoculation, we thought the infection spots transformed.The number of foci in midgut showed: foci became more with the delay of infection times. The foci were more in control than in test. Which stated the addition of optical brighteners fastened the infections of virus and enhanced the ability.The foci located the basal in midgut not in control but also in test, the results showed that the addition of optical brighteners didn’t change the spots of infection but improved the viral enhancement, in addition, Infection of hemolymph cell showed that optical brightener increased sensitivity of larvae to virus, forming more foci.In addition, We designed a test: The midgut was removed from Spodoptera exigua larvae and was cut longitudinally into halves, the midgut epithelia were cleaned and digested with dispase II ,the pelleted cells in Grace medium with 7% FBS supplemented with 100units/ml penicillin,100ug/ml streptomycin were cooled and then suspended in R18-labelled virus. Aliquots of cells were mixed 0.15%mg/ml FB-28,and kept at 4C for 30min,fusion assays were conducted: fluorescence was measured immediately at regular time-points with fluorescence spectrophotometer with an excitation wave length of 560nm and emission wave length of 590nm. the percentages of membrane fusion was calculated. By monitoring fusion using the R18 assay, We found that the fluorescent brightener 28 influenced membrane fusion of virus and midgut epithelia cells.更多还原