家蚕抗性品系NC99R抗BmNPV作用机制分析

Mechanism analysis of Anti-BmNPV resistant strain NC99R

摘要:

家蚕Bombyx mori是具有重要经济价值的鳞翅目模式昆虫，由家蚕核型多角体病毒 (Bombyx mori nucleopolyhedrovirus，BmNPV) 引起的家蚕血液型脓病每年给我国蚕桑产业造成巨大的经济损失。文中拟通过鉴定目前存在的家蚕抗病毒品系NC99R抗性特征，初步解析其抗BmNPV机制，以期为家蚕抗病品系分子机制解析提供一定参考。将对照组大造 (Dazao，DZ) 品系和实验组NC99R抗性品系家蚕幼虫分别定量添食BmNPV包涵体 (Occlusion bodies，OB)，计算DZ和NC99R品系对BmNPV的半致死剂量 (Median lethal dose，LD50)，结果显示DZ品系半致死剂量为1.2×105多角体/头，抗性品系NC99R的半致死剂量为1.8×106多角体/头，抗性水平相比对照组提高15倍左右。进一步统计分析DZ和NC99R品系口服感染1×106多角体/头和体腔注射1×106粒子/头出芽型病毒粒子 (Budded virus，BV) 后的死亡率，结果显示DZ品系死亡高峰集中在第4–6天，NC99R抗性品系死亡高峰集中在第6–8天，与对照相比延迟1–2 d。通过RT-PCR分析BmNPV DNA拷贝数，显示DZ在口服感染和体腔注射后BmNPV基因组都快速增殖，抗性品系NC99R BmNPV DNA拷贝数缓慢增加。HE染色分析显示DZ和NC99R品系在口服感染前期没有显著差异，感染到96 h，DZ中肠细胞核变大，成脱落趋势；而抗性品系NC99R，细胞核膨大，但细胞排列仍较整齐。RT-PCR分析病毒不同时期基因表达显示抗性品系NC99R在口服感染和体腔注射24 h后早期基因ie-1开始下调表达，其他时期基因随即下调表达；最后维持在较低表达水平或消失。

Abstract:

Bombyx mori is a lepidopteran insect with important economic value. Bombyx mori nucleopolyhedrovirus (BmNPV) causes huge economic loss to silkworm industry in China every year. The objective of this study is to determine the anti-BmNPV mechanism of Bombyx mori strain NC99R, and to provide a basis for understanding the molecular mechanism of the silkworm resistance strain. The normal control Dazao (DZ) strain and the NC99R resistant strain were fed with occlusion bodies (OB). The median lethal dose (LD50) analysis of the DZ and NC99R showed that the LD50 of DZ was 1.2×105 OBs/larva, while NC99R was 1.8×106 OBs/larva. The LD50 of the NC99R was about 15 times higher than the DZ. The mortality of DZ and NC99R were analyzed, which were fed with 1×106 OBs/larva and injection with 1×106 BVs/larva. The results showed that the death peak of DZ was concentrated in the 4th to 6th day. And the death peak of NC99R was concentrated in the 6th to 8th day, with a delay of 1–2 days compared with the control. The BmNPV DNA copy number showed that the BmNPV genome in DZ proliferated rapidly. The copy number of BmNPV DNA in NC99R were increased slowly after oral infection and body injection. HE staining showed that midgut tissue has no significant difference between DZ and NC99R in the early stage of oral infection. At 96 h p.i., the nucleus of DZ midgut became larger and shedding. The NC99R had enlarged nuclei, but the cells were still arranged neatly. Finally, the expression of virus genes in different periods were analyzed by RT-PCR. The results indicated that the immediate early gene ie-1 expression levels began to down-regulate after 24 h p.i.. The early, late, and extremely late genes were also down-regulated, and finally maintained at a lower expression level.