家蚕抗性品系NC99R抗BmNPV作用机制分析

Mechanism analysis of Anti-BmNPV resistant strain NC99R

家蚕Bombyxmori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(Bombyxmori 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1.2×10^5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1×10^6多角体/头和体腔注射1×10^6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24h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。

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 NC99 R,and to provide a basis for understanding the molecular mechanism of the silkworm resistance strain.The normal control Dazao(DZ)strain and the NC99 R resistant strain were fed with occlusion bodies(OB).The median lethal dose(LD50)analysis of the DZ and NC99 R showed that the LD50 of DZ was 1.2×10^5 OBs/larva,while NC99 R was 1.8×106 OBs/larva.The LD50 of the NC99 R was about 15 times higher than the DZ.The mortality of DZ and NC99 R were analyzed,which were fed with 1×10^6 OBs/larva and injection with 1×10^6 BVs/larva.The results showed that the death peak of DZ was concentrated in the 4 th to 6 th day.And the death peak of NC99 R was concentrated in the 6 th to 8 th 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 NC99 R were increased slowly after oral infection and body injection.HE staining showed that midgut tissue has no significant difference between DZ and NC99 R in the early stage of oral infection.At 96 h p.i.,the nucleus of DZ midgut became larger and shedding.The NC99 R 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.