家蚕热激蛋白Hsp 25.4对BmNPV增殖的影响
Effects of heat shock protein(Hsp25.4)of Bombyx mori in BmNPV infection
唐芬芬 1杨伟克 2张祖芸 1何超 1沈登荣 1张睿 1谢昆1
作者信息
- 1. 红河学院生物科学与农学学院,云南红河州 661199
- 2. 云南省农业科学院蚕桑蜜蜂研究所,云南红河州 661101
- 折叠
摘要
[目的]探究家蚕热激蛋白(Hsp25.4)基因在家蚕核型多角体病毒(Bombyx mori nuclear polyhedrovirus,BmNPV)侵染家蚕后的表达变化规律,明确敲低该基因的表达后对BmNPV增殖复制的影响,为深入解析BmHsp25.4蛋白在BmNPV侵染家蚕过程中发挥的作用提供参考依据.[方法]以5龄家蚕幼虫为研究对象,经口添食10 µL预先制备好的BmNPV-T3株病毒悬液(5×108 个/mL),采用实时荧光定量PCR检测BmHsp25.4基因在BmNPV侵染6 h各组织的表达情况,并选择相对表达量较高的3个组织,分析BmHsp25.4基因时空表达图谱;注射双链RNA(dsRNA)检测BmHsp25.4基因在体内的干涉效果,采用实时荧光定量PCR检测BmHsp25.4基因干涉后对BmNPV增殖复制的影响.[结果]BmNPV侵染家蚕6 h,BmHsp25.4基因在家蚕血液、中肠和脂肪体组织中的相对表达量升高;时空表达谱结果显示,BmHsp25.4基因在3个组织中的表达谱均呈先升高后降低的变化趋势,其中血液和中肠组织的相对表达量均在6 h达到峰值,而脂肪体则在BmNPV侵染12 h时相对表达量达到最高;注射dsRNA干涉后,试验组BmHsp25.4基因的相对表达量与对照组相比极显著(P<0.01,下同)降低61.81%,说明BmHsp25.4基因的相对表达量被成功干涉;BmHsp25.4基因干涉后,感染24 h试验组BmNPV在各时期增殖速度极显著高于对照组,感染48 h二者差异为显著水平(P<0.05).[结论]BmNPV侵染能够引起BmHsp25.4基因的表达,并且敲低BmHsp25.4基因的表达能影响BmNPV早期在虫体内的增殖,推测Hsp25.4蛋白在家蚕的抗病毒免疫中发挥关联功能.
Abstract
[Objective]In order to provide references for futher study on understanding the role of BmHsp25.4 protein in the process of Bombyx mori nuclearpolyhedrovirus(BmNPV)infection in silkworm,this study was explored the gene BmHsp25.4 expression in silkworm larvae infected with BmNPV,and the effects of knocking down the expression of this gene on the proliferation and replication of BmNPV were also analyzed.[Method]The 5th instar larvae of B.mori wre used as material,and fed with 10 µL prepareared BmNPV polyhedra suspension(5×108 mL-1).The quantitative real-time PCR was applied to analyze the expression of BmHsp25.4 in virous tissues of silkworm larvae infected with BmNPV for 6 h,and 3 tissues with relatively high expression levels were selected to analyze the spatiotemporal expression map of BmHsp25.4 gene.Then,the interference effect of BmHsp25.4 gene in vivo was detected by injection of the double-stranded RNA(dsRNA),and the effect of BmHsP25.4 gene interference on the proliferation and replication of BmNPV was detected by quabtivatitative real-time PCR.[Result]The BmHsp25.4 gene was up-regulated in the midgut,hemo-lymph and fat body of silkworm larvae at 6 h after the BmNPV infection and the expression profile of BmHsp25.4 in those three tissues showed a trend of rising fist and then decreasing.The expression in hemolymph and midgut reached the high-est peak at 6 h,while the expression of fat body reached the highest at 12 h after BmNPV infection.After interference with dsRNA injection,the relative expression of BmHsp25.4 gene in experimental group decreasing by 61.81%compared with the control group,the difference was extremely significant(P<0.01,the same below),indicating that the expression of BmHsp25.4 was successfully interfered.After BmHsp25.4 gege interference,the proliferation rate of BmNPV in experi-mental group was significantly higher than that in control group at 24 h after infection,and the difference was significant at 48 h after infection(P<0.05).[Conclusion]The results indicate that BmNPV infection can induce the expression of BmHsp25.4,and knockdown of BmHsp25.4 can affect the early proliferation of BmNPV in the silkworm,suggesting that BmHsp25.4 may play a related role in the antiviral immunity of silkworm.
关键词
家蚕/家蚕核型多角体病毒(BmNPV)/热激蛋白/RNA干扰Key words
Bombyx mori/Bombyx mori nuclear polyhedrovirus/Heat shock protein/RNAi引用本文复制引用
基金项目
云南省基础研究计划面上项目(2022FB226)
云南省地方本科高校基础研究联合专项(2021BA070001-070)
云南省农业科学院应用基础研究专项(YJM201710)
红河学院博士专项(BS2003)
出版年
2023
国家科技期刊平台
NSTL
万方数据