家蚕热激蛋白Hsp25.4对BmNPV增殖的影响

Effects of heat shock protein Hsp25.4 of Bombyx mori on BmNPV proliferation

【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,Bm NPV)侵染家蚕后的表达变化规律,明确敲低该基因表达后对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10μL预先制备好的BmNPV-T3株病毒悬液(5×108个/mL),对照组添食等量灭菌水,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6h达峰值,而脂肪体则在BmNPV侵染12h时相对表达量达最高;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降低61.81%(P<0.01,下同),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 ds BmHsp25.4的gp41基因在各时期相对表达量极显著高于对照ds GFP处理,感染48 h二者差异显著(P<0.05)。【结论】BmNPV侵染能引起BmHsp25.4基因的表达,并且敲低BmHsp25.4基因的表达能提高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。

【Objective】To explore the expression changes of silkworm heat shock protein(Hsp25.4)gene after infection by Bombyx mori nuclear polyhedrovirus(BmNPV),and clarify the effect of knocking down of gene expression on the proliferation and replication of BmNPV,which could provide a reference for further analysis of the role of BmHsp25.4 protein in the infection of silkworm by BmNPV.【Method】The 5th instar larvae of silkworm were used as ma‐terial,and fed with 10μL prepared BmNPV polyhedra suspension(5×108 insect/mL),control group fed with equal amounf of sterile water.The real-time fluorescence quantitative PCR was applied to analyze the expression of BmHsp25.4 gene in various tissues of silkworm larvae infected with BmNPV for 6 h,and 3 tissues with relatively high expression le-vels 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 real-time fluorescence quantitative PCR.【Result】The BmHsp25.4 gene was up-regulated in the midgut,hemolymph and fat body of silkworm larvae at 6 h after the BmNPV infection and the expression profile of BmHsp25.4 gene in those three tissues showed a trend of rising fist and then decreasing.The relative expression in hemolymph and midgut reached the highest 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 expres‐sion 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 gene was successfully inter‐fered.After BmHsp25.4 gene interference,the relative expression of gp41 gene of dsBmHsp25.4 was extremely signifi‐cantly higher than control dsGFD,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 gene,and knockdown of BmHsp25.4 gene can promote the early proliferation of BmNPV in the silkworm,suggesting that Hsp25.4 protein may play a related role in the antiviral immunity of silkworm.