【作者】 李星；

【导师】 付月君；

【作者基本信息】 山西大学 ， 生物化学与分子生物学， 2016， 硕士

【摘要】 杆状病毒(baculovirus)是一类含双链环状超螺旋DNA分子(约82-180 kbp)的病毒,因其病毒粒子呈长杆状而得名,其宿主主要是鳞翅目、膜翅目、双翅目昆虫。作为杆状病毒模式种的苜蓿银纹夜蛾核型多角体病毒(Autographa californica multiple nuclear polyhedrosis virus,AcMNPV)的DNA分子约为130 kbp,含有开放阅读框150多个。与化学农药相比,AcMNPV具有杀虫率高,对人、畜安全,不易产生抗药性等特点,但杀虫速度慢、杀虫效率低限制了AcMNPV的广泛应用。东亚钳蝎昆虫毒素BmK IT是一种兴奋型昆虫特异性毒素,包含69个氨基酸残基,作用于昆虫细胞膜上钠离子通道,导致昆虫兴奋型麻痹。相关研究表明,将蝎昆虫特异性毒素基因重组进AcMNPV基因组能够提高AcMNPV杀虫效率。本实验基于前期对BmK IT提高AcMNPV抗虫活性的研究,进一步揭示AcMNPV早、晚期启动子调控表达BmK IT对昆虫Sf9细胞和棉铃虫体的作用机制。本研究分为三部分内容:第一部分:分析了AcMNPV早、晚期启动子调控表达BmK IT对Sf9细胞凋亡蛋白P53表达及核内聚集的影响。前期我们表达纯化了P53蛋白,并制备了其多克隆抗体。用终浓度为2.28×1012 vp/mL(10 MOI)的野生型病毒AcMNPV和重组病毒AcMNPV-BmK IT(IE1)、AcMNPV-BmK IT(P10)、AcMNPV-BmK IT(PH)感染细胞6-45 h,Western blot检测到AcMNPV、AcMNPV-BmK IT(IE1)、AcMNPV-BmK IT(P10)、AcMNPV-BmK IT(PH)处理组分别在21、12、27、27h时P53表达量出现明显增加,推测细胞内P53表达受外源BmK IT表达水平的影响;接着,分别用AcMNPV、AcMNPV-BmK IT(IE1)、AcMNPV-BmK IT(P10)和AcMNPV-BmK IT(PH)感染Sf9细胞21、12、27、27h,免疫荧光法检测P53在细胞核内的聚集情况,结果表明AcMNPV、AcMNPV-BmK IT(IE1)、AcMNPV-BmK IT(P10)和AcMNPV-BmK IT(PH)处理组P53在细胞核的聚集量依次增高,由此推测重组病毒能够通过P53促进细胞凋亡。以上实验结果说明三种启动子调控BmK IT表达存在时间上差异,进而影响细胞内P53表达水平及向核内聚集的程度,加速细胞凋亡进程。第二部分:分析了AcMNPV早、晚期启动子调控表达BmK IT对Sf9细胞肌动蛋白重排的影响。用终浓度为2.28×1012 vp/mL(10MOI)的AcMNPV、AcMNPV-BmK IT(IE1)、AcMNPV-BmK IT(P10)和AcMNPV-BmK IT(PH)感染Sf9细胞6-46 h,结果表明,细胞感染病毒6-21 h时,四组病毒处理的细胞内的肌动蛋白在细胞膜附近聚集,于细胞内表面形成聚集物,随着时间的推移凝聚物逐渐增加,随后肌动蛋白逐渐向细胞核内聚集。在病毒侵染37h后,AcMNPV-BmK IT(P10)和AcMNPV-BmK IT(PH)处理组细胞核内肌动蛋白的量明显减少;在病毒侵染46h后,AcMNPV-BmK IT(IE1)处理组细胞核内肌动蛋白的量明显减少。Western blot检测结果与免疫荧光观察结果一致,即不同启动子调控表达BmK IT的重组病毒感染细胞后,均会推迟肌动蛋白向核内聚集,加快肌动蛋白出核。我们推测这种变化是由于BmK IT能够作用于细胞膜上钠离子通道,进而改变细胞内微环境,促进了细胞内凋亡相关蛋白的表达,重组病毒为适应这一变化,通过调控肌动蛋白的重排来调整其增殖的进度。第三部分:分析了AcMNPV早、晚期启动子调控表达BmK IT对棉铃虫体侵染的机制。分别用终浓度为3.16×108 PIBs/mL的AcMNPV-BmK IT(IE1)、AcMNPV-BmK IT(P10)和AcMNPV-BmK IT(PH)侵染棉铃虫1h、4h、8h、12h。RT-PCR检测到在中肠组织中,三个处理组BmK IT表达量分别在4h、8h、12h时达到最大值,并且AcMNPV-BmK IT(P10)处理组BmK IT最高表达量分别是AcMNPV-BmK IT(IE1)、AcMNPV-BmK IT(PH)处理组的1.4倍和3.2倍。RT-PCR检测到在表皮组织中,AcMNPV-BmK IT(IE1)处理组BmK IT表达量较低,推测为出芽型病毒直接侵染的结果,而在AcMNPV-BmK IT(P10)和AcMNPV-BmK IT(PH)处理组中,BmK IT表达量分别在8h、12h达到最大值。末端脱氧核苷酸转移酶介导的dUTP缺口标记法(TUNEL)检测AcMNPV早、晚期启动子调控表达BmK IT的重组病毒侵染棉铃虫后肠组织细胞凋亡情况,表明四个处理组中肠组织细胞均发生不同程度的凋亡。棉铃虫体内酚氧化酶活性测定表明,AcMNPV-BmK IT(IE1)处理组虫体酚氧化酶活性在4 h达到最大值,分别是AcMNPV,AcMNPV-BmK IT(P10)和AcMNPV-BmK IT(PH)处理组的1.45倍、1.19倍、1.32倍。AcMNPV-BmK IT(P10)处理组和AcMNPV-BmK IT(PH)处理组酚氧化酶活性在8 h达到最大值,并且AcMNPV-BmK IT(PH)处理组酚氧化酶活性高于AcMNPV-BmK IT(P10)处理组。Western blot检测了早、晚期启动子调控表达BmK IT对棉铃虫中肠组织细胞P53表达的影响,结果表明AcMNPV-BmK IT(IE1)处理组在4 h时,凋亡蛋白P53在中肠组织细胞的表达量达到最大值,而AcMNPV-BmK IT(P10)和AcMNPV-BmK IT(PH)处理组在8 h时凋亡蛋白P53在中肠组织细胞的表达量达到最大值,推测在虫体水平,BmK IT可通过P53蛋白介导组织细胞凋亡。以上实验分别在细胞和虫体水平分析了 AcMNPV早、晚期启动子调控表达BmK IT的控害机制,研究结果在为杆状病毒感染鳞翅目害虫提供可寻的普遍规律的同时,揭示出重组病毒AcMNPV-BmK IT的潜在机制,为有效改造利用AcMNPV和科学生物控害提供了依据。更多还原

【Abstract】 Baculoviruses are rod-shaped,enveloped viruses with circular double-stranded DNA genomes ranging in size from 80 to 180 kbp that specifically infect arthropods of the insect orders Lepidoptera,Hymenoptera and Diptera.Autographa californica multiple nucleopolyhe-drovirus(AcMNPV),the archetype species of the a-baculoviruses of the baculoviridae,is advantageous over traditional chemical insecticides since they have longer anti-pest effects in the field and the pests are less likely to develop resistance.However,the slow anti-insect action and the narrow killing range of insects restrict the further application of AcMNPV.Buthus martensii Karsch(BmK)is a kind of scorpions which venom contains excitatory insect toxins.BmK IT is a polypeptide of 69-72 amino acid residues,acting specifically on insects and inducing a fast excitatory contraction paralysis upon injection.Previously,we engineered BmK IT gene into the genome of AcMNPV and BmK IT was inserted to the downstream of immediate early(IE 1),late(P10)and very late(Polh or PH)promoters,respectively.Based on our previous work,this study intends to disclose anti-insect mechanism and regulation analysis of these promoters in AcMNPV-BmK IT at cellular and insect level.This study mainly divided into three parts:In the first part,results from western blot and fluorescence microscopy assay showed that AcMNPV-mediated expression of BmK IT under the IE 1,P10 and PH promoter had an effect on the expression and nuclear polymerization of P53 in the nuclei.Sf9 cells were infected with AcMNPV,AcMNPV-BmK IT(IE1),AcMNPV-BmK IT(P10)and AcMNPV-BmK IT(PH)at the concentration of 2.28×1012 vp/mL(10 MOI)for 6-45 h,respectively.Western blot analysis confirmed the time-effect relationship between the expression level and regulation of promoters.Fluorescence microscopy analysis showed that AcMNPV-mediated expression of BmK IT under the IE1,P10 or PH promoter could promote the nuclear polymerization of P53 and accelerate the apoptosis of Sf9 cells.In the second part,effect of AcMNPV-mediated expression of BmK IT under the IE 1,P10 or PH promoter on the nuclear polymerization of actin in Sf9 cells was analyzed.Sf9 cells were infected with AcMNPV,AcMNPV-BmK IT(IE1),AcMNPV-BmK IT(P10),AcMNPV-BmK IT(PH)at the concentration of 2.28×1012 vp/mL(10 MOI)for 6-46 h,respectively.The results from immunofluorescence assay and western blot showed that cells infected with these four viruses for 6-21 h,microfilaments were formed on the ventral surface and actin filaments appeared in nuclei in both treatment groups.At 37 h.p.i.(hour post infection),nuclear actin decreased in the AcMNPV-BmK IT(P10)and AcMNPV-BmK IT(PH)treatment groups.However,nuclear actin decreased in the AcMNPV-BmK IT(IE1)treatment group at 46 h.p.i..These findings showed that AcMNPV-mediated expression of BmK IT under the IE1,P10 or PH promoter delayed the nuclear polymerization of F-actin and accelerated the clearance of actin in the nuclei.We suspected that this phenomenon was due to the change of cellular microenvironment induced by the effect of BmK IT on the cellular sodium ion channels.To adapt to this change,recombinant virus adjusted its proliferation progress through regulation of actin rearrangement.In the third part,regulation analysis of AcMNPV-mediated expression of BmK IT under the IE1,P10 and PH promoter was performed in vivo.Larvae of Heliothis armigera were infected with AcMNPV,AcMNPV-BmK IT(IE1),AcMNPV-BmK IT(P10),AcMNPV-BmK IT(PH)for 1-12 h at the concentration of 3.16×108 PIBs/mL,respectively.In midgut tissue,the transcription level of BmK IT reached the top value at 4,8,12 h.p.i.in AcMNPV-BmK IT(IE1),AcMNPV-BmK IT(P10)and AcMNPV-BmK IT(PH)treatment group,respectively.Moreover,the top transcription level of BmK IT in AcMNPV-BmK IT(P10)treatment group was 1.4 and 3.2 times of that inAcMNPV-BmK IT(IE1)and AcMNPV-BmK IT(PH)group.The transcription of BmK IT was also detected in the epidermal tissue of infected larvae.The low transcription level in three treatment groups at 1 h.p.i.was detected,which suggested that a small quantity of budded viruses in the sample infected the epidermal tissue directly.In the AcMNPV-BmK IT(P10)and AcMNPV-BmK IT(PH)treatment groups,high transcription level of BmK IT was detected at 8 h and 12 h.p.i.,respectively.TUNEL assay showed that the degree of midgut tissue apoptosis in the AcMNPV-BmK IT(IE 1),AcMNPV-BmK IT(P10)and AcMNPV-BmK IT(PH)treatment groups was higher than that in the AcMNPV treatment group.The degree of midgut tissue apoptosis in the AcMNPV-BmK IT(PH)treatment group was the highest.These results suggested that the recombinant AcMNPV improved the expression of BmK IT,which would further induce apoptotic cell death in the midgut tissue and thus promoted the insecticidal efficacy.The phenoloxidase activity in haemolymph of larvae infected with AcMNPV-BmK IT(IE1)reached the top level at 4 h.p.i.,which was 1.5,1.2 and 1.3 times of that in AcMNPV,AcMNPV-BmK IT(P10)and AcMNPV-BmK IT(PH)treatment groups,respectively.Phenoloxidase activity in the AcMNPV-BmK IT(P10)and AcMNPV-BmK IT(PH)treatment groups was maximal at 8 h.p.i.,and the phenoloxidase activity in AcMNPV-BmK IT(PH)group was higher than that in AcMNPV-BmK IT(P10)group.The time-effect relationship between the insect’s humoral immunity and regulation of promoters was confirmed.It was noteworthy that the changes of phenoloxidase activity were correlated with the transcription level of BmK IT which was regulated by the IE1,PH or P10 promoter.The results from western blot showed that the expression level of P53 reached the top at 4,8 and 8 h.p.i.in AcMNPV-BmK IT(IE1),AcMNPV-BmK IT(P10)and AcMNPV-BmK IT(PH)treatment group,respectively.In this study,the effect and mechanism analysis of AcMNPV-mediated expression of BmK IT under the IE1,P10 and PH promoter on Sf9 cells and larvae of Heliothis armigera were performed.Besides uncovering the universal principles mediating baculovirus’s capacity in infecting lepidoptera pests,these results provide a theoretical basis for the development and mechanism analysis of recombinant virus biopesticides.更多还原