摘要
本研究利用澳洲雪漏斗蜘蛛毒素基因ω-ACTX-Ar1b（Ar1b）替换棉铃虫核型多角体病毒（HearNPV）的人工基因组（HZ8-bacmid）中egt基因获得的Ar1b-bacmid，通过转座将多角体基因（ph）修复到Ar1b-bacmid和HZ8-bacmid上，构建成了具有感染活性的重组病毒Ar1b-HearNPV和基因修复型对照G4株HearNPV（HZ8-HearNPV）。将二者分别转染HzAM1细胞后，在细胞水平上对比研究了Ar1b毒素基因重组的病毒与正常病毒的复制与病毒生长情况，并与野生型HearNPV G4株病毒（wt-HearNPV）对照分别感染棉铃虫幼虫，对比研究了重组病毒Ar1b-HearNPV的杀虫效果。另外，研究了野生型HearNPV感染棉铃虫后对试虫ALP活性与基因表达的影响，取得的主要结果如下：
1、通过转座将ph基因修复到Ar1b-bacmid和HZ8-bacmid上，成功构建了多角体恢复型Ar1b-HearNPV和HZ8-HearNPV人工基因组。人工基因组通过转染HzAM1细胞，证实了Ar1b-HearNPV和HZ8-HearNPV均能包装成正常的病毒粒子，且二者具有相似的病毒生长曲线，但Ar1b-HearNPV较HZ8-HearNPV的病毒DNA复制能力稍低，差异不显著。
2、重组病毒LD50和LD90生测结果显示：Ar1b-HearNPV相比HZ8-HearNPV和wt-HearNPV具有更高的杀虫毒力； LT50和LT90结果表明： Ar1b-HearNPV相比HZ8-HearNPV和wt-HearNPV具有更快的杀虫速率。
3、不同剂量感染不同龄期幼虫结果表明：高剂量（120PIBs/larva）下，Ar1b-HearNPV与wt-HearNPV和HZ8-HearNPV导致的幼虫死亡率一致，且杀虫速度最快；低剂量（30PIBs/larva）下，Ar1b-HearNPV依旧有最快的杀虫速度，只是高龄幼虫的死亡率低于wt-HearNPV和HZ8-HearNPV处理组。
4、不同龄期幼虫在不同处理下日均累积增重量测试结果表明，Ar1b-HearNPV对不同龄期的幼虫均有很好的抑制生长效果；不同龄期幼虫不同处理下日均取食量结果表明：Ar1b-HearNPV喂食的幼虫，不同龄期幼虫日均取食量均为最低的。由此可见，重组病毒能抑制幼虫的取食，同时也能抑制幼虫的生长。幼虫日累积取食总量比较结果表明：高龄幼虫被野生型病毒感染后导致幼虫累积取食量高出健康试虫；而Ar1b-HearNPV感染的幼虫，不论感染幼虫的龄期大小，取食量均显著低于两种对照病毒。
5、wt-HearNPV感染后棉铃虫中肠ALP活性研究结果表明：wt-HearNPV病毒感染棉铃虫后，引起宿主昆虫中肠ALP活性显著降低，且随着病毒感染剂量的增加、或病毒感染后时间的推移ALP活性被抑制加重。wt-HearNPV感染后棉铃虫中肠ALP表达分析结果说明：病毒的感染能抑制ALP的表达。感染后时间越长，表达量越低；ALP被抑制的时间随感染剂量的增加而提前。
In this study, A per os infectivity recombined HearNPV, Ar1b-HearNPV, was constructedby repair the polyhedra gene (ph) to Ar1b-bacmid and HZ8-bacmid. The recombinantHearNPV was identified as an improved recombinant through bioassays. The virulence, dailycumulative weight gain and daily diet consumption from different instars larvae infected byAr1b-HearNPVwas investigated. And the differences of ALP activity and ALP expressionprofile under HearNPV infection to host larvae were studied also, following were the majorcollusions:
The ph with its own promoter in the Ha.p-ph-HTb vector was repaired to Ar1b-bacmidand HZ8-bacmid facilitated by helper plasmid. The Ar1b-HearNPV and HZ8-HearNPV OBscan be found after transfection to HzAM1cell lines using, respectively. The viral growthcurve indicated that Ar1b-HearNPV could produce BVs as well as HZ8-HearNPV. The viralDNA replication curve showed that Ar1b-HearNPV DNA had less copies than HZ8-HearNPVhad in HzAM1cell lines.
The lethal dose tests showed that the Ar1b-HearNPV owend a higher virulence thanHZ8-HearNPV. The lethal time tests indicated that the Ar1b-HearNPV can speed death oflarvae.
The bioassay results of2nd,3rdand4thinstars larvae infected with high dose (120PIBs/larva) and low dose (30PIBs/larva) demonstrated that the high dose of Ar1b-HearNPVhad a similar final mortality to HZ8-HearNPV and wt-HearNPV infection. For larvae thatinfected with the low dose, Ar1b-HearNPV could kill the larvae much quicker thanHZ8-HearNPV and wt-HearNPV, although the HZ8-HearNPV and wt-HearNPV treatmentsobtained a higher final mortality.
The daily cumulative weight gain of different instar larvae showed that Ar1b-HearNPVcan restrain the development of the infectec larvae. On the other hand, compared withwt-HearNPV and HZ8-HearNPV infections, Ar1b-HearNPV infected larvae had the lowestdaily diet consumption regardless the larvae instars, while CK owned the highest one. Totalaccumulation of diet consumption analysis illustrated that Ar1b-HearNPV had the lowest dietconsumption. This kind of inhibition on diet consumption would be deceased with the rise of the larvae’s instars.
The enzyme activity and gene expression level were determined at different stages afterviral infection with different infection titer. The results indicated that the ALP activity and itsgene expression level were inhibited significantly by HearNPV infection and this inhibitionstrength was co-related with viral infection titer and the time post infection. The viral invasionto host cell possibly could be facilitated by inhibition on ALP activity by virus. One of themechanisms of ALP activity inhibition by viral infection was down regulation of ALPexpression by virus.
引文
惠丰立,夏敏,梁子安,2003.杆状病毒杀虫剂研究进展.植物保护,29(3):9-11
胡志红, Just M. V.,1997.具有生物安全性的杆状病毒杀虫剂基因工程技术的发展与前景(英文).中国病毒学,12(1):14-25
季平,张志芳,何家禄,吴祥甫, Peter H. L.,1997.慈姑蛋白酶抑制剂B基因在家蚕幼虫和蛹体中的表达.生物化学与生物物理学报,27(1):17-23
黎路林, Possee R. D.,1995.利用杆状病毒在昆虫细胞内表达苏云金杆菌以色列亚种27kDδ-内毒素.病毒学报,11(4):357-363
刘祖强,杨复华,齐义鹏,朱应,朱反修,2001.杆状病毒的垂直传播及绿色荧光蛋白在棉铃虫幼虫中的表达.昆虫学报,44(1):1-8
吕鸿声,1998.昆虫病毒的分子生物学.北京：中国农业科技出版社,614-615
马永平,欧洋,孟小平,徐进平,叶林柏,1999.杆状病毒增效蛋白研究进展.中国病毒学,14(3):185-189
彭建新,陈曲侯,1994.杆状病毒egt基因的结构功能与表达.病毒学报,10(1):93-96
陶黎明,高菊芳,2002.用于害虫治理的重组杆状病毒.世界农药,24(3):12-15
陶圣如,1994.病毒农药的发展状况和前进.浙江化工,25(1):6-8
杨林,李国清,黄葆英,王全忠,龙綮新,王珣章,2000. GST融合蛋白在杆状病毒系统中表达的可溶性研究.中国病毒学,15(2):143-148
闫庆生,欧阳晓立,李充壁,庞义,1999.昆虫杆状病毒的蜕皮甾体尿苷二磷酸葡萄糖转移酶.生物化学与生物物理进展,26(3):241-242
姚斌,范云六,曾勤,赵荣敏,1996.表达昆虫特异神经毒素AaIT基因的转基因烟草的抗虫性.生物工程学报,12(2):133-118
张俊杰,张友清,2001.重组杆状病毒杀虫剂的研究进展.中国生物防治,17(4):179-183
张耀洲,1994.家蚕和苜蓿银纹夜蛾核型多角体病毒p10基因的研究.中国科学(B辑),24(2):157-164
张忠信,孙修炼,张光裕,1998.棉铃虫核型多角体病毒对宿主昆虫的弱化作用.植物保护学报，25(3):204-208
郑本欣,钟国洪,陈业荣,2000.吊丝虫杀病毒制剂防治小菜蛾试验初报.广东农业科学,5:52-53
Adams, J. R. and McClintock, J. T.,1991. Baculoviridae. Nuclear polyhedrosis viruses.Part1. Nuclear polyhedrosis viruses of insects. Atlas of Invertebrate Viruses,87–180
Afonso C. L., Tulman E. R., Lu Z., Balinsky C. A., Moser B. A., Becnel J. J., Rock D. J.and Kutish G. F.,2001. Genome sequence of a baculovirus pathogenic for Culex nigripalpus.Journal of virology,75(22):11157-11165
Akermann, H.-W. and Smirnoff W. A.,1983. A morphological investigation of23baculoviruses. Journal of invertebrate pathology,41(3):269-280
Ayres M. D., Howard S. C., Kuzio j., Lopez-Ferber M. and Possee R. D.,1994. Thecomplete DNA sequence of Autographa californica nuclear polyhedrosis virus. Virology,202(2):586-605
Barbehenn, R. V. and Martin, M. M.1997. Permeability of the peritrophic envelope ofherbivorous insects to dextran sulfate: a test of the polyanion exclusion hypothesis. Journal ofInsect physiology,43(3):243-249
Bischoff D. S. and Slavicek J. M.,1997. Molecular analysis of an enhancin gene in theLymantria dispar nuclear polyhedrosis virus. Journal of virology,71(11):8133-8140
Bonning B. C. and Hammock B. D.,1996. Development of recombinant baculovirusesfor insect control. Aunnal review of entomology,41:191-210
Burden J. P., Hails R. S., Windass J. W., Suner M. and Cory J. S.,1999. Infectivigy,speed of kill, and productivity of a baculovirus expressing the ithc mite toxin Txp-1in secondand fourth instar larvae of Trichoplusia ni. Journal of Invertebrate Pathology,75:226-236
Carbonnell L. F., Hodge M. R., Tomalski M. D. and Miller L. K.,1988. Synthesis of agene coding for an insect-specific scorpion neurotoxin and attempts to express it usingbaculovirus vectors. Gene,73(2):409-418
Chisholm G. E. and Henner D. J.,1988. Multiple early transcripts and splicing of theAutographa californica nuclear polyhedrosis virus IE-1gene. Journal of virology.62(9):3193–3200
Duffy S.P., Young A. M., Morin B., Lucarotti C. J., Koop B. F. and Levin D. B.,2006.Sequence analysis and organization of the Neodiprion abietis nucleopolyhedrovirus genome.Journal of virology,80(14):6952-6963
Eldridge R., Horodyski F. M., Morton D. B., O’Reilly D. R., Truman J. W., Riddiford L.M. and Miller L. K.,1991. Expression of an eclosion hormone gene in insect cells usingbaculovirus vectors. Insect Biochemistry,21(3):341-345
Fan, Q., Li S., Wang L., Zhang B., Ye B., Zhao Z. and Cui L.,2007. The genomesequence of the multinucleocapsid nucleopolyhedrovirus of the Chinese oak silkwormAntheraea pernyi. Virology,366(2):304-315
Federici B. A. and Granados R. R.,1986. Ultrastructure of baculoviruses, in The biologyof baculoviruses, CRC Press: Boca Raton:61-88
Garcia-Maruniak, A., Maruniak J. E., Zanotto P. M., Doumbouya A. E., Liu J. C., MerrittT. M. and Lanoie J. S.,2004. Sequence analysis of the genome of the Neodiprion sertifernucleopolyhedrovirus. Journal of Virology,78(13):7036-7051
Gershburg E., Rivkin H. and Chjanovsky N.,1997. Expression of the Autographacalifornica nuclear polyhedrosis virus apoptotic suppressor gene p35in nonpermissiveSpodoptera littoralis cells. Journal of Virology,71(10):7593-7599
Georgievska L., Hoover K., van der Werf W., Mu oz D., Caballero P., Cory J. S. andVlak J. M.,2010. Dose dependency of time to death in single and mixed infections with awildtype and egt deletion strain of Helicoverpa armigera nucleopolyhedrovirus. Journal ofInvertebrate Pathology.104(1):44–50
Ghosh S., Parvez M. K., Banerjee K., Sarin S. K. and Hasnain S. E.,2002. Baculovirusas mammalian cell expression vector for gene therapy: an emerging strategy. Molecular Thera,6(1):5-11
Gopalakrishnan B., Muthukrishnan S. and Kramer K. J.,1995. Baculovirus-mediatedexpression of a Manduca sexta chitinase gene: Properties of the recombinant protein. InsectBiochemistry and Molecular Biology,25(2):255-265
Haas-Stapleton E. J., Washburn J. O. and Volkman L. E.,2004. P74mediates specificbinding of Autographa californica M nucleopolyhedrovirus occlusion-derived virus toprimary cellular targets in the midgut epithelia of Heliothis virescens Larvae. Journal ofVirology,78(13):6786–6791
Hammock B D., Bonning B. C., Possee R. D., Hanzlik T. N. and Maeda S.,1990.Expression and effects of the juvenile hormone esterase in a baculovirus vector. Nature,344:458-461
Wang H. Z., Deng F., Pijlman G. P., Chen X. W., Sun X. L., Vlak J. M. and Hu Z. H.,2003. Cloning of biologically active genomes from a Helicoverpa armigerasingle-nucleocapsid nucleopolyhedrovirus isolate by using a bacterial artificial chromosome.Virus Research,97:57-63
Harrap K. A.,1972. The structure of nuclear polyhedrosis viruses. Ⅰ. The inclusionbody. Virology,50(1):114–123
Hayakawa, T., Ko R., Okano K., Seong S. I. Goto C. and Meada S.,1999. Sequenceanalysis of the Xestia c-nigrum granulovirus genome. Virology,262(2):277-297
Herniou E. A., Olszewski J. A., Cory J. S., and O’Reilly D. R.,2003. The genomesequence and evolution of baculoviruses. Annual Review of Entomlogy,48:211–234.
Horton H. M. and Burand J. P.,1993. Saturable attachment sites for polyhedron-derivedbaculovirus on insect cells and evidence for entry via direct membrane fusion. Journal ofVirology,67(4):1860–1868
Hughes K.M. and Addison R.B.,1970. Two nuclear polyhedrosis viruses of theDouglas-fir tussock moth. Journal of Invertebrate Pathology,16(2):196-204
Hyink O., Dellow R. A., Olsen M. J., Caradoc-Davies K. M., Herniou E. A., Cory J. S.,O’Reilly D. R. and Ward V. K.,2002. Whole genome analysis of the Epiphyas postvittananucleopolyhedrovirus. Journal of General Virology,83(4):957-971
Ijkel W. F., van Strien E. A., Heldens J. G., Broer R., Zuidema D., Goldbach R. W. andVlak J. M.,1999. Sequence and organization of the Spodoptera exigua multicapsidnucleopolyhedrovirus genome. Journal of General Virology,80(12):3289-3304
Inceoglu B., Lango J., Wu J., Hawkins P., Southern J. and Hammock B. D.,2001.Isolation and characterization of a novel type of neurotoxic peptide from the venom of theSouth African scorpion Parabuthus transvaalicus (Buthidae) European Journal ofBiochemisitry/FEBS.,268(20):5407-5413
Jakubowska A. K., Petters S. A., Ziemnicka J., Vlak J. M. and van Oers M. M.,2006.Genome sequence of an enhancin gene-rich nucleopolyhedrovirus (NPV) from Agrotissegetum: collinearity with Spodoptera exigua multiple NPV. Journal of General Virology,87(3):537-51
Jehle J. A., Blissard G. W., Bonning B. C., Cory J. S., Herniou E. A., Rohrmann G. F.,Theilmann D. A., Thiem S. M. and Vlak J. M.,2006. On the classification and nomenclatureof baculoviruses: a proposal for revision. Archives of Virology,151(7):1257-1266
Korth K. L. and Leavings C. S.,1993. Baculovirus expression of the maizemitochondrial protein URF13confers insecticidal activity in cell cultures and larvae.Proceedings of the National Academy of Sciences of the United States of America,90(8):3388-3392
Kuzio J., Pearson M. N., Harwood S. H., Funk C. J., Evans J. T., Slavicek J. M. andRohrmann G. F.,1999. Sequence and analysis of the genome of a baculovirus pathogenic forLymantria dispar. Virology,253(1):17-34
Lange M. and Jehle J. A.,2003. The genome of the Cryptophlebia leucotretagranulovirus. Virology,317(2):220-36
Larsson R.,1984. Insect pathological investigations on Swedish Thysanura: A nuclearpolyhedrosis virus of the bristletail Dilta hibernica. Journal of Invertebrate Pathology,44(2):172–177
Lauzon H. A., Jamieson P. B., Krell P. J. and Arif B. M.,2005. Gene organization andsequencing of the Choristoneura fumiferana defective nucleopolyhedrovirus genome. Journalof General Virology,86(4):945-961
Lee S. Y., Qu X., Chen W., Poloumienko A., MacAfee N., Morin B., Lucarotti C. andKrause M.,1997. Insecticidal activity of a recombinant baculovirus containing an antisensec-myc fragment. Journal of General Virology,78(2):273-281
Lepore L. S., Roelvink P. R. and Granados R. R.,1996. Enhancin, the granulosis virusprotein that facilitates nucleopolyhedrovirus (NPV) infections, is a metalloprotease. Journalof Invertebrate Pathology,68(2):131-140
Luque T., Finch R., Crook N., O’Reilly D. R. and Wingstanley D. The completesequence of the Cydia pomonella granulovirus genome. Journal of General Virology,82(10):2531-2547
Maeda S.,1989. Increased insecticidal effect by a recombinant baculovirus carrying asynthetic diuretic hormone gene. Biochemical and Biophysical research Communications,165(3):117-118
Maeda S.,1995. Further development of recombinant baculovirus insecticides. CurrentOpinion in Biotechnology,6(3):313-319
Martignoni M. E. and Iwai P. J.,1986. A catalog of viral diseases of insects, mites, andticks. USDA Forest Service PNW-195,4th ed.,51
Merrywheather A. T., Weyer U., Harris M. P., Hirst M., Booth T. and Possee R. D.,1990.Journal of General Virology,71(7):1535-1544
Murphy F. A., Fauquet C. M., Bishop D. H., Ghabrial S. A., Jarvis A. W., Martelli G. P.,Mayo M. P. and Summers M. D.,1995. Classification and nomenclature of viruses: Sixthreport of the international committee for the taxonomy of viruses. Arch. Virol. Suppl.,10:1–586
Nakai M., Goto C., Kang W., Shikata M., Luque T. and Kunimi Y.,2003. Genomesequence and organization of a nucleopolyhedrovirus isolated from the smaller tea tortrix,Adoxophyes honmai. Virology,316(1):171-183
O’Reilly D. R.,1995. Baculovirus-encoded ecdysteroid UDP-glucosyltransferases.Insect Biochemistry and Molecular Biology,25(5):541-550
O’Reilly D. R. and Miller L. K.,1989. A baculovirus blocks insect molting by producingecdysteroid UDP-glucosyl transferase. Science,8:1110-1112
Pang Y., Frutos R. and Federici B. A.,1992. Synthesis and toxicity of full-length andtruncated bacterial CryIVD mosquitocidal proteins expressed in lepidopteran cells using abaculovirus vector. Journal of General Virology,73(1):89-101
Pearson M. N. and Rohrmann G. F.,2002. Transfer, incorporation, and substitution ofenvelope fusion proteins among members of the Baculoviridae, Orthomyxoviridae, andMetaviridae (insect retrovirus) families. Journal of Viology,76(11):5301-5304
Ribeiro B. M. and Crook N. E.,1993. Expression of full-length and truncated forms ofcrystal protein genes from Bacillus thuringiensis subsp. kurstaki in a baculovirus andpathogenicity of the recombinant viruses. Journal of General Virology,62(2):121-130
Roelvinkm P. W., Corsaro B. G. and Granados R. R.,1995. Characterization of theHelicoverpa armigera and Pseudaletia unipuncta granulovirus enhancin genes. Journal ofGeneral Virology,76(1):2693-2705
Rohrmann G.F.,2011. Baculovirus molecular biology (Second Edition).Http://www.ncbi.nlm.nih.gov/books/NBK49500/Copyright:1-39
Slack J. and Arlf B. M.,2007. The baculovruses occlusion-derived virus: virion structureand function. CRC Press: Elsevier Inc:1-64
Stewart L. M., Hirst M., López Ferber M., Merryweather A. T., Cayley P. J. and Possee R.D.,1991. Construction of an improved baculovirus insecticide containing an insect-specifictoxin gene. Nature,352(6330):85-88
Terra W. R. and Ferreira C.,1994. Insect digestive enzymes: Properties,compartmen-talization and function. Comparative biochemistry and physiology. Part B.Biochemistry&molecular biology,109:1–62
Theilmann D. A. and Stewart S.,1992. Tandemly repeated sequence at the3’ end of theIE-2gene of the baculovirus Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus isan enhancer element. Virology,187(1):97–106
Tinsley T. W. and Kelly D. C.,1985. Taxonomy and nomenclature of insect pathogenicviruses. Vol.3. Academic Press:3-25
Tomalski M. D., King T. P. and Miller L. K.,1993. Expression of hornet genes encodingvenom allergen antigen5in insects. Archives of Insect Biochemistry and Physology,22(3-4):303-313
Uwo M. F., Ui-Tei K., Park P. And Takeda M.,2002. Replacement of midgut epithliumin the greater wax moth, Galleria mellonela, during larval-pupal moult. Cell and TissueResearch,308(2):319-331.
Volkman L. E. and Goldsmith P. A.,1985. Mechanism of neutralization of buddedAutographa californica nuclear polyhedrosis virus by a monoclonal antibody: Inhibition ofentry by adsorptive endocytosis. Virology,143(1):185-195
Wang Y. Q., Wang J., Li J. Liu Q., Huang S. D., Wang D.,2010. Bacterial expression andpurification of an active ω-Atracotoxin-Ar1b from spider Atrax robustus. AgriculturalSciences in China,9:1292-1298
Williams G. V. and Faulkner P.,1997. Cytological changes and viral morphogenesisduring Baculovirus infection. Plenum Press, New York:61–108.
Wormleaton S., Kuzio J. and Winstanley D.,2003. The complete sequence of theAdoxophyes orana granulovirus genome. Virology,311(2):350-365
Xeros N.,1952. Cytoplasmic polyhedral virus diseases. Nature,170(4338):1073
Xiao H. and Qi Y.,2007. Genome sequence of Leucania seperata nucleopolyhedrovirus.Virus Genes,35(3):845-856
Young J. C., Mackinnon E. A. and Faulkner P.,1993. The architecture of the virogenicstroma in isolated nuclei of Spodoptera frugiperda cells in vitro infected by Autographacalifornica nuclear polyhedrosis virus. Journal of Structural Biology,110(2):141–153
Zanotto P. M., Kessing B. D. and Maruniak J. E.,1993. Phylogenetic interrelationshipsamong baculoviruses: evolutionary rates and host associations. Journal of InvertebratePathoogyl,62(2):147-164