斜纹夜蛾重组杆状病毒的构建及生物学活性测定
摘要
斜纹夜蛾(Spodoptera litura)是一种广泛分布的、危害严重的农作物和绿化植物的重要害虫。由于长期大量的化学药剂的使用,斜纹夜蛾对有机氯、有机磷及氨基甲酸酯、拟除虫菊酯等多类杀虫剂产生了不同程度的抗药性。大量使用化学杀虫剂对生态环境造成很大的污染,非靶标物种常受到危害如甲壳类和鱼类,甚至危害到人类健康。杆状病毒杀虫剂却具有较好的宿主专一性,对非靶标动植物无影响。但是野生型杆状病毒用作杀虫剂也存在很多不足之处,如杀虫谱窄、杀虫速度慢等缺点。应用分子生物技术对野生病毒基因组进行改造,成为研究新型、高效、安全的生物杀虫剂的研究热点。
本研究在构建重组病毒转移载体的基础上,对具有强毒力的野生型斜纹夜蛾核型多角体病毒(SpltMNPVⅡ)进行了改造,主要工作成果如下:1.克隆并分析了苜蓿银纹夜蛾(AcMNPV)基因组中的类芋螺毒素基因(Ac-ctx-like)。2.构建了重组病毒转移载体psk-Δegt-Pph–egfp和psk-Δegt-Pph-ctx-Pp10-egfp。3.经脂质体介导的共转染及一系列的筛选获得了一株重组斜纹夜蛾核型多角体病毒SpltMNPV-Δegt-Pph-egfp。4.对SpltMNPV-Δegt-Pph-egfp进行了生物学活性测定。该重组病毒可使半致死时间明显缩短,是一株有效的重组杆状病毒。研究结果为进一步研究与开发重组斜纹夜蛾核型多角体病毒生物杀虫剂打下了基础。
Spodoptera litura is one of the serious pests around the world, attacking crop and greening plant. Owing to a great quantity of chemical insecticide been used for a long time, Spodoptera litura has resistance to organochlorine pesticide, organophosphorus pesticide, carbamic acid ester pesticide, pyrethroid insecticides. At the sametime, chemical pesticide polluted the environment. Fish and shellfish, even human, was hurt by it. Baculovirus has host specify, and it has no effect on nontarget animals. However, it has some disadvantages. For example, low speed of killing and narrow insecticidal spectrum. In order to solve the above problems one of the most popular approach is to improve the insecticidal properties of SPltMNPV by means of genetie engineering and moleeular biology.
In this research, SpltMNPVⅡwas reformed. The main conclusions are as follows:
1. Cloning and analysis of ctx-like gene, coming from AcMNPV.
2. Recombinant baculovirus transfer vectors psk-Δegt-Pph–egfp and psk-Δegt- Pph-ctx-Pp10-egfp had been constructed successfully.
3. A recombinant baculovirus SpltMNPV-Δegt-Pph-egfp was gained after cotransfection and repeatedly screen.
4. Biological assay was been done about the recombinant baculovirus SpltMNPV-Δegt-Pph-egfp. It reduced the LT50 effectively.
The results lay the foundation for the improvement and development of this virus insecticide.
本研究在构建重组病毒转移载体的基础上,对具有强毒力的野生型斜纹夜蛾核型多角体病毒(SpltMNPVⅡ)进行了改造,主要工作成果如下:1.克隆并分析了苜蓿银纹夜蛾(AcMNPV)基因组中的类芋螺毒素基因(Ac-ctx-like)。2.构建了重组病毒转移载体psk-Δegt-Pph–egfp和psk-Δegt-Pph-ctx-Pp10-egfp。3.经脂质体介导的共转染及一系列的筛选获得了一株重组斜纹夜蛾核型多角体病毒SpltMNPV-Δegt-Pph-egfp。4.对SpltMNPV-Δegt-Pph-egfp进行了生物学活性测定。该重组病毒可使半致死时间明显缩短,是一株有效的重组杆状病毒。研究结果为进一步研究与开发重组斜纹夜蛾核型多角体病毒生物杀虫剂打下了基础。
Spodoptera litura is one of the serious pests around the world, attacking crop and greening plant. Owing to a great quantity of chemical insecticide been used for a long time, Spodoptera litura has resistance to organochlorine pesticide, organophosphorus pesticide, carbamic acid ester pesticide, pyrethroid insecticides. At the sametime, chemical pesticide polluted the environment. Fish and shellfish, even human, was hurt by it. Baculovirus has host specify, and it has no effect on nontarget animals. However, it has some disadvantages. For example, low speed of killing and narrow insecticidal spectrum. In order to solve the above problems one of the most popular approach is to improve the insecticidal properties of SPltMNPV by means of genetie engineering and moleeular biology.
In this research, SpltMNPVⅡwas reformed. The main conclusions are as follows:
1. Cloning and analysis of ctx-like gene, coming from AcMNPV.
2. Recombinant baculovirus transfer vectors psk-Δegt-Pph–egfp and psk-Δegt- Pph-ctx-Pp10-egfp had been constructed successfully.
3. A recombinant baculovirus SpltMNPV-Δegt-Pph-egfp was gained after cotransfection and repeatedly screen.
4. Biological assay was been done about the recombinant baculovirus SpltMNPV-Δegt-Pph-egfp. It reduced the LT50 effectively.
The results lay the foundation for the improvement and development of this virus insecticide.
引文
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[1] Francisco J.R.Pinedo, Flavio Moscardi, Teresa Luque, Julie A.Oszewski, and Bergmann M. Ribeiroa. Inactivation of the ecdysteroid UDP-glucosyltransferase (egt) gene of Anticarsia gemmatalis nucleo polyhedro virus(AgMNPV) improves its virulence towards its insect host[J]. Biological Control. 2003, 27: 336–344
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[1] Francisco J.R.Pinedo, Flavio Moscardi, Teresa Luque, Julie A.Oszewski, and Bergmann M. Ribeiroa. Inactivation of the ecdysteroid UDP-glucosyltransferase (egt) gene of Anticarsia gemmatalis nucleopolyhedrovirus(AgMNPV) improves its virulence towards its insect host[J]. Biological Control. 2003, 27: 336–344.
[2] O’Reilly D R ,Miller L K. Regulation of expression of a baculovirus ecdysteroid UDP-glucosyltransferase gene[J]. J Virol,1990 ,64:1321-1328.
[2] Graham M., Nicholson. Fighting the global pest problem[J]. Toxicon. 2007, 49: 413–422.
[3] Brooks, E.M., Hines, E.R., Viral biopesticides for heliothine control—fact or fiction[J]. Today’s Life. Sci. 1999. 1: 38–44.
[4] Casida, J.E., Quistad, G.B., Golden age of insecticide research: past, present, orfuture[J] Annu. Rev. Entomol. 1998. 43,1-16.
[5] Van Wijngaarden, R.P., Brock, T.C., Van den Brink, P.J., Threshold levels for effects of insecticides in freshwater ecosystems: a review. Ecotoxicology. 2005.14, 355–380.
[6]丁锦华,苏建亚.农业昆虫学[M ].北京:中国农业出版社, 2002: 281.
[7]李靖,江国良等.桃园斜纹夜蛾发生情况及防治措施[J].四川农业科技,2010, 9: 53.
[8]周新伟,邓金花,顾俊荣,等.斜纹夜蛾在草坪上的发生特点及防治技术[J].江苏农业科学,2009,6: 188-189.
[9]马赵江,周小军,戴为光,等.金华菜区斜纹夜蛾发生规律与防治技术研究[J].长江蔬菜,2007,53-54.
[10]韩群营,黄明生,董方友,等. 2009年武汉市蔡甸区斜纹夜蛾暴发原因及综防对策[J].安徽农学通报,2010,16(14): 124.
[11]周晓梅,黄炳球.斜纹夜蛾抗药性及其防治对策的研究进展[J].昆虫知识, 2002, 39 ( 2) : 98-102.
[12]百度百科. http://baike.baidu.com/view/3845645.htm,2010.
[13]胡远扬.昆虫病毒研究的回顾与展望[J].中国病毒学,2004,19(3):303-308.
[14] Inceoglu ,A.B ., Kamita, S.G., Hinton ,A.C., et al. Recombinant baculoviruses for insect control[J]. Pest Management Science , 2001, 57 : 981–98.
[15]中国农药信息网.农药有效成分http://www.chinapesticide.gov.cn/ service/aspx,2010.
[16] Moscardi ,F. Assessment of the application of baculoviruses forcontrol of Lepidoptera[J]. Annu Rev Entomol, 1999, 44:257-289.
[17] Sun X L, Chen XW, Zhang Z X, et al. Bollworm responses torelease of genetically modified Helicoverpa armigera nucleopoly2 hedroviruses in cotton [J]. Journal of Invertebrate Pathology, 2002, 81(2): 63–69.
[18] David Grzywacza, Wilfred L. Mushobozib, Mark Parnella, Flavia Jolliffec, Kenneth Wilson, Evaluation of Spodoptera exempta nucleopolyhedrovirus (SpexNPV) for the field control of African armyworm (Spodoptera exempta) in Tanzania[J], Crop Protection 2008, 27: 17–24.
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[1] Francisco J.R.Pinedo, Flavio Moscardi, Teresa Luque, Julie A.Oszewski, and Bergmann M. Ribeiroa. Inactivation of the ecdysteroid UDP-glucosyltransferase (egt) gene of Anticarsia gemmatalis nucleo polyhedro virus(AgMNPV) improves its virulence towards its insect host[J]. Biological Control. 2003, 27: 336–344
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[1] Francisco J.R.Pinedo, Flavio Moscardi, Teresa Luque, Julie A.Oszewski, and Bergmann M. Ribeiroa. Inactivation of the ecdysteroid UDP-glucosyltransferase (egt) gene of Anticarsia gemmatalis nucleopolyhedrovirus(AgMNPV) improves its virulence towards its insect host[J]. Biological Control. 2003, 27: 336–344.
[2] O’Reilly D R ,Miller L K. Regulation of expression of a baculovirus ecdysteroid UDP-glucosyltransferase gene[J]. J Virol,1990 ,64:1321-1328.