杀虫剂亚致死浓度对褐飞虱保幼激素水解酶及类胰岛素基因的影响研究

发布时间：2018-01-31 12:47

本文关键词： 褐飞虱茚虫威氯虫苯甲酰胺类胰岛素基因保幼激素酯酶基因保幼激素环氧水解酶基因保幼激素二醇激酶基因　出处：《扬州大学》2017年硕士论文　论文类型：学位论文

【摘要】：褐飞虱 Nilaparvata lugens(Stal)(Hemiptera:Delphacidae)属半翅目,飞虱科,是水稻上的重要害虫。茚虫威和氯虫苯甲酰胺作为两种新型杀虫剂,在生产实践中被广泛使用。前期研究表明亚致死浓度的茚虫威和氯虫苯甲酰胺能够抑制褐飞虱的生殖。褐飞虱的生殖受许多因素影响,其中保幼激素对卵巢发育起重要作用,而保幼激素收受到水解酶的调控。胰岛素是昆虫重要的内分泌激素,对昆虫生理生化和生殖有调控作用,同时也与保幼激素有密切联系。本文通过克隆得到了保幼激素二醇激酶Nljhdk基因的全长,分析了该基因在短翅型褐飞虱不同龄期的表达差异,并利用RNAi研究其与保幼激素酯酶Nljhe和环氧水解酶Nljheh的关系,以及亚致死浓度茚虫威和氯虫苯甲酰胺对褐飞虱4个类胰岛素基因NlIlp1-4及3种保幼激素水解酶基因mRNA相对表达水平的影响,进一步揭示这两种药剂抑制生殖的机理。利用RT-PCR技术克隆了褐飞虱保幼激素二醇激酶基因684 bp cDNA(Nljhdk)。Nljhdk开放阅读框长558 bp,编码185个氨基酸,预测蛋白分子质量21.57 kDa,等电点为4.54。多重序列比对发现Nljhdk与天蚕Antheraea yamamai(KC127671)、桔小实绳Bactrocera dorsalis(JN256054)、家蚕Bombyx mori(AY363308)、马铃薯甲虫 Leptinotarsa decemlineata(KP295467)、烟草天蛾Manduca sexta(AJ430670)、亚洲玉米螟 Ostrinia furnacalis(HM626514)、小菜蛾 Plutella xylostella(JN410823)和斜纹夜蛾Spodoptera litura(KP192493)JHDK基因的氨基酸序列同源性分别为43.65%、55.68%、41.62%、61.08%、51.35%、48.65%、43.78%和45.95%。应用qPCR对Nljhdk表达模式分析发现,Nljhdk在若虫和成虫各个发育阶段都有表达,在4龄时表达水平最低,在5龄时有最高表达,并且在雄成虫中高于雌成虫。利用RNAi技术,发现干扰Nljhdk对Nljhe没有明显影响,但能够提高Nljheh的表达水平。茚虫威对褐飞虱的亚致死效应研究发现,茚虫威亚致死浓度能够显著抑制卵巢内NlIlp2-4的表达水平,20 mg/L茚虫威时能够显著上调NlIlp1的相对表达量10.67%;Nljhe表达水平随着浓度(3,5,10和20 mg/L)的增加,比对照分别显著上升了 52.92%、38.82%、105.5%和79.44%;Nljheh相对表达量相比对照组被显著抑制,1、3、5、10和20 mg/L浓度茚虫威处理后显著降低了 34.09%、30.49%、48.21%、45.21%和42.49%;Nljhdk在1和3 mg/L浓度时显著上升了 39.62%和59.32%,其余浓度处理没有显著差异。氯虫苯甲酰胺亚致死浓度处理褐飞虱后,雌虫卵巢内NlIlp1表达水平在15 mg/L浓度时比对照显著上升了 26.88%;NlIlp2表达水平在3.75 mg/L处理浓度比对照显著下降7.9%,但在7.5、30和60 mg/L时比对照显著上升了 20.13%、28.12%和47.77%;NlIlp3相对表达量只有在最高浓度60 mg/L时有明显的升高,比对照组高153.27%;NlIlp4相对表达量在3.75 mg/L时比对照上升了 9.62%,而在7.5、15、30和60 mg/L浓度时分别比对照上升了 51.24%、69.77%、52.63%和 95.17%。15、30 和 60 mg/L 的氯虫苯甲酰胺处理后,褐飞虱卵巢内Nljhe相对表达量分别比对照显著上升了 25.77%、24.12%和72.45%。Nljheh的相对表达量在3.75、15和60 mg/L氯虫苯甲酰胺处理后比对照显著上升了 16.18%、23.09%和65.00%。15和60 mg/L的氯虫苯甲酰胺处理后,褐飞虱卵巢内Mljhdk的相对表达量分别比对照显著上升了 48.8%和8.51%,但其他浓度处理后,Nljhdk的相对表达量反而下降。本文深入探究了茚虫威和氯虫苯甲酰胺抑制褐飞虱生殖的机制,为稻田合理使用这两种药剂提供了理论基础。
[Abstract]:The brown planthopper Nilaparvata lugens (Stal) (Hemiptera:Delphacidae) belong to Hemiptera, Delphacidae, is an important pest on rice. Indoxacarb and chlorantraniliprole as two kinds of new pesticides, is widely used in the production practice. Previous studies showed that sublethal concentrations of indoxacarb and chlorantraniliprole can inhibit brown planthopper the reproduction of brown planthopper. Reproduction is affected by many factors, including juvenile hormone plays an important role in ovarian development and juvenile hormone to accept hydrolase regulation. Insulin is an important hormone of insects, regulate insect physiology and reproduction, but also closely linked with the juvenile hormone. Is obtained in this paper. The cloning of full-length jhdk gene Nljhdk, analyzes the difference between the expression of the gene in brachypterous BPH at different ages, and the use of RNAi to study with the juvenile hormone esterase and Nljhe The relationship between Nljheh epoxide hydrolase, and sublethal concentrations of indoxacarb and chlorantraniliprole on BPH relative expression level influence NlIlp1-4 of 4 types of insulin gene and 3 juvenile hormone hydrolase gene mRNA, further reveal the mechanism of the two kinds of agents that inhibit reproduction. RT-PCR technology was used to clone BPH juvenile hormone diol 684 BP cDNA kinase gene (Nljhdk).Nljhdk long open reading frame of 558 BP, encoding 185 amino acids. The predicted molecular mass of 21.57 kDa and isoelectric point of 4.54. multiple sequence comparison showed that Nljhdk and Antheraea Yamamai (KC127671), a small orange solid rope Bactrocera dorsalis (JN256054), Bombyx mori (AY363308), Bombyx mori potato the beetle Leptinotarsa decemlineata (KP295467), Manduca sexta (AJ430670) of Manduca sexta, Asian corn borer (HM626514), Ostrinia furnacalis Plutella xylostella (JN410823) of diamondback moth and oblique Noctuid Spodoptera litura (KP192493) JHDK gene amino acid sequence homology were 43.65%, 55.68%, 41.62%, 61.08%, 51.35%, 48.65%, 43.78% and 45.95%. based on qPCR Nljhdk expression pattern analysis showed that Nljhdk developed in the nymph and adult stages are expressed in 4 at the age of the lowest level of expression, the highest expression at the age of 5, and higher than that of female adults in male adults. The use of RNAi technology, found that the interference of Nljhdk has no obvious effect on Nljhe, but it can increase the expression level of Nljheh. Indoxacarb on Nilaparvatalugens sublethal effects of the indene worm can inhibit the expression of WIA lethal concentration level of NlIlp2-4 in the ovary. 20 mg/L indoxacarb can up regulate the expression of NlIlp1 relative amount of 10.67%; the expression level of Nljhe with concentration (3,5,10 and 20 mg/L) increased than in the control group were significantly increased by 52.92%, 38.82%, 105.5% and 79.44%; Nljheh The relative expression was significantly inhibited compared with the control group, 1,3,5,10 and mg/L 20 concentration of indoxacarb treatment decreased significantly after 34.09%, 30.49%, 48.21%, 45.21% and 42.49%; 1 and 3 Nljhdk in the concentration of mg/L significantly increased by 39.62% and 59.32%, no significant differences in other concentrations. Chlorantraniliprole sublethal concentration. The brown planthopper, the expression level at the concentration of 15 mg/L significantly increased 26.88% NlIlp1 female ovaries; the expression level of NlIlp2 in 3.75 concentration of mg/L significantly decreased by 7.9%, but in 7.5,30 and 60 mg/L significantly increased by 20.13%, 28.12% and 47.77%; the relative expression of NlIlp3 was significantly increased only in the the highest concentration of 60 mg/L, 153.27% higher than the control group; the relative expression of NlIlp4 in 3.75 mg/L higher than the control by 9.62%, and 60 in 7.5,15,30 and mg/L concentration respectively increased by 51.24%, 69.77%, 52.63% 95.17%.15,30 and mg/L and 60 chlorantraniliprole, brown planthopper in the ovaries of the relative expression of Nljhe were significantly increased by 25.77% and 24.12%, the relative expression amount of 72.45%.Nljheh in 3.75,15 and mg/L 60 chlorantraniliprole after treatment significantly increased by 16.18%, 23.09% and 65.00%.15 and 60 mg/L chloride worm benzamide treatment, the relative expression of Mljhdk in ovary of Brown Planthopper were significantly increased by 48.8% and 8.51%, but with other treatments, the relative expression of Nljhdk decreased. This paper explores the mechanism of indoxacarb and chlorantraniliprole inhibiting BPH reproduction, provides a theoretical basis for the in rational use of these two agents.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.112.3

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