本文關(guān)鍵詞： 褐飛虱 茚蟲(chóng)威 氯蟲(chóng)苯甲酰胺 類(lèi)胰島素基因 保幼激素酯酶基因 保幼激素環(huán)氧水解酶基因 保幼激素二醇激酶基因　出處：《揚(yáng)州大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：褐飛虱 Nilaparvata lugens(Stal)(Hemiptera:Delphacidae)屬半翅目,飛虱科,是水稻上的重要害蟲(chóng)。茚蟲(chóng)威和氯蟲(chóng)苯甲酰胺作為兩種新型殺蟲(chóng)劑,在生產(chǎn)實(shí)踐中被廣泛使用。前期研究表明亞致死濃度的茚蟲(chóng)威和氯蟲(chóng)苯甲酰胺能夠抑制褐飛虱的生殖。褐飛虱的生殖受許多因素影響,其中保幼激素對(duì)卵巢發(fā)育起重要作用,而保幼激素收受到水解酶的調(diào)控。胰島素是昆蟲(chóng)重要的內(nèi)分泌激素,對(duì)昆蟲(chóng)生理生化和生殖有調(diào)控作用,同時(shí)也與保幼激素有密切聯(lián)系。本文通過(guò)克隆得到了保幼激素二醇激酶Nljhdk基因的全長(zhǎng),分析了該基因在短翅型褐飛虱不同齡期的表達(dá)差異,并利用RNAi研究其與保幼激素酯酶Nljhe和環(huán)氧水解酶Nljheh的關(guān)系,以及亞致死濃度茚蟲(chóng)威和氯蟲(chóng)苯甲酰胺對(duì)褐飛虱4個(gè)類(lèi)胰島素基因NlIlp1-4及3種保幼激素水解酶基因mRNA相對(duì)表達(dá)水平的影響,進(jìn)一步揭示這兩種藥劑抑制生殖的機(jī)理。利用RT-PCR技術(shù)克隆了褐飛虱保幼激素二醇激酶基因684 bp cDNA(Nljhdk)。Nljhdk開(kāi)放閱讀框長(zhǎng)558 bp,編碼185個(gè)氨基酸,預(yù)測(cè)蛋白分子質(zhì)量21.57 kDa,等電點(diǎn)為4.54。多重序列比對(duì)發(fā)現(xiàn)Nljhdk與天蠶Antheraea yamamai(KC127671)、桔小實(shí)繩Bactrocera dorsalis(JN256054)、家蠶Bombyx mori(AY363308)、馬鈴薯甲蟲(chóng) Leptinotarsa decemlineata(KP295467)、煙草天蛾Manduca sexta(AJ430670)、亞洲玉米螟 Ostrinia furnacalis(HM626514)、小菜蛾 Plutella xylostella(JN410823)和斜紋夜蛾Spodoptera litura(KP192493)JHDK基因的氨基酸序列同源性分別為43.65%、55.68%、41.62%、61.08%、51.35%、48.65%、43.78%和45.95%。應(yīng)用qPCR對(duì)Nljhdk表達(dá)模式分析發(fā)現(xiàn),Nljhdk在若蟲(chóng)和成蟲(chóng)各個(gè)發(fā)育階段都有表達(dá),在4齡時(shí)表達(dá)水平最低,在5齡時(shí)有最高表達(dá),并且在雄成蟲(chóng)中高于雌成蟲(chóng)。利用RNAi技術(shù),發(fā)現(xiàn)干擾Nljhdk對(duì)Nljhe沒(méi)有明顯影響,但能夠提高Nljheh的表達(dá)水平。茚蟲(chóng)威對(duì)褐飛虱的亞致死效應(yīng)研究發(fā)現(xiàn),茚蟲(chóng)威亞致死濃度能夠顯著抑制卵巢內(nèi)NlIlp2-4的表達(dá)水平,20 mg/L茚蟲(chóng)威時(shí)能夠顯著上調(diào)NlIlp1的相對(duì)表達(dá)量10.67%;Nljhe表達(dá)水平隨著濃度(3,5,10和20 mg/L)的增加,比對(duì)照分別顯著上升了 52.92%、38.82%、105.5%和79.44%;Nljheh相對(duì)表達(dá)量相比對(duì)照組被顯著抑制,1、3、5、10和20 mg/L濃度茚蟲(chóng)威處理后顯著降低了 34.09%、30.49%、48.21%、45.21%和42.49%;Nljhdk在1和3 mg/L濃度時(shí)顯著上升了 39.62%和59.32%,其余濃度處理沒(méi)有顯著差異。氯蟲(chóng)苯甲酰胺亞致死濃度處理褐飛虱后,雌蟲(chóng)卵巢內(nèi)NlIlp1表達(dá)水平在15 mg/L濃度時(shí)比對(duì)照顯著上升了 26.88%;NlIlp2表達(dá)水平在3.75 mg/L處理濃度比對(duì)照顯著下降7.9%,但在7.5、30和60 mg/L時(shí)比對(duì)照顯著上升了 20.13%、28.12%和47.77%;NlIlp3相對(duì)表達(dá)量只有在最高濃度60 mg/L時(shí)有明顯的升高,比對(duì)照組高153.27%;NlIlp4相對(duì)表達(dá)量在3.75 mg/L時(shí)比對(duì)照上升了 9.62%,而在7.5、15、30和60 mg/L濃度時(shí)分別比對(duì)照上升了 51.24%、69.77%、52.63%和 95.17%。15、30 和 60 mg/L 的氯蟲(chóng)苯甲酰胺處理后,褐飛虱卵巢內(nèi)Nljhe相對(duì)表達(dá)量分別比對(duì)照顯著上升了 25.77%、24.12%和72.45%。Nljheh的相對(duì)表達(dá)量在3.75、15和60 mg/L氯蟲(chóng)苯甲酰胺處理后比對(duì)照顯著上升了 16.18%、23.09%和65.00%。15和60 mg/L的氯蟲(chóng)苯甲酰胺處理后,褐飛虱卵巢內(nèi)Mljhdk的相對(duì)表達(dá)量分別比對(duì)照顯著上升了 48.8%和8.51%,但其他濃度處理后,Nljhdk的相對(duì)表達(dá)量反而下降。本文深入探究了茚蟲(chóng)威和氯蟲(chóng)苯甲酰胺抑制褐飛虱生殖的機(jī)制,為稻田合理使用這兩種藥劑提供了理論基礎(chǔ)。
[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.

【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S435.112.3

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