【摘要】：吡蟲啉是新煙堿類殺蟲劑,該產(chǎn)品殺蟲譜廣,低毒高效,應(yīng)用廣泛。粘質(zhì)沙雷氏菌廣泛分布于自然界,是水和土壤中的常居菌群,對(duì)黃脛小車蝗(Oedaleus infernalis Saussure)有很高的毒力,有較好的生防效果。東亞飛蝗Locusta migratoria manilensis(Meyen)作為一種模式昆蟲,在組織觀察及模型構(gòu)建,殺蟲菌株及殺蟲活性物質(zhì)篩選等方面應(yīng)用廣泛。為初步探究昆蟲中腸解毒、免疫、消化相關(guān)基因?qū)Σ煌瑒┝窟料x啉的響應(yīng)機(jī)制,以及吡蟲啉和粘質(zhì)沙雷氏菌對(duì)昆蟲血淋巴免疫系統(tǒng)的影響,本研究以東亞飛蝗為實(shí)驗(yàn)材料,開(kāi)展了相關(guān)的研究工作。一、高低劑量吡蟲啉處理下東亞飛蝗中腸轉(zhuǎn)錄組的測(cè)序及數(shù)據(jù)分析通過(guò)生物測(cè)定,本研究分別選用吡蟲啉LD10(0.037 mg/蟲)和LD80(4.11 mg/蟲)作為低劑量和高劑量,處理東亞飛蝗成蟲(雌雄各半)。無(wú)菌解剖取樣,RNA提取檢測(cè)合格后,利用Illumina Hiseq2000平臺(tái)測(cè)序。組裝結(jié)果總共Unigene 59331個(gè),平均長(zhǎng)度 747 nt,N50 達(dá)到 1187 nt。Unigene 功能注釋,注釋到 NR,NT,Swiss-Prot,KEGG,COG,GO 庫(kù)的 Unigene 分別是 23201 個(gè)、11141 個(gè)、18837 個(gè)、16709 個(gè)、9818個(gè)、11585個(gè),所有注釋上的Unigene是25531個(gè)。進(jìn)一步分析顯示,注釋為解毒酶的Unigene受低劑量吡蟲啉影響更多,影響最顯著的是細(xì)胞色素P450s(差異表達(dá)個(gè)數(shù)/測(cè)得總數(shù)=47.19%)。解毒酶中差異表達(dá)Unigene最多的是P450s的6 family,CarEs的A clade和GSTs的sigma class,而葡萄糖醛酸轉(zhuǎn)移酶UGTs大部分Unigene在吡蟲啉處理后呈下調(diào)趨勢(shì)。免疫相關(guān)基因在高低劑量吡蟲啉處理后,響應(yīng)Unigene數(shù)量差距不明顯。溶菌酶、雙翅肽、Apolipophorin Ⅲ、轉(zhuǎn)鐵蛋白在高低劑量處理中均呈下調(diào)趨勢(shì),僅防御素在高劑量處理中特異上調(diào)。消化酶基因在不同劑量的吡蟲啉處理中,差異表達(dá)的基因種類更為豐富和復(fù)雜。11個(gè)編碼糜蛋白酶(chymotrypsin)的unigenes中,9個(gè)在吡蟲啉處理后出現(xiàn)差異表達(dá),其中8個(gè)為顯著下調(diào)。二、高低劑量吡蟲啉處理后東亞飛蝗中腸轉(zhuǎn)錄組部分差異表達(dá)基因的定量驗(yàn)證本研究綜合unigene長(zhǎng)度、差異表達(dá)倍數(shù)等因素,選擇部分解毒酶、免疫相關(guān)基因進(jìn)行定量驗(yàn)證。定量結(jié)果顯示,在選擇驗(yàn)證的23個(gè)基因中,76.08%結(jié)果與轉(zhuǎn)錄組一致,驗(yàn)證了轉(zhuǎn)錄組的準(zhǔn)確性。在細(xì)胞色素P450s基因中,共選擇了 6個(gè)基因分別在高低劑量吡蟲啉處理后進(jìn)行了驗(yàn)證,與轉(zhuǎn)錄組結(jié)果一致的占到了 75.00%。定量結(jié)果顯示CYP6K1(CL4043C2)表達(dá)差異最為顯著,在高劑量吡蟲啉處理后上升了 10.07倍,在低劑量吡蟲啉處理后上升了 9.32倍。CYP6BQ37(U27188)在高劑量吡蟲啉處理后上升了 6.68倍,而低劑量處理和對(duì)照不存在顯著差異,表現(xiàn)出劑量依賴性誘導(dǎo)高表達(dá);與此相反,CYP6HQ1(CL659C2)在低劑量處理后上升了 6.54,而高劑量處理與對(duì)照間不存在顯著差異,同樣表現(xiàn)出劑量依賴性誘導(dǎo)高表達(dá)。在谷胱甘肽S-轉(zhuǎn)移酶(GSTs)中,共選擇了 4個(gè)基因進(jìn)行了驗(yàn)證,與轉(zhuǎn)錄組結(jié)果一致的占到了 75.00%。其中,GSTS2(CL1173C2)在高劑量吡蟲啉處理后上調(diào)了 9.00倍,而低劑量處理和對(duì)照不存在顯著差異;GSTD5(U33519)、GST03(CL3618C4)和 GSTS1(CL4610C5)在低劑量吡蟲啉處理后分別上調(diào)了 10.34、8.34和3.04倍,而高劑量處理與對(duì)照間不存在顯著差異,均表現(xiàn)出劑量依賴性誘導(dǎo)高表達(dá)。在羧酸酯酶(CarEs)中,共選擇了 4個(gè)基因進(jìn)行了驗(yàn)證,與轉(zhuǎn)錄組結(jié)果一致的占到了 50.00%。CesD1(U30920)和CesA20(U6549)高劑量吡蟲啉處理后分別上調(diào)7.03倍和13.32倍,而低劑量處理和對(duì)照不存在顯著差異;而CesA3(CL3747C1)在低劑量吡蟲啉處理后上調(diào)了 4.50倍,而高劑量處理與對(duì)照間不存在顯著差異。CesA8(U12483)高、低劑量吡蟲啉處理后分別上調(diào)3.37倍和5.46倍。在免疫防御相關(guān)基因中,共選擇了 9個(gè)基因進(jìn)行了驗(yàn)證,與轉(zhuǎn)錄組結(jié)果一致的占到了 88.89%。結(jié)果顯示,Mucin-5(CL2786C1)基因差異表達(dá)量最高,高劑量吡蟲啉處理后上調(diào)12.2倍,低劑量吡蟲啉處理后上調(diào)9.23倍;PGRP1(CL2306C2)在高劑量處理后上調(diào)11.67倍,低劑量處理后上調(diào)7.96倍;Serpin4(CL2600C2)在高劑量吡蟲啉處理后上調(diào)7.18倍,低劑量吡蟲啉處理后上調(diào)10.08倍。Defensin-1(U43570)在高劑量吡蟲啉處理后上調(diào)5.17倍,而低劑量處理和對(duì)照不存在顯著差異。Diptericin(U8926)在高低劑量吡蟲啉處理后均下調(diào),分別為對(duì)照的0.25倍和0.12倍。三、吡蟲啉、粘質(zhì)沙雷氏菌對(duì)東亞飛蝗血淋巴免疫通路基因的調(diào)控為了更好的了解吡蟲啉對(duì)東亞飛蝗免疫影響,以及免疫系統(tǒng)的響應(yīng)機(jī)制,本研究用低劑量吡蟲啉和粘質(zhì)沙雷氏菌(實(shí)驗(yàn)室分離的一株高致病菌)飼喂東亞飛蝗。采用熒光定量分析方法,分別檢測(cè)了血淋巴中IMD信號(hào)通路和Toll信號(hào)通路的2個(gè)關(guān)鍵基因,IMD信號(hào)通路的關(guān)鍵基因?yàn)?PGRP-LE和Relish;Toll信號(hào)通路的關(guān)鍵基因?yàn)?GNBP3和MyD88。并分別檢測(cè)了 IMD信號(hào)通路的產(chǎn)物之一:雙翅肽(diptericin)和Toll信號(hào)通路的產(chǎn)物之一:防御素(defensin)。粘質(zhì)沙雷氏菌處理后,IMD信號(hào)通路上的Relish上調(diào)了 9.50倍,PGRP-LE上調(diào)了 6.06倍,但是IMD信號(hào)通路的產(chǎn)物之一Diptericin卻沒(méi)有出現(xiàn)顯著差異。粘質(zhì)沙雷氏菌處理后,Toll信號(hào)通路的GNBP3上調(diào)了 4.94倍,但產(chǎn)物之一的Defensin-1顯著下調(diào)。在低劑量吡蟲啉處理后,東亞飛蝗血淋巴免疫防御的IMD信號(hào)通路的Relish和通路產(chǎn)物Diptericin分別上調(diào)了 6.77倍和7.88倍。但是,吡蟲啉處理對(duì)Toll信號(hào)通路的GNBP3和MyD88均沒(méi)有顯著影響,而Toll信號(hào)通路產(chǎn)物Defensin-1顯著下調(diào)。綜合中腸和血淋巴兩部分結(jié)果,不同劑量吡蟲啉對(duì)同一免疫組織的誘導(dǎo)情況不同,暗示昆蟲在不同劑量吡蟲啉脅迫下響應(yīng)機(jī)制不同。同濃度吡蟲啉對(duì)不同組織的免疫誘導(dǎo)也有很大差別,暗示不同組織在接觸殺蟲劑后,采取了不同的應(yīng)對(duì)策略,同一抗菌肽在不同組織中的作用可能有所差別。吡蟲啉能夠誘導(dǎo)東亞飛蝗血淋巴的IMD信號(hào)通路,而對(duì)Toll信號(hào)通路誘導(dǎo)效果不明顯,暗示吡蟲啉與真菌或革蘭氏陽(yáng)性菌復(fù)配使用能夠起到更好的殺蟲效果。本研究推測(cè)不同殺蟲劑能夠誘導(dǎo)昆蟲特定的免疫通路,為殺蟲真菌、革蘭氏陽(yáng)性菌、革蘭氏陰性菌與農(nóng)藥的復(fù)配提供了新的思路和理論依據(jù)。
[Abstract]:Imidacloprid is a new nicotine insecticide. It has a broad spectrum of insecticides, low toxicity, high efficiency and wide application. Serratia marcescens is widely distributed in nature. It is a common microbial community in water and soil. It has high toxicity to Oedaleus infernalis Saussure and good biocontrol effect. Locusta migratoria manilensis (Meyen) As a model insect, imidacloprid has been widely used in tissue observation, model construction, screening of insecticidal strains and insecticidal active substances. In order to preliminarily explore the response mechanism of insect midgut detoxification, immunity and digestion related genes to different doses of imidacloprid, and the effects of imidacloprid and Serratia marcescens on the immune system of insect hemolymph, this study was conducted. 1. Sequencing and data analysis of midgut transcriptome of Locusta migratoria migratoria under high and low doses of imidacloprid were analyzed by bioassay. Imidacloprid LD10 (0.037 mg/worm) and LD80 (4.11 mg/worm) were used as low doses and high doses to treat adult Locusta migratoria migratoria migratoria (male and female, respectively). Aseptic anatomy sampling, RNA extraction and detection of qualified, using Illumina Hiseq2000 platform sequencing. The assembly results of a total of 59331 Unigene, an average length of 747 nt, N50 to 1187 nt. Unigene functional annotations, annotated to NR, NT, Swiss-Prot, KEGG, COG, GO libraries Unigene is 23201, 11141, 18837, 16709, 9818, 11585 Further analysis showed that the effect of low-dose imidacloprid on Unigene was more significant than that of cytochrome P450s (the number of differential expression/total number measured = 47.19%). Most Unigene s of UGTs were down-regulated after imidacloprid treatment. There was no significant difference in the number of immune-related genes responding to Unigene between high and low doses of imidacloprid treatment. Lysozyme, dipeptide, Apolipophorin III and transferrin were down-regulated in high and low doses of imidacloprid treatment. Defensins were only down-regulated in high doses of imidacloprid treatment. Digestive enzyme genes were more abundant and complex in different doses of imidacloprid treatment. Nine of the 11 unigenes encoding chymotrypsin were differentially expressed after imidacloprid treatment, and eight of them were significantly down-regulated. Second, the midgut transcription group of migratory locusts in East Asia was treated with high and low doses of imidacloprid. Quantitative validation of some differentially expressed genes by using UniGene length and multiple of differentially expressed genes, partial detoxification enzymes were selected and Immuno-related genes were quantitatively validated. A total of 6 genes were selected and validated after high and low doses of imidacloprid treatment, accounted for 75.00% of the transcripts. The quantitative results showed that the expression of CYP6K1 (CL4043C2) was the most significant difference, which increased 10.07 times after high doses of imidacloprid treatment and 9.32 times after low doses of imidacloprid treatment. In contrast, CYP6HQ1 (CL659C2) increased by 6.54 after low-dose treatment, but there was no significant difference between high-dose treatment and control, which also showed dose-dependent induction of high expression. Four genes of glutathione S-transferase (GSTs) were selected for validation, which accounted for 75.00% of the total. GSTS2 (CL1173C2) was up-regulated by 9.00 times after high-dose imidacloprid treatment, but there was no significant difference between low-dose imidacloprid treatment and control group; GSTD5 (U33519), GST03 (CL3618C4) and GSTS1 (CL4610C5) were down-regulated at low-dose. Imidacloprid treatment up-regulated 10.34, 8.34 and 3.04 times respectively, but there was no significant difference between the high-dose treatment and the control, showing dose-dependent induction of high expression. CesA3 (CL3747C1) was up by 4.50 times after low-dose imidacloprid treatment, but there was no significant difference between high-dose imidacloprid treatment and control. CesA8 (U12483) was up by 3.37 times and 5.46 times after low-dose imidacloprid treatment. Nine of the defense-related genes were identified, accounting for 88.89% of the total. The results showed that Mucin-5 (CL2786C1) gene was up-regulated by 12.2 times after high-dose imidacloprid treatment, 9.23 times after low-dose imidacloprid treatment, and PGRP1 (CL2306C2) was up-regulated by 11.67 times and down-regulated by low-dose imidacloprid treatment. Serpin 4 (CL2600C2) increased by 7.18 times after high dose imidacloprid treatment and 10.08 times after low dose imidacloprid treatment. Defensin-1 (U43570) increased by 5.17 times after high dose imidacloprid treatment, but there was no significant difference between low dose imidacloprid treatment and control. Diptericin (U8926) increased by 7.18 times after high and low dose imidacloprid treatment. Imidacloprid and Serratia marcescens were used to regulate the hemolymph immune pathway genes of migratory locusts in order to better understand the immune effects of Imidacloprid on migratory locusts in East Asia and the response mechanism of the immune system. Two key genes of IMD signaling pathway and Toll signaling pathway in hemolymph were detected by fluorescence quantitative analysis. PGRP-LE and Relish were the key genes of IMD signaling pathway, GNBP3 and MyD88 were the key genes of Toll signaling pathway. After treatment with Serratia marcescens, Reish in IMD signaling pathway was up-regulated by 9.50 times and PGRP-LE by 6.06 times, but Diptericin, one of the products of IMD signaling pathway, was not significantly different. After treatment with Serratia marcescens, GNBP3 in Toll signaling pathway was up-regulated. After treatment with low dose imidacloprid, Relish and Diptericin of IMD signaling pathway of hemolymph immune defense were up-regulated by 6.77 and 7.88 times, respectively. However, imidacloprid treatment had no significant effect on GNBP3 and MyD88 of Toll signaling pathway, while Toll signaling pathway was up-regulated by 7.88 times. The results of midgut and hemolymph showed that different doses of imidacloprid could induce the same immune tissue differently, suggesting that the response mechanism of insects was different under different doses of imidacloprid stress. Imidacloprid can induce IMD signaling pathways in the haemolymph of migratory locusts in East Asia, but not Toll signaling pathways, suggesting that the combination of Imidacloprid with fungi or Gram-positive bacteria can achieve better insecticidal efficacy. It is speculated that different insecticides can induce specific immune pathways of insects, which provides new ideas and theoretical basis for the combination of insecticidal fungi, Gram-positive bacteria, Gram-negative bacteria and pesticides.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S482.3

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