吡虫啉处理对东亚飞蝗解毒和防卫基因表达的影响
发布时间：2018-09-07 16:00
【摘要】：吡虫啉是新烟碱类杀虫剂,该产品杀虫谱广,低毒高效,应用广泛。粘质沙雷氏菌广泛分布于自然界,是水和土壤中的常居菌群,对黄胫小车蝗(Oedaleus infernalis Saussure)有很高的毒力,有较好的生防效果。东亚飞蝗Locusta migratoria manilensis(Meyen)作为一种模式昆虫,在组织观察及模型构建,杀虫菌株及杀虫活性物质筛选等方面应用广泛。为初步探究昆虫中肠解毒、免疫、消化相关基因对不同剂量吡虫啉的响应机制,以及吡虫啉和粘质沙雷氏菌对昆虫血淋巴免疫系统的影响,本研究以东亚飞蝗为实验材料,开展了相关的研究工作。一、高低剂量吡虫啉处理下东亚飞蝗中肠转录组的测序及数据分析通过生物测定,本研究分别选用吡虫啉LD10(0.037 mg/虫)和LD80(4.11 mg/虫)作为低剂量和高剂量,处理东亚飞蝗成虫(雌雄各半)。无菌解剖取样,RNA提取检测合格后,利用Illumina Hiseq2000平台测序。组装结果总共Unigene 59331个,平均长度 747 nt,N50 达到 1187 nt。Unigene 功能注释,注释到 NR,NT,Swiss-Prot,KEGG,COG,GO 库的 Unigene 分别是 23201 个、11141 个、18837 个、16709 个、9818个、11585个,所有注释上的Unigene是25531个。进一步分析显示,注释为解毒酶的Unigene受低剂量吡虫啉影响更多,影响最显著的是细胞色素P450s(差异表达个数/测得总数=47.19%)。解毒酶中差异表达Unigene最多的是P450s的6 family,CarEs的A clade和GSTs的sigma class,而葡萄糖醛酸转移酶UGTs大部分Unigene在吡虫啉处理后呈下调趋势。免疫相关基因在高低剂量吡虫啉处理后,响应Unigene数量差距不明显。溶菌酶、双翅肽、Apolipophorin Ⅲ、转铁蛋白在高低剂量处理中均呈下调趋势,仅防御素在高剂量处理中特异上调。消化酶基因在不同剂量的吡虫啉处理中,差异表达的基因种类更为丰富和复杂。11个编码糜蛋白酶(chymotrypsin)的unigenes中,9个在吡虫啉处理后出现差异表达,其中8个为显著下调。二、高低剂量吡虫啉处理后东亚飞蝗中肠转录组部分差异表达基因的定量验证本研究综合unigene长度、差异表达倍数等因素,选择部分解毒酶、免疫相关基因进行定量验证。定量结果显示,在选择验证的23个基因中,76.08%结果与转录组一致,验证了转录组的准确性。在细胞色素P450s基因中,共选择了 6个基因分别在高低剂量吡虫啉处理后进行了验证,与转录组结果一致的占到了 75.00%。定量结果显示CYP6K1(CL4043C2)表达差异最为显著,在高剂量吡虫啉处理后上升了 10.07倍,在低剂量吡虫啉处理后上升了 9.32倍。CYP6BQ37(U27188)在高剂量吡虫啉处理后上升了 6.68倍,而低剂量处理和对照不存在显著差异,表现出剂量依赖性诱导高表达;与此相反,CYP6HQ1(CL659C2)在低剂量处理后上升了 6.54,而高剂量处理与对照间不存在显著差异,同样表现出剂量依赖性诱导高表达。在谷胱甘肽S-转移酶(GSTs)中,共选择了 4个基因进行了验证,与转录组结果一致的占到了 75.00%。其中,GSTS2(CL1173C2)在高剂量吡虫啉处理后上调了 9.00倍,而低剂量处理和对照不存在显著差异;GSTD5(U33519)、GST03(CL3618C4)和 GSTS1(CL4610C5)在低剂量吡虫啉处理后分别上调了 10.34、8.34和3.04倍,而高剂量处理与对照间不存在显著差异,均表现出剂量依赖性诱导高表达。在羧酸酯酶(CarEs)中,共选择了 4个基因进行了验证,与转录组结果一致的占到了 50.00%。CesD1(U30920)和CesA20(U6549)高剂量吡虫啉处理后分别上调7.03倍和13.32倍,而低剂量处理和对照不存在显著差异;而CesA3(CL3747C1)在低剂量吡虫啉处理后上调了 4.50倍,而高剂量处理与对照间不存在显著差异。CesA8(U12483)高、低剂量吡虫啉处理后分别上调3.37倍和5.46倍。在免疫防御相关基因中,共选择了 9个基因进行了验证,与转录组结果一致的占到了 88.89%。结果显示,Mucin-5(CL2786C1)基因差异表达量最高,高剂量吡虫啉处理后上调12.2倍,低剂量吡虫啉处理后上调9.23倍;PGRP1(CL2306C2)在高剂量处理后上调11.67倍,低剂量处理后上调7.96倍;Serpin4(CL2600C2)在高剂量吡虫啉处理后上调7.18倍,低剂量吡虫啉处理后上调10.08倍。Defensin-1(U43570)在高剂量吡虫啉处理后上调5.17倍,而低剂量处理和对照不存在显著差异。Diptericin(U8926)在高低剂量吡虫啉处理后均下调,分别为对照的0.25倍和0.12倍。三、吡虫啉、粘质沙雷氏菌对东亚飞蝗血淋巴免疫通路基因的调控为了更好的了解吡虫啉对东亚飞蝗免疫影响,以及免疫系统的响应机制,本研究用低剂量吡虫啉和粘质沙雷氏菌(实验室分离的一株高致病菌)饲喂东亚飞蝗。采用荧光定量分析方法,分别检测了血淋巴中IMD信号通路和Toll信号通路的2个关键基因,IMD信号通路的关键基因为:PGRP-LE和Relish;Toll信号通路的关键基因为:GNBP3和MyD88。并分别检测了 IMD信号通路的产物之一:双翅肽(diptericin)和Toll信号通路的产物之一:防御素(defensin)。粘质沙雷氏菌处理后,IMD信号通路上的Relish上调了 9.50倍,PGRP-LE上调了 6.06倍,但是IMD信号通路的产物之一Diptericin却没有出现显著差异。粘质沙雷氏菌处理后,Toll信号通路的GNBP3上调了 4.94倍,但产物之一的Defensin-1显著下调。在低剂量吡虫啉处理后,东亚飞蝗血淋巴免疫防御的IMD信号通路的Relish和通路产物Diptericin分别上调了 6.77倍和7.88倍。但是,吡虫啉处理对Toll信号通路的GNBP3和MyD88均没有显著影响,而Toll信号通路产物Defensin-1显著下调。综合中肠和血淋巴两部分结果,不同剂量吡虫啉对同一免疫组织的诱导情况不同,暗示昆虫在不同剂量吡虫啉胁迫下响应机制不同。同浓度吡虫啉对不同组织的免疫诱导也有很大差别,暗示不同组织在接触杀虫剂后,采取了不同的应对策略,同一抗菌肽在不同组织中的作用可能有所差别。吡虫啉能够诱导东亚飞蝗血淋巴的IMD信号通路,而对Toll信号通路诱导效果不明显,暗示吡虫啉与真菌或革兰氏阳性菌复配使用能够起到更好的杀虫效果。本研究推测不同杀虫剂能够诱导昆虫特定的免疫通路,为杀虫真菌、革兰氏阳性菌、革兰氏阴性菌与农药的复配提供了新的思路和理论依据。
[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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S482.3
[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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S482.3
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