大豆蚜抗高效氯氟氰菊酯的分子机制及差异蛋白质组学分析

发布时间：2018-09-10 05:52

【摘要】：大豆蚜(Aphis glycines Matsumura)属于半翅目(Hemiptera)蚜科(Ahpididae),以刺吸式口器为害大豆和野生大豆,并且可以传播植物病毒,造成重大的经济损失,是重要的大豆害虫。高效氯氟氰菊酯作为一种有效杀虫剂,由于长期应用防治大豆蚜,已经产生抗药性。昆虫抗药性形成的机制包括表皮穿透力降低,解毒代谢作用增强和靶标敏感度降低,其中解毒代谢抗性和靶标敏感度降低是导致昆虫产生抗药性的主要机制。本研究以大豆蚜为研究对象,从生物化学、分子生物学和蛋白质组学角度揭示大豆蚜对高效氯氟氰菊酯的抗性机制,对有效防治大豆蚜,开展抗药性治理具有现实意义。在实验室通过多代抗性筛选,建立遗传背景相同的大豆蚜高效氯氟氰菊酯敏感品系(CSS)和抗性品系(CRR),CRR品系抗性倍数为43.42倍。建立了高效氯氟氰菊酯与其他杀虫剂的交互抗性谱,为田间合理使用农药提供了实验依据。在大豆蚜CRR品系和CSS品系中加入酶的相关抑制剂,研究增效剂对高效氯氟氰菊酯的增效作用。在建立稳定抗药性品系基础上,通过对比分析两个品系羧酸酯酶活力;应用q RT-PCR技术,揭示了CRR品系羧酸酯酶m RNA转录水平的变化,结果表明羧酸酯酶过量表达与抗性产生相关。酯酶活性增高和细胞色素P450活性增强是昆虫产生抗性的主要机制,通过对细胞色素P450表达量的分析,明确了其表达量的增加是大豆蚜对高效氯氟氰菊酯产生抗性的重要因素。钠离子通道作为菊酯类杀虫剂的作用靶标,通过对其基因IIS4-S6的克隆与序列分析,探讨了钠离子通道相关基因与大豆蚜产生抗性的关系。在此基础上,应用差异蛋白质组技术,比较了大豆蚜高效氯氟氰菊酯CSS品系和CRR品系蛋白表达的差异,对抗药性响应蛋白进行了分析。交互抗性毒力测定结果显示,大豆蚜高效氯氟氰菊酯抗性品系对毒死蜱产生中等水平的交互抗性(11.66倍),与乙酰甲胺磷产生低水平交互抗性(8.20倍),与顺式氰戊菊酯产生中等水平交互抗性(13.83倍),与氟氯氰菊酯产生中等水平交互抗性(9.64倍),与氯氰菊酯产生较高水平交互抗性(37.23倍),与联苯菊酯产生低水平交互抗性(4.81倍),与灭多威产生中等水平交互抗性(9.32倍),与克百威产生中等水平交互抗性(14.60倍),与溴虫氰、吡虫啉、啶虫脒、丁醚脲和阿维菌素无交互抗性。增效剂研究结果表明,在高效氯氟氰菊酯中加入TPP、DEF、PBO增效剂,大豆蚜CRR品系增效系数分别达到了5.85、23.00和40.59;对于CSS品系增效系数为0.26、0.35和3.00,结果显示增效剂对抗性品系作用明显,说明大豆蚜对高效氯氟氰菊酯产生抗性与酯酶相关。酯酶动力学测定结果表明,抗性品系酶活比率是敏感品系的1.405倍,CSS品系和CRR品系间羧酸酯酶比活力存在极显著差异(p0.01)。利用q RT-PCR技术对大豆蚜羧酸酯酶表达量进行了分析,CRR品系是CSS品系的5.87倍,CRR品系和CSS品系羧酸酯酶基因m RNA转录水平差异显著。除此之外,对大豆蚜细胞色素P450氧化酶家族基因进行了测定,在大豆蚜高效氯氟氰菊酯抗性品系中CYP6A13-like,CYP6A2-like,CYP6A14-like和Cytochrome b-c1基因表达量显著增加。钠离子通道基因IIS4、IIS5、IIS6的克隆和测序发现,核苷酸序列中包含了kdr和super-kdr位点,如果大豆蚜抗性品系钠离子通道基因序列相对应的位点发生了突变,说明这两个位点跟大豆蚜对高效氯氟氰菊酯的kdr和super-kdr相关,为研究钠离子通道与大豆蚜对高效氯氟氰菊酯抗性机制奠定了一定的理论基础。利用双向电泳(2-DE)技术,对大豆蚜CSS品系和CRR品系蛋白质差异表达情况进行了研究,2-DE图谱分析结果表明,共检测到36个蛋白丰度差异表达变化在2倍以上的蛋白点,有24个蛋白得到了有效鉴定,包括微管结合蛋白、肌动蛋白、表皮蛋白、果糖1,6-二磷酸醛缩酶、烯醇酶、热激蛋白等,部分抗药性响应蛋白在大豆蚜抗高效氯氟氰菊酯中发挥重要的作用。
[Abstract]:Soybean aphids (Aphis glycines Matsumura) belong to the family Hemiptera aphidae, which infects soybeans and wild soybeans with pricking mouthparts and can transmit plant viruses, causing significant economic losses. As an effective insecticide, cyhalothrin has been used for a long time to control soybean aphids. The mechanisms of insect resistance include decreased penetration of the epidermis, increased detoxification and metabolism, and decreased target sensitivity. The main mechanisms leading to insect resistance are decreased detoxification and metabolism resistance and target sensitivity. To reveal the resistance mechanism of soybean aphids to high-efficiency cyhalothrin is of practical significance for effective control of soybean aphids and drug resistance control. A high-efficiency cyhalothrin-sensitive strain (CSS) and a resistant strain (CRR) of soybean aphids with the same genetic background were established in the laboratory through multi-generation resistance screening. The cross-resistance spectrum of high-efficiency cyhalothrin and other insecticides was established to provide experimental basis for rational use of pesticides in the field. The synergistic effect of synergists on high-efficiency cyhalothrin was studied by adding enzyme-related inhibitors to the CRR and CSS strains of soybean aphid. Carboxylesterase activity of CRR strain was detected by Q RT-PCR. The results showed that overexpression of carboxylesterase was related to resistance. Increased esterase activity and increased cytochrome P450 activity were the main mechanisms of insect resistance. The expression of cytochrome P450 was analyzed. The increase of its expression level is an important factor for the resistance of soybean aphids to high-efficiency cyhalothrin. Sodium channel is the target of pyrethroid insecticides. Through cloning and sequence analysis of its gene IIS4-S6, the relationship between the genes related to sodium channel and the resistance of soybean aphids to cyhalothrin was discussed. The difference of protein expression between high-performance cyhalothrin resistant strains CSS and CRR of soybean aphids was compared with that of soybean aphids. The results of cross-resistance toxicity test showed that high-performance cyhalothrin resistant strains of soybean aphids had moderate cross-resistance to chlorpyrifos (11.66 times) and low cross-resistance to acetamidophos (11.66 times). Horizontal cross-resistance (8.20 times), moderate cross-resistance (13.83 times) to cis-fenvalerate, moderate cross-resistance (9.64 times) to cyhalothrin, high cross-resistance (37.23 times) to cypermethrin, low cross-resistance (4.81 times) to bifenthrin, and moderate cross-resistance (9.64 times) to methomyl. The results of synergist study showed that the synergistic coefficients of CRR strain of soybean aphid reached 5.85, 23.00 and 40.59 respectively when TPP, DEF and PBO synergists were added to high-efficiency cyhalothrin. The results showed that the synergist had significant effect on the resistance of soybean aphid to cyhalothrin, indicating that the resistance of soybean aphid to cyhalothrin was related to esterase. The esterase kinetics analysis showed that the ratio of esterase activity of resistant strain was 1.405 times higher than that of sensitive strain, and the specific activity of carboxylesterase was significantly different between CSS strain and CR strain (p0.05). The expression of carboxylesterase in soybean aphid was analyzed by Q RT-PCR. CRR strain was 5.87 times higher than CSS strain. The transcription level of carboxylesterase gene m RNA was significantly different between CRR strain and CSS strain. The expression levels of CYP6A13-like, CYP6A2-like, CYP6A14-like and CYtochrome b-c1 genes were significantly increased. Cloning and sequencing of sodium channel genes IIS4, IIS5 and IIS6 showed that the nucleotide sequence contained KDR and super-kdr loci. If the corresponding sites of the sodium channel gene sequence of soybean aphid resistant strain were mutated, the two loci could be explained. The site was correlated with KDR and super-kdr of high-efficiency Cyhalothrin in soybean aphids, which laid a theoretical foundation for studying the mechanism of resistance of sodium channel to high-efficiency Cyhalothrin in soybean aphids. The results showed that 36 protein abundances were more than 2-fold differentially expressed, and 24 proteins were identified effectively, including tubule-binding protein, actin, epidermal protein, fructose-1,6-diphosphate aldolase, enolase, heat shock protein, etc. Some resistance-responsive proteins played an important role in the resistance of soybean aphid to beta-cyhalothrin. The purpose.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.651

【相似文献】