【摘要】：大豆蚜(Aphis glycines Matsumura)屬于半翅目(Hemiptera)蚜科(Ahpididae),以刺吸式口器為害大豆和野生大豆,并且可以傳播植物病毒,造成重大的經(jīng)濟損失,是重要的大豆害蟲。高效氯氟氰菊酯作為一種有效殺蟲劑,由于長期應用防治大豆蚜,已經(jīng)產(chǎn)生抗藥性。昆蟲抗藥性形成的機制包括表皮穿透力降低,解毒代謝作用增強和靶標敏感度降低,其中解毒代謝抗性和靶標敏感度降低是導致昆蟲產(chǎn)生抗藥性的主要機制。本研究以大豆蚜為研究對象,從生物化學、分子生物學和蛋白質(zhì)組學角度揭示大豆蚜對高效氯氟氰菊酯的抗性機制,對有效防治大豆蚜,開展抗藥性治理具有現(xiàn)實意義。在實驗室通過多代抗性篩選,建立遺傳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品系抗性倍數(shù)為43.42倍。建立了高效氯氟氰菊酯與其他殺蟲劑的交互抗性譜,為田間合理使用農(nóng)藥提供了實驗依據(jù)。在大豆蚜CRR品系和CSS品系中加入酶的相關(guān)抑制劑,研究增效劑對高效氯氟氰菊酯的增效作用。在建立穩(wěn)定抗藥性品系基礎(chǔ)上,通過對比分析兩個品系羧酸酯酶活力;應用q RT-PCR技術(shù),揭示了CRR品系羧酸酯酶m RNA轉(zhuǎn)錄水平的變化,結(jié)果表明羧酸酯酶過量表達與抗性產(chǎn)生相關(guān)。酯酶活性增高和細胞色素P450活性增強是昆蟲產(chǎn)生抗性的主要機制,通過對細胞色素P450表達量的分析,明確了其表達量的增加是大豆蚜對高效氯氟氰菊酯產(chǎn)生抗性的重要因素。鈉離子通道作為菊酯類殺蟲劑的作用靶標,通過對其基因IIS4-S6的克隆與序列分析,探討了鈉離子通道相關(guān)基因與大豆蚜產(chǎn)生抗性的關(guān)系。在此基礎(chǔ)上,應用差異蛋白質(zhì)組技術(shù),比較了大豆蚜高效氯氟氰菊酯CSS品系和CRR品系蛋白表達的差異,對抗藥性響應蛋白進行了分析。交互抗性毒力測定結(jié)果顯示,大豆蚜高效氯氟氰菊酯抗性品系對毒死蜱產(chǎn)生中等水平的交互抗性(11.66倍),與乙酰甲胺磷產(chǎn)生低水平交互抗性(8.20倍),與順式氰戊菊酯產(chǎn)生中等水平交互抗性(13.83倍),與氟氯氰菊酯產(chǎn)生中等水平交互抗性(9.64倍),與氯氰菊酯產(chǎn)生較高水平交互抗性(37.23倍),與聯(lián)苯菊酯產(chǎn)生低水平交互抗性(4.81倍),與滅多威產(chǎn)生中等水平交互抗性(9.32倍),與克百威產(chǎn)生中等水平交互抗性(14.60倍),與溴蟲氰、吡蟲啉、啶蟲脒、丁醚脲和阿維菌素無交互抗性。增效劑研究結(jié)果表明,在高效氯氟氰菊酯中加入TPP、DEF、PBO增效劑,大豆蚜CRR品系增效系數(shù)分別達到了5.85、23.00和40.59;對于CSS品系增效系數(shù)為0.26、0.35和3.00,結(jié)果顯示增效劑對抗性品系作用明顯,說明大豆蚜對高效氯氟氰菊酯產(chǎn)生抗性與酯酶相關(guān)。酯酶動力學測定結(jié)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品系間羧酸酯酶比活力存在極顯著差異(p0.01)。利用q RT-PCR技術(shù)對大豆蚜羧酸酯酶表達量進行了分析,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轉(zhuǎn)錄水平差異顯著。除此之外,對大豆蚜細胞色素P450氧化酶家族基因進行了測定,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基因表達量顯著增加。鈉離子通道基因IIS4、IIS5、IIS6的克隆和測序發(fā)現(xiàn),核苷酸序列中包含了kdr和super-kdr位點,如果大豆蚜抗性品系鈉離子通道基因序列相對應的位點發(fā)生了突變,說明這兩個位點跟大豆蚜對高效氯氟氰菊酯的kdr和super-kdr相關(guān),為研究鈉離子通道與大豆蚜對高效氯氟氰菊酯抗性機制奠定了一定的理論基礎(chǔ)。利用雙向電泳(2-DE)技術(shù),對大豆蚜CSS品系和CRR品系蛋白質(zhì)差異表達情況進行了研究,2-DE圖譜分析結(jié)果表明,共檢測到36個蛋白豐度差異表達變化在2倍以上的蛋白點,有24個蛋白得到了有效鑒定,包括微管結(jié)合蛋白、肌動蛋白、表皮蛋白、果糖1,6-二磷酸醛縮酶、烯醇酶、熱激蛋白等,部分抗藥性響應蛋白在大豆蚜抗高效氯氟氰菊酯中發(fā)揮重要的作用。
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S435.651

【相似文獻】

相關(guān)期刊論文 前10條

1 吳炳芝,孫毅民,張傳文;吡蟲啉防治大豆蚜蟲試驗初報[J];黑龍江農(nóng)業(yè)科學;2001年03期

2 苗進,吳孔明,李國勛;大豆蚜的研究進展[J];大豆科學;2005年02期

3 劉振勇,李唯實;大豆蚜蟲發(fā)生原因及防治措施[J];作物雜志;2005年02期

4 ;黑龍江大豆蚜蟲將中等偏重發(fā)生[J];農(nóng)藥市場信息;2006年16期

5 劉健;趙奎軍;;大豆蚜的生物學防治技術(shù)[J];昆蟲知識;2007年02期

6 李春紅;解春霞;;淺談大豆蚜防治技術(shù)[J];化工之友;2007年17期

7 袁國慶;;大豆蚜發(fā)生規(guī)律及防治技術(shù)[J];農(nóng)業(yè)科技通訊;2008年02期

8 孫艷華;唐成霞;許麗艷;;大豆蚜蟲的發(fā)生與防治[J];種子世界;2008年06期

9 劉興龍;李新民;劉春來;王克勤;王爽;劉宇;;大豆蚜研究進展[J];中國農(nóng)學通報;2009年14期

10 李長鎖;于涵;馬躍;;大豆蚜發(fā)生規(guī)律及防治措施[J];現(xiàn)代化農(nóng)業(yè);2009年10期

相關(guān)會議論文 前3條

1 孫雅杰;高月波;;大豆蚜田間種群消長與蚜害防治[A];當代昆蟲學研究——中國昆蟲學會成立60周年紀念大會暨學術(shù)討論會論文集[C];2004年

2 郭文英;喬格俠;任炳忠;;大豆蚜線粒體基因組序列測定與分析[A];北京昆蟲學會通訊（第23期）[C];2011年

3 宋淑云;晉齊鳴;楊敏芝;張偉;李紅;沙洪林;;白僵菌對大豆蚜的寄生性研究[A];農(nóng)業(yè)生物災害預防與控制研究[C];2005年

相關(guān)重要報紙文章 前6條

1 商丘市農(nóng)業(yè)局 謝幸華;大豆蚜[N];河南科技報;2005年

2 安徽省植�？傉�;安徽局部地區(qū)大豆蚜蟲數(shù)量偏多[N];農(nóng)資導報;2006年

3 劉忠林 記者 孟寶林;科學防治病蟲害 大豆水稻是重點[N];牡丹江日報;2007年

4 王春雨 高增雙;三江平原罕見旱情該引發(fā)何樣思考？[N];中國社會報;2007年

5 徐仁吉;夏季農(nóng)田要注意防治病蟲害[N];四平日報;2009年

6 市植檢植保站 劉振勇;今年我市農(nóng)作物主要生物災害發(fā)生趨勢分析[N];黑河日報;2010年

相關(guān)博士學位論文 前4條

1 王玲;大豆蚜氣味結(jié)合蛋白的結(jié)合特性及組織定位[D];東北農(nóng)業(yè)大學;2014年

2 畢銳;大豆蚜抗高效氯氟氰菊酯的分子機制及差異蛋白質(zhì)組學分析[D];吉林大學;2016年

3 楊帥;大豆蚜對吡蟲啉的抗性監(jiān)測及抗性機理研究[D];東北農(nóng)業(yè)大學;2012年

4 張瑩;大豆蚜的飛行生物學及對寄生蜂的傳播潛力[D];中國農(nóng)業(yè)科學院;2009年

相關(guān)碩士學位論文 前10條

1 張拓;大豆蚜熱休克蛋白70基因的克隆、原核表達與定量分析[D];東北農(nóng)業(yè)大學;2013年

2 劉興龍;黑龍江大豆蚜對大豆危害及產(chǎn)量損失的研究[D];中國農(nóng)業(yè)科學院;2013年

3 李冉;基于線粒體基因的不同地理種群大豆蚜遺傳分化研究[D];東北農(nóng)業(yè)大學;2016年

4 李長鎖;哈爾濱地區(qū)大豆蚜越冬和遷飛擴散習性的研究[D];東北農(nóng)業(yè)大學;2008年

5 鞠靜;利用熒光定量PCR技術(shù)分析捕食性天敵對大豆蚜的控害作用[D];東北農(nóng)業(yè)大學;2010年

6 戴長春;大豆蚜（Aphis glycines Matsumura）種群動態(tài)及天敵控制作用研究[D];東北農(nóng)業(yè)大學;2005年

7 張俊杰;大豆抗蚜資源篩選及大豆蚜生物型鑒定初探[D];上海交通大學;2013年

8 陳曉慧;大豆蚜對溫度和寄主植物的適應性研究[D];東北農(nóng)業(yè)大學;2015年

9 楊帥;大豆蚜（Aphis glycines Matsumura）不同地理種群生態(tài)適應性研究[D];東北農(nóng)業(yè)大學;2009年

10 張樺;抗高效氯氟氰菊酯大豆蚜羧酸酯酶生化及分子機制研究[D];吉林大學;2013年

，

本文編號：2233544