朱砂葉螨對丁氟螨酯抗性機制研究
發(fā)布時間：2018-12-14 07:29
【摘要】：朱砂葉螨(Tetranychus cinnabarinus)是一種能夠危害多種作物的世界性害螨,由于殺螨劑大量持續(xù)不科學(xué)使用以及該螨繁殖力強、時代周期短、活動范圍小等生物學(xué)特點,其抗藥性問題尤為嚴重。丁氟螨酯(Cyflumetofen)是日本開發(fā)的苯酰乙腈類新型殺螨劑,該藥殺螨活性高、持效性好、對非靶標生物安全,2013年在我國登記銷售。本研究在室內(nèi)篩選出朱砂葉螨對丁氟螨酯抗性品系(CyR)和敏感品系(CyS)的基礎(chǔ)上,著重研究了朱砂葉螨對丁氟螨酯產(chǎn)生抗性的生理生化及分子機制,主要研究內(nèi)容和結(jié)果如下：1.CyR品系經(jīng)過室內(nèi)連續(xù)64代的篩選,LCso值由2.19mg/L上升到42.99mg/L,相對于CyS品系抗性倍數(shù)(Rf)達到54.42倍,而相對于室內(nèi)相對敏感品系(SS)抗性倍數(shù)為19.63倍。2.三種增效劑PBO、DEM和DEF分別單獨與藥劑混用及共同與藥劑混用對丁氟螨酯抗性品系第43代(CyR43)的增效比分別為2.15、3.77、1.76和3.62,以DEM增效作用最大,其次是PBO。酶活性測定表明CyR43品系的多功能氧化酶(MFOs)、谷胱甘肽-S-轉(zhuǎn)移酶(GSTs)和羧酸酯酶(CarEs)三種解毒酶的比活力分別是CyS品系的1.85、7.20和1.95倍,差異均達顯著水平,說明三種解毒酶均不同程度參與朱砂葉螨對丁氟螨酯抗藥性。DEM增效作用最顯著及GSTs活性升高倍數(shù)最大一致表明GSTs是朱砂葉螨對丁氟螨酯產(chǎn)生代謝抗性最主要的解毒酶。3.丁氟螨酯的作用靶標是線粒體電子傳遞鏈復(fù)合物Ⅱ(SQR),對CyR49(Rf=31.19)、CyR54 (Rf=43.38)和CYR64(Rf=54.42)的SQR活性測定表明：CyR品系較CyS品系的SQR活性降低,且隨著抗性倍數(shù)升高,CyR49、CyR54和CyR64的SQR活性占CyS的SQR活性百分比(CyR/CyS)逐漸降低,分別為89%、81%和58%。酶活性離體抑制實驗結(jié)果表明丁氟螨酯對CyS及CyR64的抑制中濃度(IC50)分別為23.141±0.173nmol/L和63.207±1.900nmol/L,的不敏感性指數(shù)(R IC50/S IC50)是CyS的2.73倍。說明CyR品系SQR活性及敏感性相較CyS品系降低可能是朱砂葉螨對丁氟螨酯產(chǎn)生抗性的原因。4.利用RT-PCR及PCR技術(shù),克隆獲得5條朱砂葉螨SQR基因,分別為SDHA, SDHB, SDHC, SDHD和SDH5 (Genbank登錄號依次為：KP686429, KP686430, KP686431, KP686432和KP686433),序列分析表明CyR品系的5條SQR基因均未發(fā)生氨基酸水平的突變。采用熒光實時定量PCR (qPCR)檢測5條SQR基因表達情況,結(jié)果顯示5條基因均是在成螨期的表達量高于其他螨態(tài),并且CyR品系中的表達量低于CyS品系,其中SDHA、SDHB及SDH5這3條基因的下調(diào)均具有顯著差異。以上研究表明可能在目前抗性水平下尚未形成氨基酸突變介導(dǎo)的靶標抗性,SQR活性及敏感性降低的分子機制是SQR三條關(guān)鍵基因表達下調(diào),猜測可能存在某些亞基基因表達下調(diào)造成SQR這類復(fù)合物蛋白分子構(gòu)型變異而引起靶標敏感性降低。5.通過HPLC測定朱砂葉螨CyR和CyS品系的ATP含量,結(jié)果顯示CyR64品系的ATP含量顯著低于CyS品系,這可能是由于CyR品系SQR活性及基因表達量均顯著低于CyS品系,所以導(dǎo)致CyR品系的ATP生成量低于CyS品系。用10mg/L丁氟螨酯分別處理CyS和CyR64品系4h后,發(fā)現(xiàn)CyS品系A(chǔ)TP含量顯著降低,而CyR64品系A(chǔ)TP含量并無顯著變化,這一方面可能由于CyR品系的代謝解毒酶活性高于CyS品系,因此CyR對丁氟螨酯的解毒代謝能力更強,使得CyR對丁氟螨酯的耐受性更高；二是CyR品系SQR對丁氟螨酯敏感性降低,從而增加了耐受性。
[Abstract]:Tetranychus cinnabarinus is a worldwide pest that can harm a variety of crops. Cyflumeetofen is a new type of anti-killing agent developed by Japan, which is high in killing activity, good in efficacy, and safe for non-target organisms, and is registered and sold in China in 2013. On the basis of screening of the resistance strain (CyR) and the sensitive strain (CyS) of Cinnabaris, the physiological and biochemical and molecular mechanism of the resistance of the cinnabar to the difluoro-methyl ester was studied. The main contents and results were as follows: 1. The Lso value of the CyR strain was increased from 2.19mg/ L to 42.99mg/ L, and the resistance multiple (Rf) of the CyS line was 54. 42 times, while the resistance to the relative sensitive line (SS) in the room was 19.63 times. The synergistic ratio of three potentiators, PBO, DEM and DEF, to the third generation (CyR43) of the D-fluorophonate-resistant strain, respectively, was 2.15, 3.77, 1.76 and 3.62, respectively, with the best effect of DEM, followed by PBO. The activity of the enzyme showed that the specific activity of the multifunctional oxidase (MFOs), glutathione S-transferase (GSTs) and acid esterase (CarEs) of the CyR43 strain was 1.85, 7.20 and 1.95 times, respectively, and the difference was significant. It is indicated that the three detoxification enzymes are involved in the drug resistance of the cinnabar leaf and the cinnabar. The most significant and consistent increase of GSTs activity shows that GSTs is the most important detoxification enzyme for the metabolic resistance of cinnabar. The SQR activity of CyR49 (Rf = 31. 19), CyR54 (Rf = 43. 38) and CYR64 (Rf = 54. 42) showed that the SQR activity of CyR49 (Rf = 31. 19), CyR54 (Rf = 43. 38) and CYR64 (Rf = 54. 42) showed that the SQR activity of CyR strain was lower than that of CyS strain, and with the increase of resistance multiple, CyR49, The SQR activity of CyR54 and CyR64 (CyR/ CyS) decreased gradually, 89%, 81% and 58%, respectively. The results of the inhibition of the activity of the enzyme showed that the concentration of D _ (50) in the inhibition of CyS and CyR64 (IC50) was 23.141-0.173nmol/ L and 66.3-207-1.900nmol/ L, and the non-sensitivity index (R-50/ S-50) of CyS was 2.73-fold higher than that of CyS. The reason that the CyS strain of the CyR strain and the sensitive phase of CyR strain may be lower than that of CyS strain may be the reason for the resistance of Cinnabaris to D. The SQR gene of 5 Cinnabaris were obtained by RT-PCR and PCR, and SDHA, SDHB, SDHC, SDHD and SDH5 (Genbank accession number were KP686429, KP686430, KP686431, KP686432 and KP686433). The expression of five SQR genes was detected by fluorescence real-time quantitative PCR (qPCR). The results showed that the expression of five genes was higher than that of the other five genes, and the expression of the CyR strain was lower than that of the CyS strain, among which the down-regulation of the three genes of SDHA, SDHB and SDH5 was significantly different. The above studies have shown that the molecular mechanism that may not form an amino acid mutation-mediated target resistance at the present resistance level, the SQR activity and the sensitivity reduction is down-regulated by three key gene expression of SQR, It is speculated that there may be a decrease in the sensitivity of the target due to the variation in the molecular configuration of the SQR complex protein due to the down-regulation of some subunit genes. The ATP content of CyR and CyS lines was determined by HPLC. The results showed that the ATP content of CyR 64 strain was significantly lower than that of CyS strain, which could result in lower ATP production of CyR strain than that of CyS strain. After 4 h of CyS and CyR64 strain, the ATP content of CyS strain was significantly reduced and the ATP content of CyR64 strain was not changed significantly. and the sensitivity of the CyR strain SQR to the butyrofluorophenyl ester is reduced, and the tolerance is increased.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S433.7
[Abstract]:Tetranychus cinnabarinus is a worldwide pest that can harm a variety of crops. Cyflumeetofen is a new type of anti-killing agent developed by Japan, which is high in killing activity, good in efficacy, and safe for non-target organisms, and is registered and sold in China in 2013. On the basis of screening of the resistance strain (CyR) and the sensitive strain (CyS) of Cinnabaris, the physiological and biochemical and molecular mechanism of the resistance of the cinnabar to the difluoro-methyl ester was studied. The main contents and results were as follows: 1. The Lso value of the CyR strain was increased from 2.19mg/ L to 42.99mg/ L, and the resistance multiple (Rf) of the CyS line was 54. 42 times, while the resistance to the relative sensitive line (SS) in the room was 19.63 times. The synergistic ratio of three potentiators, PBO, DEM and DEF, to the third generation (CyR43) of the D-fluorophonate-resistant strain, respectively, was 2.15, 3.77, 1.76 and 3.62, respectively, with the best effect of DEM, followed by PBO. The activity of the enzyme showed that the specific activity of the multifunctional oxidase (MFOs), glutathione S-transferase (GSTs) and acid esterase (CarEs) of the CyR43 strain was 1.85, 7.20 and 1.95 times, respectively, and the difference was significant. It is indicated that the three detoxification enzymes are involved in the drug resistance of the cinnabar leaf and the cinnabar. The most significant and consistent increase of GSTs activity shows that GSTs is the most important detoxification enzyme for the metabolic resistance of cinnabar. The SQR activity of CyR49 (Rf = 31. 19), CyR54 (Rf = 43. 38) and CYR64 (Rf = 54. 42) showed that the SQR activity of CyR49 (Rf = 31. 19), CyR54 (Rf = 43. 38) and CYR64 (Rf = 54. 42) showed that the SQR activity of CyR strain was lower than that of CyS strain, and with the increase of resistance multiple, CyR49, The SQR activity of CyR54 and CyR64 (CyR/ CyS) decreased gradually, 89%, 81% and 58%, respectively. The results of the inhibition of the activity of the enzyme showed that the concentration of D _ (50) in the inhibition of CyS and CyR64 (IC50) was 23.141-0.173nmol/ L and 66.3-207-1.900nmol/ L, and the non-sensitivity index (R-50/ S-50) of CyS was 2.73-fold higher than that of CyS. The reason that the CyS strain of the CyR strain and the sensitive phase of CyR strain may be lower than that of CyS strain may be the reason for the resistance of Cinnabaris to D. The SQR gene of 5 Cinnabaris were obtained by RT-PCR and PCR, and SDHA, SDHB, SDHC, SDHD and SDH5 (Genbank accession number were KP686429, KP686430, KP686431, KP686432 and KP686433). The expression of five SQR genes was detected by fluorescence real-time quantitative PCR (qPCR). The results showed that the expression of five genes was higher than that of the other five genes, and the expression of the CyR strain was lower than that of the CyS strain, among which the down-regulation of the three genes of SDHA, SDHB and SDH5 was significantly different. The above studies have shown that the molecular mechanism that may not form an amino acid mutation-mediated target resistance at the present resistance level, the SQR activity and the sensitivity reduction is down-regulated by three key gene expression of SQR, It is speculated that there may be a decrease in the sensitivity of the target due to the variation in the molecular configuration of the SQR complex protein due to the down-regulation of some subunit genes. The ATP content of CyR and CyS lines was determined by HPLC. The results showed that the ATP content of CyR 64 strain was significantly lower than that of CyS strain, which could result in lower ATP production of CyR strain than that of CyS strain. After 4 h of CyS and CyR64 strain, the ATP content of CyS strain was significantly reduced and the ATP content of CyR64 strain was not changed significantly. and the sensitivity of the CyR strain SQR to the butyrofluorophenyl ester is reduced, and the tolerance is increased.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S433.7
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