朱砂叶螨对丁氟螨酯抗性机制研究
发布时间：2018-12-14 07:29
【摘要】：朱砂叶螨(Tetranychus cinnabarinus)是一种能够危害多种作物的世界性害螨,由于杀螨剂大量持续不科学使用以及该螨繁殖力强、时代周期短、活动范围小等生物学特点,其抗药性问题尤为严重。丁氟螨酯(Cyflumetofen)是日本开发的苯酰乙腈类新型杀螨剂,该药杀螨活性高、持效性好、对非靶标生物安全,2013年在我国登记销售。本研究在室内筛选出朱砂叶螨对丁氟螨酯抗性品系(CyR)和敏感品系(CyS)的基础上,着重研究了朱砂叶螨对丁氟螨酯产生抗性的生理生化及分子机制,主要研究内容和结果如下：1.CyR品系经过室内连续64代的筛选,LCso值由2.19mg/L上升到42.99mg/L,相对于CyS品系抗性倍数(Rf)达到54.42倍,而相对于室内相对敏感品系(SS)抗性倍数为19.63倍。2.三种增效剂PBO、DEM和DEF分别单独与药剂混用及共同与药剂混用对丁氟螨酯抗性品系第43代(CyR43)的增效比分别为2.15、3.77、1.76和3.62,以DEM增效作用最大,其次是PBO。酶活性测定表明CyR43品系的多功能氧化酶(MFOs)、谷胱甘肽-S-转移酶(GSTs)和羧酸酯酶(CarEs)三种解毒酶的比活力分别是CyS品系的1.85、7.20和1.95倍,差异均达显著水平,说明三种解毒酶均不同程度参与朱砂叶螨对丁氟螨酯抗药性。DEM增效作用最显著及GSTs活性升高倍数最大一致表明GSTs是朱砂叶螨对丁氟螨酯产生代谢抗性最主要的解毒酶。3.丁氟螨酯的作用靶标是线粒体电子传递链复合物Ⅱ(SQR),对CyR49(Rf=31.19)、CyR54 (Rf=43.38)和CYR64(Rf=54.42)的SQR活性测定表明：CyR品系较CyS品系的SQR活性降低,且随着抗性倍数升高,CyR49、CyR54和CyR64的SQR活性占CyS的SQR活性百分比(CyR/CyS)逐渐降低,分别为89%、81%和58%。酶活性离体抑制实验结果表明丁氟螨酯对CyS及CyR64的抑制中浓度(IC50)分别为23.141±0.173nmol/L和63.207±1.900nmol/L,的不敏感性指数(R IC50/S IC50)是CyS的2.73倍。说明CyR品系SQR活性及敏感性相较CyS品系降低可能是朱砂叶螨对丁氟螨酯产生抗性的原因。4.利用RT-PCR及PCR技术,克隆获得5条朱砂叶螨SQR基因,分别为SDHA, SDHB, SDHC, SDHD和SDH5 (Genbank登录号依次为：KP686429, KP686430, KP686431, KP686432和KP686433),序列分析表明CyR品系的5条SQR基因均未发生氨基酸水平的突变。采用荧光实时定量PCR (qPCR)检测5条SQR基因表达情况,结果显示5条基因均是在成螨期的表达量高于其他螨态,并且CyR品系中的表达量低于CyS品系,其中SDHA、SDHB及SDH5这3条基因的下调均具有显著差异。以上研究表明可能在目前抗性水平下尚未形成氨基酸突变介导的靶标抗性,SQR活性及敏感性降低的分子机制是SQR三条关键基因表达下调,猜测可能存在某些亚基基因表达下调造成SQR这类复合物蛋白分子构型变异而引起靶标敏感性降低。5.通过HPLC测定朱砂叶螨CyR和CyS品系的ATP含量,结果显示CyR64品系的ATP含量显著低于CyS品系,这可能是由于CyR品系SQR活性及基因表达量均显著低于CyS品系,所以导致CyR品系的ATP生成量低于CyS品系。用10mg/L丁氟螨酯分别处理CyS和CyR64品系4h后,发现CyS品系ATP含量显著降低,而CyR64品系ATP含量并无显著变化,这一方面可能由于CyR品系的代谢解毒酶活性高于CyS品系,因此CyR对丁氟螨酯的解毒代谢能力更强,使得CyR对丁氟螨酯的耐受性更高；二是CyR品系SQR对丁氟螨酯敏感性降低,从而增加了耐受性。
[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.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：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.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433.7
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