朱砂叶螨P-糖蛋白与阿维菌素抗性关系研究

发布时间：2018-03-28 18:46

  本文选题：阿维菌素　切入点：朱砂叶螨　出处：《西南大学》2015年硕士论文

【摘要】：朱砂叶螨(Tetranychus cinnabarinus)是一种世界性害螨,因其世代周期短、繁殖能力强、受药机会多、近亲交配率高等特点而极易产生抗药性。阿维菌素是一类具有杀虫、杀螨和杀线虫活性的大环内酯化合物,随着使用频率的不断增加,已有不少害虫对其产生了抗药性。P-糖蛋白(P-glycoprotein, Pgp)是ABC(ATP-binding cassette)转运蛋白超家族成员之一,主要功能是利用其与ATP的结合和水解供能进行底物的跨膜转运,保护组织免受内源性代谢物和外源性有毒物质的伤害。近年来已有研究表明许多寄生虫和昆虫对阿维菌素产生抗药性与PgP有关。PgP引起的抗性也称MDR (multidrug resistance)。本论文重点研究朱砂叶螨对阿维菌素的抗药性与P-糖蛋白的相关关系,获得了以下主要研究结果：1.室内抗性筛选获得了朱砂叶螨阿维菌素抗性品系(AbR,抗性倍数超过20倍),经PgP特异性抑制剂维拉帕米处理12 h后,阿维菌素对朱砂叶螨敏感品系(SS)和AbR的毒力分别增大了19.91%和74.51%,初步说明PgP在阿维菌素抗性品系中作用更明显,与阿维菌素抗性形成有关。2.朱砂叶螨P-糖蛋白ATP酶动力学测定结果为：Km分别为1.835 ± 0.142 mM(SS)和1.595 ± 0.353 mM (AbR), Vmax分别为0.097 ± 0.036μmol/mg/min (SS)和0.108 ± 0.054μmol/mg/min (AbR); Km和Vmax在敏感和抗性品系间无显著差异。3.对朱砂叶螨P-糖蛋白ATP酶进行活性测定,结果发现该酶在AbR的活性显著高于SS；用低剂量阿维菌素(LC30)分别对朱砂叶螨SS及AbR品系的雌成螨进行诱导后,ATP酶活性在SS中没有显著变化,但在AbR中表现为活性在诱导后显著升高。4.克隆获得两条朱砂叶螨P-糖蛋白基因,分别命名为TcPgpl和TcPgp2。其中TcPgp1 cDNA序列全长3885 bp,编码1295个氨基酸；TcPgp2 cDNA序列全长3879bp,编码1293个氨基酸。两条基因皆具有Walker-A、C-motif、Walker-B、D-loop和H-loop等特征序列。5.序列分析表明SS和AbR品系中TcPgp1和TcPgp2基因不存在氨基酸水平差异。RT-qPCR分析结果表明TcPgp1在各螨态的表达量差异不显著,而TcPgp2在卵和幼螨的表达量显著高于若螨和成螨; TcPgp1和TcPgp2的表达量在敏感和抗性品系之间均没有显著性差异。药剂诱导实验表明,SS品系TcPgp1和TcPgp2的表达量均没有显著变化,但AbR品系TcPgp1和TcPgp2的表达量在诱导后均显著
[Abstract]:Tetranychus cinnabarinus (Tetranychus cinnabarinus) is a worldwide pest. Acaricidal and nematicidal macrolides, with the increasing frequency of use, many pests have developed resistance to them. P-glycoprotein (Pgp) is a member of the ABC(ATP-binding cassette-based transporter superfamily. Its main function is to utilize its binding with ATP and hydrolytic energy supply to carry out the transmembrane transport of the substrate. To protect tissues from endogenous metabolites and exogenous toxic substances. Recent studies have shown that resistance of many parasites and insects to avermectin is associated with resistance to PgP. PGP is also known as MDR multidrug resistance. To study the relationship between the resistance of Tetranychus cinnabarinus to abamectin and P- glycoprotein, The main results of this study were as follows: 1. The resistant strain of abamectin of Tetranychus cinnabinus was selected in laboratory, and the resistance ratio was more than 20 times. The strain was treated with verapamil, a specific inhibitor of PgP, for 12 h. The virulence of avermectin to AbR and AbR increased by 19.91% and 74.51%, respectively, which indicated that PgP was more effective in avermectin resistant strains. The results of kinetic analysis of P- glycoprotein ATP enzyme of Tetranychus cinnabinae were: 1.835 卤0.142 mm ATP and 1.595 卤0.353 mm ATP, Vmax 0.097 卤0.036 渭 mol/mg/min and 0.108 卤0.054 渭 mol/mg/min, respectively, but km and Vmax were not found in sensitive and resistant lines. The activity of P-glycoprotein ATP enzyme in Tetranychus cinnabarinus was determined. The results showed that the activity of this enzyme in AbR was significantly higher than that in SS.After induced by low dose avermectin (Avermectin), SS and female adult mites of AbR strain had no significant change in SS. But in AbR, the activity increased significantly after induction. 4. Two P- glycoprotein genes of Tetranychus cinnabarinus were cloned. They were named TcPgpl and TcPgp2 respectively. The TcPgp1 cDNA sequence was 3885 BP in length, encoding 1295 amino acids TcPgp2 cDNA sequence in 3879 BP, encoding 1293 amino acids. Both of the two genes had the characteristic sequences of Walker-Agna C-motifen Walker-BAND-loop and H-loop. The sequence analysis showed that TcPgp1 and H-loop in SS and AbR strains. There was no amino acid level difference in TcPgp2 gene. RT-qPCR analysis showed that there was no significant difference in the expression of TcPgp1 among mites. However, the expression of TcPgp2 in eggs and juvenile mites was significantly higher than that of mites and adults, and there was no significant difference in the expression of TcPgp1 and TcPgp2 between sensitive and resistant lines. The results of medicament induction showed that there was no significant change in the expression of TcPgp1 and TcPgp2 in SS strains. However, the expression of TcPgp1 and TcPgp2 in AbR lines were significant after induction.
【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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