二斑叶螨多重抗性品系解毒酶基因表达模式解析  

Analysis of Detoxification Enzyme Genes in the Multiple Pesticide-Resistant Strain of Tetranychus urticae

作　　者：周兴隆[1] 宋丽雯[1] 杨顺义[1] 李静静[1,2] 王进军[3] 张新虎[1] 沈慧敏[1] 

机构地区：[1]甘肃农业大学草业学院/草业生态系统省部共建教育部重点实验室/中-美草地畜牧业可持续发展中心,兰州730070 [2]甘肃省小陇山林业实验局云坪林场,甘肃天水741020 [3]西南大学植物保护学院昆虫学及害虫控制工程重点实验室,重庆400716

出　　处：《中国农业科学》2016年第9期1696-1704,共9页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31260442);国家公益性行业(农业)科研专项(201103020)

摘　　要：【目的】二斑叶螨(Tetranychus urticae)是一种重要的农业害螨,由于个体小、繁殖快等特点,极易产生抗药性,论文从生理生化和分子水平探讨二斑叶螨m RNA水平相对表达量的变化,旨在明确二斑叶螨对混剂的多重抗性机制,为该螨的综合治理提供依据。【方法】在温度(25±l)℃,相对湿度60%±5%,光周期L﹕D=16 h﹕8 h的室内条件下,于盆栽豇豆苗上饲养不接触任何药剂的二斑叶螨敏感品系(SS)和用螺螨酯、甲氰菊酯、阿维菌素以其单剂的致死中浓度(LC50)为汰选浓度混合连续汰选的多重抗性品系(Mp-R);Mp-R品系用药4—5次待其种群扩增后,参照FAO推荐的叶片残毒法进行室内毒力测定一次,计算其LC50,求出抗性倍数(RR),并记为一个汰选周期;连续汰选3个周期后,逐渐增加汰选浓度,用Polo Plus软件求其毒力回归方程、LC50、抗性指数及卡方值;采用生化分析法测定选育50代的二斑叶螨SS与Mp-R品系卵、幼螨、若螨、雄成螨、雌成螨的谷胱甘肽S转移酶(GSTs)、羧酸酯酶(Car Es)、多功能氧化酶(MFOs)的活性,以ELFn为内参基因,采用RT-q PCR技术以比较Ct值的方法计算二斑叶螨Mp-R品系中10个与抗性相关的解毒酶基因的表达量。【结果】在室内经50代抗性选育,二斑叶螨对阿维菌素、甲氰菊酯及螺螨酯的LC50分别达到1 103.55、5 993.33和2 345.62 mg?L-1,抗性倍数分别为603.03、167.65和51.77倍。卵中Mp-R品系MFOs比活力显著高于SS品系,GSTs、Car Es比活力差异不显著;其他发育阶段Mp-R品系的GSTs、Car Es比活力显著高于SS品系,MFOs比活力则差异不显著;Mp-R品系雌成螨的GSTs、Car Es比活力显著高于其他发育阶段,卵的MFOs比活力显著高于其他发育阶段。与敏感品系相比,二斑叶螨Tu GSTd05、Tu GSTd06与Tu GSTd09基因表达量在Mp-R品系各发育阶段显著上调1.80倍以上,Tu GSTd01表达量在若螨期显著上调1.63倍,其他发育阶段差异不显著;CYP392E10�【Objective】Tetranychus urticae is an important mite in agricultural production, which is easy to produce resistance to pesticide because its small size and rapid propagation. The objective of this study is to discuss the change of m RNA relative transcript level, find out multiple resistant mechanism of T. urticae and provide a theoretical basis for its integrated management（prevention and control）. 【Method】SS and Mp-R strain of T. urticae were raised in laboratory. Mp-R strain was continuously selected with median lethal concentration（LC50） of mixture of spirodiclofen, fenpropathrin and abamectin. After spraying insecticide for 4-5 times, biological assay was done referring to leaf-residue method which recommended by FAO, LC50 and resistance ratio were calculated, and a selected cycle was recorded. After 3 cycles, acaricides concentrations were gradually increased, then toxicity equation, LC50, resistance ratio, and chi-square value were calculated by Polo Plus software. The activities of Car Es, GSTs and MFOs were measured by biochemical analysis in egg, larva, nymph and adult of SS and Mp-R strains of T. urticae. Ten ditoxifying genes expression which were related to resistance were measured with RT-q PCR technology using ELFn as reference gene through comparing Ct value method.【Result】After 50 generations of selection, LC50 of T. urticae to abamectin, spirodiclofen and fenpropathrin reached 1 103.55, 5 993.33 and 2 345.62 mg·L-1, and the resistance ratios were 603.03, 167.65 and 51.77 times, respectively. Activities of MFOs in egg of Mp-Rstrain increased significantly compared with the SS strain, but Car Es and GSTs activities had no significant difference. Car Es and GSTs activities at other stages of Mp-R strain increased significantly compared with the SS strain, but MFOs activity did not increase. In Mp-R strain, Car Es and GSTs activities of female adult mite and MFOs activity in egg increased significantly compared with other stages, and compared with the SS strain, the expression o

关 键 词：二斑叶螨 多重抗性 解毒酶基因 RT-q PCR 

分 类 号：S433.7[农业科学—农业昆虫与害虫防治]