二斑叶螨多重抗性品系最优内参基因的筛选及CYP392A亚家族基因的表达分析

›› 2015, Vol. 58 ›› Issue (11): 1229-1236.doi:

二斑叶螨多重抗性品系最优内参基因的筛选及CYP392A亚家族基因的表达分析

周兴隆¹, 杨顺义¹, 郝雨¹, 王进军², 张新虎¹, 沈慧敏^1,*

（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. 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）

出版日期:2015-11-20 发布日期:2015-11-20
作者简介:周兴隆, 男, 1985年生, 甘肃定西人, 硕士研究生, 研究方向为害虫（螨）抗药性, E-mail: zhouxl94@163.com

Selection of the most suitable reference genes and expression profiling of CYP392A subfamily genes in the multi-pesticide resistant strain of Tetranychus urticae (Acari: Tetranychidae)

ZHOU Xing-Long¹, YANG Shun-Yi ¹ , HAO Yu¹ , WANG Jin-Jun ², ZHANG Xin-Hu ¹, SHEN Hui-Min^1,*

(1. The Sino-U.S. Centers for Grazingland Ecosystem Sustainability, Key Laboratory of Grassland Ecosystem Education Ministry, College of Prataculture, Gansu Agricultural University, Lanzhou 730070, China; 2. China Key Laboratory of Entomology and Pest Control Engineering, College of Plant Protection, Southwest University, Chongqing 400716, China)

Online:2015-11-20 Published:2015-11-20

摘要/Abstract

摘要： 【目的】筛选出二斑叶螨Tetranychus urticae Koch抗甲氰菊酯、阿维菌素及螺螨酯混剂的实时定量PCR最优内参基因。【方法】选取5.8S rRNA, α-tubulin, TBP, β-actin, ELFn, RPL13a, GAPDH和SDHA 8个候选内参基因，以GeNorm, BestKeeper和Normfinder 3个软件分析这8个基因在二斑叶螨多重抗性品系中的表达稳定性, 并以筛选的内参基因分析二斑叶螨P450酶系CYP392A亚家族基因的表达水平。【结果】经GeNorm, BestKeeper和Normfinder 3个软件综合评价确定ELFn基因为二斑叶螨敏感品系(susceptible strain, SS)和多重抗性品系(multi-pesticide resistant strain, Mp-R)各发育阶段的最优内参基因。以ELFn为内参基因对二斑叶螨CYP392A亚家族16个基因表达量进行分析，结果表明：经多重抗性选育40代后，Mp-R品系卵期CYP392A1表达量显著上调；CYP392A16基因在各发育阶段表达量极显著高于SS品系相应发育阶段；其他基因表达量在敏感品系和抗性品系之间差异不显著。【结论】筛选出了SS和MpR品系中各发育阶段最佳内参基因为EFLn；Mp-R品系CYP392A亚家族16个基因的表达量在幼螨和若螨阶段低于卵与成螨阶段，其中CYP392A16基因在二斑叶螨多重抗性的形成中起主要作用。该结果为二斑叶螨多重抗性研究奠定了一定基础。

关键词: 二斑叶螨, 多重抗性, 内参基因, CYP392A亚家族, 基因表达量, RT-qPCR

Abstract: 【Aim】 This study aims to select suitable reference genes in the multi-pesticide (abamectin, fenpropathrin and spirodiclofen) resistant strain (Mp-R) of T. urticae by quantitative real-time PCR (RT-qPCR). 【Methods】 Eight candidate reference genes including 5.8S rRNA, α-tubulin, TBP,β-actin , ELFn, RPL13a, GAPDH and SDHA were chosen. The stability of these candidate reference genes was investigated by using three softwares (GeNorm, NormFinder and BestKeeper). Then the expression of CYP392A subfamily gene in P450 enzyme system of T. urticae was analyzed by using different reference genes. 【Results】 EFLn was confirmed the most suitable reference gene in the sensitive strain (SS) and the multi-pesticide resistant strain (Mp-R) of T. urticae by geNorm, NormFinder and BestKeeper softwares. The expression level of CYP392A1 was up-regulated significantly in the eggs of the Mp-R strain, and the expression levels of CYP392A16 gene in various developmental stages of the Mp-R strain was significantly higher than the same stage of the sensitive strain of T. urticae after 40-generation selection with the multi-pesticides. The expression levels of CYP392A16 gene in various developmental stages of the Mp-R strain was significantly higher than those in the same stage of the sensitive strain of T. urticae. The expression levels of other genes were not significantly different between the Mp-R strain and the sensitive strain. 【Conclusion】 EFLn gene is confirmed the most suitable reference gene in the multi-pesticide resistant strain and the susceptible strain of T. urticae. This work is contributable to the foundation for future gene expression studies in the resistant strain of T. urticae.

Key words: Tetranychus urticae, resistance, CYP392A, gene expression level, RT-qPCR

周兴隆, 杨顺义, 郝雨, 王进军, 张新虎, 沈慧敏. 二斑叶螨多重抗性品系最优内参基因的筛选及CYP392A亚家族基因的表达分析[J]. , 2015, 58(11): 1229-1236.

ZHOU Xing-Long, YANG Shun-Yi , HAO Yu , WANG Jin-Jun , ZHANG Xin-Hu , SHEN Hui-Min. Selection of the most suitable reference genes and expression profiling of CYP392A subfamily genes in the multi-pesticide resistant strain of Tetranychus urticae (Acari: Tetranychidae)[J]. , 2015, 58(11): 1229-1236.

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