两种方法在二斑叶螨电压门控钠离子通道基因突变检测中的应用

doi:10.11686/cyxb20140517

摘要/Abstract

摘要：

靶标突变是二斑叶螨对拟除虫菊酯类药剂产生高抗性的重要原因,而用于靶标突变检测的方法较多,有必要寻找一种经济、灵敏且有效的方法进行突变检测。本研究根据二斑叶螨电压门控钠离子通道(voltage-gated sodium channels,VGSCs)基因上已报道的3个点突变(L1022V、A1215D和F1538I),采用PCR产物直接测序和限制性片段长度多态性聚合酶链反应(polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP)两种不同的方法对二斑叶螨田间种群(WW-R和LZ-R)进行靶标VGSCs基因突变检测。PCR产物直接测序结果显示,与SS种群相比,WW-R和LZ-R种群VGSCs基因均存在两种氨基酸突变A1215D和F1538I,但没有发现存在F1022V突变,其中A1215D突变发生在结构域ⅡS6-ⅢS1连接处,F1538I突变发生在结构域ⅢS6螺旋处,而采用PCR-RFLP方法只检测出A1215D突变。两种不同的方法对田间采集的二斑叶螨种群VGSCs基因突变位点检测的比较,为抗菊酯类药剂二斑叶螨种群抗性分子监测技术的建立和抗性治理奠定了基础。

Abstract:

Target mutation produces high levels of resistance to pyrethroid insecticides in the spider mite Tetranychus urticae. However, as there are many techniques for detecting target mutations it would be advantageous to identify methods that are both economical and effective. Three mutations (L1022V, A1215D and F1538I) have been identified in the voltage-gated sodium channels (VGSCs) of T. urticae. We have tested the ability of two different methods, PCR product direct sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), to detect these VGSC gene mutations in two field strains (WW-R and LZ-R) of T. urticae. The results of PCR product direct sequencing showed that the A1215D and F1538I mutations existed in the VGSC genes of both the WW-R and LZ-R strains, but that the F1022V mutation was not detected. The A1215D mutation was located between domains IIS6 and IIIS1 of the VGSCs, while the F1538I mutation was identified in domain IIIS6. The PCR-RFLP method, however, proved able to identify only the A1215D mutation. Our testing of these two methods shows that detecting VGSC mutations in field strains of T. urticae has the potential to develop molecular-based monitoring that can be used to manage the species’ resistance to pyrethroid insecticides.

中图分类号:

S433.7

杨顺义,岳秀利,王进军,沈慧敏,郭金梅,沈一凡,周兴隆. 两种方法在二斑叶螨电压门控钠离子通道基因突变检测中的应用[J]. 草业学报, 2014, 23(5): 153-160.

YANG Shun-yi,YUE Xiu-li,WANG Jin-jun,SHEN Hui-min,GUO Jin-mei,SHEN Yi-fan,ZHOU Xing-long. Testing two methods to detect voltage-gated sodium channels gene mutations in Tetranychus urticae[J]. Acta Prataculturae Sinica, 2014, 23(5): 153-160.

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