草业学报
草業學報
초업학보
PRATACULTURAL SCIENCE
2014年
5期
153-160
,共8页
杨顺义%岳秀利%王进军%沈慧敏%郭金梅%沈一凡%周兴隆
楊順義%嶽秀利%王進軍%瀋慧敏%郭金梅%瀋一凡%週興隆
양순의%악수리%왕진군%침혜민%곽금매%침일범%주흥륭
二斑叶螨%甲氰菊酯%靶标突变%直接测序法%PCR-RFLP法
二斑葉螨%甲氰菊酯%靶標突變%直接測序法%PCR-RFLP法
이반협만%갑청국지%파표돌변%직접측서법%PCR-RFLP법
Tetranychusurticae%fenpropathrin%target mutation%direct sequencing%PCR-RFLP
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 sodi-um 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。
靶標突變是二斑葉螨對擬除蟲菊酯類藥劑產生高抗性的重要原因，而用于靶標突變檢測的方法較多，有必要尋找一種經濟、靈敏且有效的方法進行突變檢測。本研究根據二斑葉螨電壓門控鈉離子通道（voltage-gated sodi-um channels，VGSCs）基因上已報道的3箇點突變（L1022V、A1215D和F1538I），採用PCR產物直接測序和限製性片段長度多態性聚閤酶鏈反應（polymerase chain reaction-restriction fragment length polymorphism，PCR-RFLP）兩種不同的方法對二斑葉螨田間種群（WW-R和LZ-R）進行靶標VGSCs 基因突變檢測。PCR產物直接測序結果顯示，與SS種群相比，WW-R和LZ-R種群VGSCs基因均存在兩種氨基痠突變A1215D和F1538I，但沒有髮現存在F1022V突變，其中A1215D突變髮生在結構域ⅡS6-ⅢS1連接處，F1538I突變髮生在結構域ⅢS6螺鏇處，而採用PCR-RFLP方法隻檢測齣A1215D突變。兩種不同的方法對田間採集的二斑葉螨種群VGSCs 基因突變位點檢測的比較，為抗菊酯類藥劑二斑葉螨種群抗性分子鑑測技術的建立和抗性治理奠定瞭基礎。
파표돌변시이반협만대의제충국지류약제산생고항성적중요원인，이용우파표돌변검측적방법교다，유필요심조일충경제、령민차유효적방법진행돌변검측。본연구근거이반협만전압문공납리자통도（voltage-gated sodi-um channels，VGSCs）기인상이보도적3개점돌변（L1022V、A1215D화F1538I），채용PCR산물직접측서화한제성편단장도다태성취합매련반응（polymerase chain reaction-restriction fragment length polymorphism，PCR-RFLP）량충불동적방법대이반협만전간충군（WW-R화LZ-R）진행파표VGSCs 기인돌변검측。PCR산물직접측서결과현시，여SS충군상비，WW-R화LZ-R충군VGSCs기인균존재량충안기산돌변A1215D화F1538I，단몰유발현존재F1022V돌변，기중A1215D돌변발생재결구역ⅡS6-ⅢS1련접처，F1538I돌변발생재결구역ⅢS6라선처，이채용PCR-RFLP방법지검측출A1215D돌변。량충불동적방법대전간채집적이반협만충군VGSCs 기인돌변위점검측적비교，위항국지류약제이반협만충군항성분자감측기술적건립화항성치리전정료기출。
Target mutation produces high levels of resistance to pyrethroid insecticides in the spider mite Tet-ranychusurticae.However,as there are many techniques for detecting target mutations it would be advanta-geous 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 frag-ment 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.urticaehas the potential to develop molecular-based monitoring that can be used to manage the species’resistance to pyrethroid insecticides.
