Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (21): 4542-4549.doi: 10.3864/j.issn.0578-1752.2013.21.017

• PLANT PROTECTION • Previous Articles     Next Articles

Selection of Reference Genes and Study of the Expression Levels of Detoxifying Enzymes of Tetranychus urticae (Acari: Tetranychidae)

 YUE  Xiu-Li-1, GAO  Xin-Ju-1, WANG  Jin-Jun-2, 吕Juan-Juan-1 , SHEN  Hui-Min-1   

  1. 1.College of Prataculture, Gansu Agricultural University, Lanzhou 730070
    2.College of Plant Protection, Southwest University, Chongqing 400716)
  • Received:2013-04-15 Online:2013-11-01 Published:2013-06-05

PDF

660

Cited

8

Abstract: 【Objective】The objective of this study is to select suitable reference genes for studying the expression levels of detoxifying enzymes on fenpropathrin susceptible (SS) and resistant strains (Fe-R) of Tetranychus urticae by using quantitative real-time PCR (RT-qPCR).【Method】The mRNA expression stability of eight candidate reference genes 5.8S rRNA, α-tubulin, β-actin, ELF, GAPDH, RPL13a, SDHA and TBP of T. urticae were detected by RT-qPCR, the relative expression of the main detoxifying enzymes CCEs, GSTs and P450s were analyzed. GeNorm and NormFinder softwares were used to analyze and evaluate the data for reference genes and comparison of Ct value method was used for the calculation of relative expression levels.【Result】α-tubulin was one of the most stable genes among the eight reference genes. When α-tubulin was used as a reference gene, the expression levels of TuGSTo1, TuGSTd1and CYP3D2 in Fe-R strain were significantly higher than that in SS strain while TuCCE1 was downregulated.【Conclusion】The gene α-tubulin can be used as an ideal reference in both SS and Fe-R strains of T. urticae. Futhermore, the elevated relative expression levels of TuGSTo1, TuGSTd1 and CYP3D2 may be the molecular mechanism of T. urticae that with high resistance to fenpropathrin.

Key words: reference gene , RT-qPCR , CCEs , GSTs , P450s , Tetranychus urticae

[1] Grbic M, Van Leeuwen T, Clark R M, Rombauts S, Rouze P, Grbic V, Osborne E J, Dermauw W, Phuong C T N, Ortego F, Hernandez-Crespo P, Diaz I, Martinez M, Navajas M, Sucena E, Magalhaes S, Nagy L, Pace R M, Djuranovic S, Smagghe G, Iga M, Christiaens O, Veenstra J A, Ewer J, Villalobos R M, Hutter J L, Hudson S D, Velez M, Yi S V, Zeng J, Pires-daSilva A, Roch F, Cazaux M, Navarro M, Zhurov V, Acevedo G, Bjelica A, Fawcett J A, Bonnet E, Martens C, Baele G, Wissler L, Sanchez-Rodriguez A, Tirry L, Blais C, Demeestere K, Henz S R, Gregory T R, Mathieu J, Verdon L, Farinelli L, Schmutz J, Lindquist E, Feyereisen R, Van de Peer Y. The genome of Tetranychus urticae reveals herbivorous pest adaptations. Nature, 2011, 479(7374): 487-492.

[2] Ginzinger D G. Gene quantification using real-time quantitative PCR: An emerging technology hits the mainstream. Experimental Hematology, 2002, 30(6): 503-512.

[3] Radonic A, Thulke S, Mackay I M, Landt O, Siegert W, Nitsche A. Guideline to reference gene selection for quantitative real-time PCR. Biochemical and Biophysical Research Communications, 2004, 313(4): 856-862.

[4] Bustin S A. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. Journal of Molecular Endocrinology, 2002, 29(1): 23-39.

[5] Shen G M, Jiang H B, Wang X N, Wang J J. Evaluation of endogenous references for gene expression profiling in different tissues of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae). Bmc Molecular Biology, 2010, 11: 76.

[6] Niu J Z, Dou W, Ding T B, Yang L H, Shen G M, Wang J J. Evaluation of suitable reference genes for quantitative RT-PCR during development and abiotic stress in Panonychus citri (McGregor) (Acari: Tetranychidae). Molecular Biology Reports, 2012, 39(5): 5841-5849.

[7] Sun W, Jin Y, He L, Lu W C, Li M. Suitable reference gene selection for different strains and developmental stages of the carmine spider mite, Tetranychus cinnabarinus, using quantitative real-time PCR. Journal of Insect Science, 2010, 10(208): 1-12.

[8] Yang X, Buschman L L, Zhu K Y, Margolies D C. Susceptibility and detoxifying enzyme activity in two spider mite species (Acari: Tetranychidae) after selection with three insecticides. Journal of Economic Entomology, 2002, 95(2): 399-406.

[9] Van Leeuwen T, Tirry L. Esterase-mediated bifenthrin resistance in a multiresistant strain of the two-spotted spider mite, Tetranychus urticae. Pest Management Science, 2007, 63(2): 150-156.

[10] Dermauw W, Wybouw N, Rombauts S, Menten B, Vontas J, Grbic M, Clark R M, Feyereisen R, Van Leeuwen T. A link between host plant adaptation and pesticide resistance in the polyphagous spider mite Tetranychus urticae. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110(2): 113-122.

[11] Demaeght P, Dermauw W, Tsakireli D, Khajehali J, Nauen R, Tirry L, Vontas J, Lummen P, Van Leeuwen T. Molecular analysis of resistance to acaricidal spirocyclic tetronic acids in Tetranychus urticae: CYP392E10 metabolizes spirodiclofen, but not its corresponding enol. Insect Biochemistry Molecular Biology, 2013, 43(6): 544-554.

[12] 高新菊, 沈慧敏. 二斑叶螨对甲氰菊酯的抗性选育及解毒酶活力变化. 昆虫学报, 2011, 54(1): 64-69.

Gao X J, Shen H M. Resistance slection with fenpropathrin and the change of detoxification enzyme activities in Tetranychus urticae Koch (Acari: Tetranychidae). Acta Entomologica Sinica, 2011, 54(1): 64-69. (in Chinese)

[13] 孙伟. 药剂胁迫下朱砂叶螨酯酶基因的表达特征[D]. 重庆: 西南大学, 2010.

Sun W. Expression characteristic of esterase genes suffering the stress of pesticide in Tetranychus cinnabarinus (Boisduval)[D]. Chongqing: Southwest University, 2010. (in Chinese)

[14] Pfaffl M W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research, 2001, 29(9): 2002-2007.

[15] Boda E, Pini A, Hoxha E, Parolisi R, Tempia F. Selection of reference genes for quantitative real-time RT-PCR studies in mouse brain. Journal of Molecular Neuroscience, 2009, 37(3): 238-253.

[16] Scharlaken B, de Graaf D C, Goossens K, Brunain M, Peelman L J, Jacobs F J. Reference gene selection for insect expression studies using quantitative real-time PCR: The head of the honeybee, Apis mellifera, after a bacterial challenge. Journal of Insect Science, 2008, 8(33): 1-10.

[17] Chi X Y, Hu R B, Yang Q L, Zhang X W, Pan L J, Chen N, Chen M N, Yang Z, Wang T, He Y A, Yu S L. Validation of reference genes for gene expression studies in peanut by quantitative real-time RT-PCR. Molecular Genetics and Genomics, 2012, 287(2): 167-176.

[18] Henn D, Bandner-Risch D, Perttunen H, Schmied W, Porras C, Ceballos F, Rodriguez-Losada N, Schafers H J. Identification of reference genes for quantitative RT-PCR in ascending aortic aneurysms. Plos One, 2013, 8(1): e54132.

[19] Andersen C L, Jensen J L, Orntoft T F. Normalization of real-time quantitative reverse transcription-PCR data: A model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Research, 2004, 64(15): 5245-5250.

[20] 杨丽红. 柑橘全爪螨Panonychus citri (McGregor) 对热胁迫的响应机制研究[D]. 重庆: 西南大学, 2011.

Yang L H. Study on mechanisms of Panonychus citri (McGregor) in response to thermal stress[D]. Chongqing: Southwest University, 2011. (in Chinese)

[21] Teng X L, Zhang Z, He G L, Yang L W, Li F. Validation of reference genes for quantitative expression analysis by real-time RT-PCR in four lepidopteran insects. Journal of Insect Science, 2012, 12(60): 1-17.

[22] Lord J C, Hartzer K, Toutges M, Oppert B. Evaluation of quantitative PCR reference genes for gene expression studies in Tribolium castaneum after fungal challenge. Journal of Microbiological Methods, 2010, 80(2): 219-221.

[23] Argyropoulos D, Psallida C, Spyropoulos C G. Generic normalization method for real-time PCR-Application for the analysis of the mannanase gene expressed in germinating tomato seed. FEBS Journal, 2006, 273(4): 770-777.

[24] Yan H, Jia H, Wang X, Gao H, Guo X, Xu B. Identification and characterization of an Apis cerana cerana Delta class glutathione S-transferase gene (AccGSTD) in response to thermal stress. Naturwissenschaften, 2013, 100(2): 153-163.

[25] 蒋红波. 嗜卷书虱P450 基因的分子生物学特性及其异源表达研究[D]. 重庆: 西南大学, 2010.

Jiang H B. Molecular characterizations and heterologous expression of P450 genes in the psocid, Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae)[D]. Chongqing: Southwest University, 2010. (in Chinese)

[26] Van Leeuwen T, Dermauw W, Grbic M, Tirry L, Feyereisen R. Spider mite control and resistance management: does a genome help? Pest Management Science, 2013, 69(2): 156-159.
[1] QIU YiLei,WU Fan,ZHANG Li,LI HongLiang. Effects of Sublethal Doses of Imidacloprid on the Expression of Neurometabolic Genes in Apis cerana cerana [J]. Scientia Agricultura Sinica, 2022, 55(8): 1685-1694.
[2] GAO XingXiang,ZHANG YueLi,AN ChuanXin,LI Mei,LI Jian,FANG Feng,ZHANG ShuangYing. Investigation and Analysis of Weed Community Succession in Winter Wheat Field of Shandong Province [J]. Scientia Agricultura Sinica, 2021, 54(24): 5230-5239.
[3] XIAO Fang,LI Jun,WANG HaoQian,ZHAI ShanShan,CHEN ZiYan,GAO HongFei,LI YunJing,WU Gang,ZHANG XiuJie,WU YuHua. Establishment and Application of A Duplex ddPCR Method to Quantify the NK603/zSSIIb Copy Number Ratio in Transgenic Maize NK603 [J]. Scientia Agricultura Sinica, 2021, 54(22): 4728-4739.
[4] MuKang LUO,XuChao JIA,RuiFen ZHANG,Lei LIU,LiHong DONG,JianWei CHI,YaJuan BAI,MingWei ZHANG. Phenolic Content, Bioavailability and Antioxidant Activity of Carambola [J]. Scientia Agricultura Sinica, 2020, 53(7): 1459-1472.
[5] JU PengJu,NING Lei,GE LinHao,XU ChengJie,SHI HuaWei,LIANG KaiGe,MA Liang,LIU TaoRan,CHEN Ming,SUN DaiZhen. Analysis of Foreign Gene Copy Number in Transgenic Wheat by Optimized Digital PCR [J]. Scientia Agricultura Sinica, 2020, 53(10): 1931-1939.
[6] WAN DongLi,HOU XiangYang,DING Yong,REN WeiBo,WANG Kai,LI XiLiang,WAN YongQing. Response and the Expression of Pi-Responsive Genes in Leymus chinensis Under Inorganic Phosphate Treatment [J]. Scientia Agricultura Sinica, 2019, 52(23): 4215-4227.
[7] WenJian YANG,HaoLiang PU,LiuQing WANG,QiuHui HU,Fei PEI. Quality Change and Bacteria Succession of Dried Carrot Stored at Different Water Activities [J]. Scientia Agricultura Sinica, 2019, 52(20): 3661-3671.
[8] LIU FanQi,WAN GuiJun,ZENG LuYing,LI ChunXu,PAN WeiDong,CHEN FaJun. Selection of Stable Internal Reference Genes for Transcript Expression Analyses in Laodelphax striatellus Under Near-Zero Magnetic Field [J]. Scientia Agricultura Sinica, 2019, 52(19): 3346-3356.
[9] GE Bo, WANG BaoBao, GUO Cheng, SUN SuLi, CHEN GuoKang, WANG XiaoMing, ZHU ZhenDong, DUAN CanXing. Composition and quantitative analysis of Fusarium species in maize rhizosphere soil [J]. Scientia Agricultura Sinica, 2018, 51(19): 3683-3693.
[10] HE YuJuan, JU Di, WANG Yue, YANG XueQing, WANG XiaoQi . Compositive and Inductive Expression Patterns of Protease Inhibitor Genes OsLTPL164 and OsLTPL151 in Rice (Oryza sativa) [J]. Scientia Agricultura Sinica, 2018, 51(12): 2311-2321.
[11] TianBo DING, XiaoBei LIU, Jie LI, KeKe WEI, Dong CHU. Development of a Real-Time Fluorescent Quantitative PCR Method for the Detection of Tomato chlorosis virus and Its Application [J]. Scientia Agricultura Sinica, 2018, 51(10): 2013-2022.
[12] PENG FuZhi, RAN MaoLiang, WENG Bo, LI Zhi, DONG LianHua, CHEN Bin. Validation of Reference Genes for Quantitative RT-PCR Analysis in Porcine Testis Tissues [J]. Scientia Agricultura Sinica, 2017, 50(15): 3033-3041.
[13] YANG Dan, LI Qing, WANG GuiXi, MA QingHua, ZHU LiQuan. Reference Genes Selection and System Establishment for Real-time qPCR Analysis in Ping’ou Hybrid Hazelnut (C. heterophylla Fisch. × C. avellana L.) [J]. Scientia Agricultura Sinica, 2017, 50(12): 2399-2410.
[14] ZHOU Xing-long, SONG Li-wen, YANG Shun-yi, LI Jing-jing, WANG Jin-jun, ZHANG Xin-hu, SHEN Hui-min. Analysis of Detoxification Enzyme Genes in the Multiple Pesticide-Resistant Strain of Tetranychus urticae [J]. Scientia Agricultura Sinica, 2016, 49(9): 1696-1704.
[15] LIU Xiao-jian, SUN Ya-wen, CUI Miao, MA En-bo, ZHANG Jian-zhen. Molecular Characteristics and Functional analysis of Trehalase Genes in Locusta migratoria
 
[J]. Scientia Agricultura Sinica, 2016, 49(22): 4375-4386.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd