Molecular Detection of Toxicodynamic and Metabolic Factors Associated with the Pyrethroid and Carbamate Resistance in Myzus persicae (Sulzer)
복숭아혹진딧물의 피레스로이드와 카바메이트계 살충제 저항성요인의 분자적 진단

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dc.description학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부, 2012. 8. 이시혁.-
dc.description.abstractThe green peach aphid (Myzus persicae) is an economically serious pest of agricultural and horticultural crops all over the world and developed resistance almost all kinds of insecticides. In this study, target site mutations were investigated by sequencing the voltage-gated sodium channel (vgsc) and acetylcholinesterase (ace) genes in 7 and 10 local strains collected in 2010 and 2011, respectively. In addition, leaf dip bioassay was conducted to determine LC50 values of bifenthrin and methomyl. Based on the mutation survey, quantitative sequencing (QS) was employed for investigation of resistance allele frequencies in local strains. Mutation survey of the vgsc revealed that M918L and L1014F mutations were present in local strains. Especially, M918L mutation showed a higher correlation than L1014F between the resistance allele frequency and actual resistance level. Thus, QS using newly identified M918L mutation should facilitate the detection and monitoring of pyrethroid resistance levels in the field.
As putative mutations for target site insensitivity to carbamates, frequencies of A301S and S431F mutations in ace were investigated by sequencing. However, only S431F mutation was revealed in local strains. QS was employed to detect S431F mutation frequency. Correlation analysis of mutation allele frequency versus actual resistance level revealed that S431F mutation itself does not appear to play a significant role.
To identify whether higher expression of M. persicae carboxylesterase (CbE) E4 is due to gene duplication, gene copy number was determined by quantitative real-time PCR. To determine CbE E4 quantity, Western blotting in conjunction with activity staining were conducted. Correlation analysis was carried out to investigate the possible connection between CbE expression level and its gene copy number, activity and protein quantity. Qualitative changing from point mutation was considered and mutation survey was conducted by PCR. However, new mutation G134C was revealed in oxyanion hole. To predict the functions of this mutation, altered CbE E4 3D structure was predicted and correlation analysis was conducted using sequence chromatogram of G134C mutation. Finally, we expect that altered CbE E4 might play a significant role to hydrolyze and/or sequestrate the carbamate insecticide in M. persicae.
dc.description.tableofcontentsTABLE OF CONTENTS
1. Myzus persicae 1
2. Insecticide resistance monitoring 2
3. Carboxylesterase (CbE) as co-resistance factor 3

Pyrethroid resistance monitoring in local strains of Myzus persicae and characterization of the voltage-gated sodium channel gene 4
Abstract 5
1. Introduction 6
2. Materials and methods 7
2.1 M. persicae strain 7
2.2 Pyrethroid resistance monitoring 7
2.2.1 Local strain genotyping for the detection of resistance mutation 8
2.2.2 Bioassay of local strains by bifenthrin 9
2.2.3 Establishment of QS protocol for the detection of resistance allele frequency 10
2.2.4 Correlation analysis between the vgsc mutation frequency and bifenthrin resistance ratios. 11
2.2.5 Analysis of exon-intron structure in partial genomic DNA fragment of vgsc 11
2.2.6 Analysis of vgsc gene copy number ratio by quantitative real-time PCR(qPCR) 12

3. Results 13
3.1 Bifenthrin resistance level in local strains 13
3.2 Genotypes of the vgsc mutation sites 14
3.3 Establishment of QS protocol 15
3.4 Correlation between the two vgsc mutations and bifenthrin resistance levels 18
3.5 Exon-intron structure of vgsc genomic DNA fragments 22
3.6 vgsc gene copy number ratio 22
4. Discussion 23

Carbamate resistance monitoring in local strains of M. persicae and characterization of the acetylcholinesterase gene 25
Abstract 26
1. Introduction 27
2. Materials and methods 28
2.1 M. persicae strain 28
2.2 Carbamate resistance monitoring 28
2.2.1 Local strain genotyping for the detection of acetylcholinesterase mutation 28
2.2.2 Bioassay of local strains by methomyl 29
2.2.3 Establishment of the QS protocol to predict resistance allele frequency 30
2.2.4 Correlation analysis between the ace mutation frequency and methomyl resistance ratios 31
3. Results 32
3.1 Methomyl resistance level in local strains 32
3.2 Genotypes of the ace mutation sites 32
3.3 Establishment of QS protocol 38
3.4 Correlation between the ace mutation and methomyl resistance levels 38
4. Discussion 39

Characterization of carboxylesterase (E4) as a nonspecific metabolic resistance factor in M. persicae 41
Abstract 42
1. Introduction 43
2. Materials and methods 44
2.1 Quantitative real-time PCR (qCR) of Carboxylesterase (CbE) E4 gene 44
2.2 Generation of anti-CbE E4 polyclonal antibody 45
2.3 Activity staining and Western blotting 46
2.4 Mutation survey of CbE E4 gene of local strains 47
2.5 3D structure modeling 47
2.6 Correlation between insecticide resistance ratio, gene copy number and expression levels 47
3. Results 48
3.1 Gene copy number of CbE E4 in local strains 48
3.2 CbE E4 expression levels in local strains 48
3.3 Comparison of CbE E4 sequences among local strains 50
3.4 Three-dimensional (3D) modeling of CbE E4 55
3.5 Determination of correlation factor related with insecticide resistance 56
4. Disscussion 57
dc.format.extent1117350 bytes-
dc.publisher서울대학교 대학원-
dc.subjectMyzus persicae-
dc.subjectM. persicae-
dc.subjectinsecticide resistance-
dc.subjectVoltage-gated sodium channel-
dc.subjectquantitative sequencing-
dc.titleMolecular Detection of Toxicodynamic and Metabolic Factors Associated with the Pyrethroid and Carbamate Resistance in Myzus persicae (Sulzer)-
dc.title.alternative복숭아혹진딧물의 피레스로이드와 카바메이트계 살충제 저항성요인의 분자적 진단-
dc.contributor.AlternativeAuthorJungHun Oh-
dc.contributor.affiliation농업생명과학대학 농생명공학부-
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Agricultural Biotechnology (농생명공학부)Theses (Master's Degree_농생명공학부)
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