[보고서]전사체 분석을 통한 왕담배나방 살충제 저항성 기작 구명

김주일

전사체 분석을 통한 왕담배나방 살충제 저항성 기작 구명
Identification fo insecticide resistance mechanism in Helicoverpa armigera based on transcriptome analysis 원문보기

보고서 정보
주관연구기관	국립식량과학원 National Institute of Crop Science
연구책임자	김주일
참여연구자	권민 , David, G. Heckel
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2019-02
과제시작연도	2018
주관부처	농촌진흥청 Rural Development Administration(RDA)
등록번호	TRKO201900017377
과제고유번호	1395053874
사업명	국제농업기술협력(R&D)
DB 구축일자	2019-11-16
키워드	왕담배나방.살충제 저항성.전사체분석.분자 마커.진단.Helicoverpa armigera.insecticide resistance.transcriptome analysis.molecular marker.diagnosis.
DOI	https://doi.org/10.23000/TRKO201900017377

초록 ▼

연구개발목표
○ 왕담배나방의 효율적인 방제를 위한 살충제 저항성 관리기술 개발
- 전사체 분석을 통한 왕담배나방의 살충제 저항성 기작 구명, 살충제 저항성 마커개발, 분자진단법을 개발, 적용하여 저항성 관리 체계를 구축하고자 함

주요연구내용
○ 계통별, 교배를 통한 F1 hybrid의 생물검정을 통한 왕담배나방의 살충제 저항성 검정
○ 전사체분석을 통한 살충제 저항성 관련 유전자 탐색, 변이 추적, 발현 패턴 분석
○ 분자 마커를 기반으로 한 분자진단법 개발, 현장 적용

연구개발성과
○ 저항성 특이적 과발현 유전자(P450, esterase 등) 탐색, 저항성 특이적 유전자
(CYP337B3, 2012 MPI-CE보고; CYP321, 본 과제를 통하여 새로운 저항성 관련 유전자) 확인
○ 나트륨 채널에서 브라질 저항성 계통 특이적 돌연변이 확인(V937G, Q960H)
○ 고리매개등온증폭법(LAMP)를 이용한 CYP337B3 유전자 진단법 개발
→ 국내에는 대부분의 야외 집단에서 CYP337B3가 존재 확인
○ 새로운 저항성 관련 유전자 CYP321 탐색, 기능 분석, LAMP 활용 저항성 유전자 진단법 개발
→ 국내외 야외 집단에서 CYP321 유전자가 존재함을 확인
→ 다양한 국내 집단을 채집하여 확인, 현장 적용 가능성 제고
○ CYP337B3는 중도 저항성에 관여, CYP321은 고도 저항성에 관여하는 것으로 판단

결과활용계획
○ 개발한 LAMP법을 활용하여 국내 왕담배나방 살충제 저항성 관리에 활용
○ 전사체 분석을 통한 조직별, 계통별 유전자의 발현 양상 결과 등은 추가 기초 연구에 활용
○ 왕담배나방의 lncRNA 분석 등 추가 연구 결과 등은 후속 연구에 접목, 활용 예정

(출처 : 요약서 4p)

Abstract ▼

Purpose&Contents
Identification of insecticide resistance mechanism in H. armigera based on transcriptome analysis and development of IRM program

Results
- Analysis of insecticide resistance mechanism of H. armigera using transcriptome analysis
- Development of molecular diagnosis method (LAMP) for resistance strain specific gene CYP337B3, CYP321
- Development of molecular diagnostics (LAMP) of CYP321
- Finding ways for insecticide resistance management (IRM) strategy via international symposium in Korea
- Establishment of an international network for IRM

Expected Contribution
- Utilizing the developed molecular diagnostics method for pesticide resistance management → Reduction of pesticide abuse and reduction of farm management cost
- Maintaining a friendly relationship with MPI-CE and expecting follow-up research project

(출처 : Summary 5p)

목차 Contents

표지 ... 1
제 출 문 ... 2
보고서 요약서 ... 4
국 문 요 약 문 ... 5
Summary ... 5
목차 ... 6
제 1 장 사업의 개요 ... 7
제1절 연구 개발 목적 ... 7
제2절 협력 사업의 필요성 ... 7
제3절 사업 범위 ... 8
제 2 장 국내외 기술개발 현황 ... 9
제 3 장 사업 수행 내용 및 결과 ... 10
제1절 왕담배나방 살충제 저항성 관련 MPI-CE, RDA 연구 ... 10
제2절 MPI-CE 왕담배나방 대량 사육 및 교배 기술 ... 14
제3절 왕담배나방 전사체 분석 연구 ... 17
제4절 왕담배나방 살충제 저항성 관여 유전자 선발 및 변이 분석 ... 30
제5절 왕담배나방 살충제 저항성 유전자 연관 분석 ... 34
제6절 왕담배나방 살충제 저항성 분자 진단법 개발 ... 37
제7절 주요 성과 요약 ... 44
제 4 장 목표달성도 및 관련분야 기여도 ... 47
제1절 : 목표대비 달성도 ... 47
제2절 : 정량적 성과(논문게재, 특허출원, 기타)를 기술 ... 47
제 5 장 사업 결과의 활용 계획 ... 48
제 6 장 연구 과정에서 수집한 해외 과학 기술 정보 ... 49
제 7 장 연구 개발 결과의 보안 등급 ... 49
제 8 장 국가과학기술종합정보시스템에 등록한 연구시설·장비 현황 ... 49
제 9 장 연구개발과제 수행에 따른 연구실 등의 안전조치 이행실적 ... 49
제 10 장 협력사업의 대표적 사업실적 ... 50
제 11 장 기타사항 ... 50
제 12 장 참고문헌 ... 51
끝페이지 ... 59

표/그림 (41)

표 Representative picture of corn damage from larval stage of Helicoverpa armigera
표 Population dynamics of four major lepidopteran pests during 2012 to 2015 by species specific sex pheromone traps. Traps were set first of April in every year
표 Dates of first collection of four major lepidopteran pests were figure out during 2012 to 2015 by species specific sex pheromone traps. Traps were set first of April in every year
표 Detailed information on corn cultivation such as planting and harvesting and survey results of lepidopteran pest damage
표 Insecticide susceptibility of 3rdinstar Helicoverpa armigera against 16 commercial insecticides in recommended concentration
표 Representative rearing methods for larval stage of Helicoverpa armigera using artificial diet in MPI-CE. First to third instar larvae were rearing in Petri dish (11cm diameter, A). After 3rd instar, each larva was transfer to 1 OZ single individual rearing cup, B and C). About 7 to 10 days after transfer, early 3rd instar larva (C) develop to 5th or 6th instar (D)
표 Representative rearing methods for pupal (A) and adult (B) stage of Helicoverpa armigera and single pair mating (C and D) strategy in MPI-CE. Until mid-stage of pupa, remained artificial diet was helpful for keeping the relative humidity inside of individual rearing cup (A). Around mid- and late stage of pupa, remained artificial diet has to get rid of that because of fungi infection and add some wet cotton to keep the relative humidity inside of individual rearing cup (B). Each mating pair (male and female adult) transfer 15 OZ paper cup for single pair mating (right: male and left: female, C) and their mating behavior (D)
표 Raw data summary from illumina based RNAseq
표 Trimmed data summary from illumina based RNAseq
표 Mapping result to GCF_002156985.1 (Helicoverpa armigera)
표 Hierarchical clustering result of RNAseq from two TWB strains (insecticide susceptible and resistant stains), Kor-R (Korean resistant strain) and bA43 (Brazilian resistant strain), Helicoverpa armigera
표 The number of differentially expressed transcripts in insecticide resistant strains (TWB-R, Kor-R, Kor-T and bA43) compared to that of susceptible strain (TWB-S) Helicoverpa armigera
표 Diagram of differentially expressed transcripts in insecticide resistant strain compared to that of susceptible strain (TWB-S)
표 Expression pattern comparisons of cytochrome P450 gene family from a insecticide susceptible and three resistant strains. A, Heat map showed differentialy expressed genes and B, phylogenetic relationship of cytochrome P450 genes from Helicoverpa armigera
표 Expression pattern comparisons of esterase gene family from a insecticide susceptible and three resistant strains. A, Heat map showed differentialy expressed genes and B, phylogenetic relationship of esterase genes from Helicoverpa armigera
표 Expression pattern comparisons of ABC transporter gene family from a insecticide susceptible and three resistant strains. A, Heat map showed differentialy expressed genes and B, phylogenetic relationship of ABC transporter genes from Helicoverpa armigera
표 Expression pattern comparisons of Glutathione S-transferase (GST) gene family from a insecticide susceptible and three resistant strains. A, Heat map showed differentialy expressed genes and B, phylogenetic relationship of Glutathione S-transferase (GST) genes from Helicoverpa armigera
표 Expression pattern comparisons of UDP glucuronosyltransferase (UGT) gene family from a insecticide susceptible and three resistant strains. A, Heat map showed differentialy expressed genes and B, phylogenetic relationship of UDP glucuronosyltransferase (UGT) genes from Helicoverpa armigera
표 Pipeline to identify the long non-coding RNA, lncRNA form Helicoverpa armigera
표 Some characteristic analysis of identified lncRNAs from Helicoverpa armigera
표 LncRNA expression comparisons via MA plot
표 The number of differentially expressed lncRNAs in insecticide resistant strains (TWB-R, Kor-R, Kor-T and bA43) compared to that of susceptible strain (TWB-S) Helicoverpa armigera
표 GO, gene ontology analysis results of lncRNAs using Blast2GO (level 3, A and B) and heat map for GO based 828 detoxification related lncRNAs
표 Expression level comparisons of highly expressed lncRNAs (over 3,000 FPKM values) in insecticide susceptible and resistant strains (D), selected theree lncRNA secondary RNA structure (E) and heat map of cuticular protein expression in insecticide susceptible and resistant strains (F)
표 Bioassay results with or without synergist, piperonyl butoxide (PBO) in 3rd instar larvae of Helicoverpa armigera. Bioassay was tested using TWB-S (insecticide susceptible line), TWB-R (insecticide resistant line): A or hybrid from Kor-R and TWB-R: B. Mortality was checked in 48hrs after treatment of deltamethrin
표 Maximum likelihood phylogenetic analysis of P450 genes form Helicoverpa armigera. CYP337B subfamily clustered with CYP321 subfamily
표 An example of HPLC results of insecticide (Deltamehtrin) breakdown rate of candidate genes that possibly involved in insecticide resistance including CYP321 gene from Helicoverpa armigera. KO(Kor-R, CYP321), Br(bA43, CYP321), NJ(TWB3, CYP337B3), NTG(negative control)
표 Alignments of sodium channel genes from three strains of Helicoverpa armigera such as insecticide susceptible strain, TWB (LOC110384245); Korean resistant strain (KY247120, KY247121) and Brazilian resistant strain bA43 (MG674159). V937G and Q960H mutations were detected in between ⅡS 4 and ⅡS 5
표 Mutation survey in para-homologous sodium channel gene from TWB-S and Kor-R strain of Helicoverpa armigera and compared their sequence with that of H. zea (V421M, V421A, V421G, I951V, and L1029H) associated with pyrethroid insecticide resistance. V421 (A), I951 (B) and L1029 (C). Shaded residues indicate transmembrane regions. The abbreviations for insect species and GenBank accession numbers for homologous sequences are as follows: Helze Helicoverpa zea, GU574730; hscp Heliothis virescens, AF072493 (Park et al. 1999); BmNav Bombyx mori , EU822499 (Shao et al., 2009); Pluxy Plutella xylostella, AB265177 (Sonoda et al., 2008); Musdo Musca domestica, X96668 (Williamson et al., 1996); Blage Blatella germanica, U73584 (Dong, 1997); para Drosophila melanogaster , M32078 (Loughney et al., 1989). (Modified from Hopkins and Pietrantonio, 2010. IBMB)
표 An example of mass mating (A and B) for rearing in resistant strain and single pair mating (C and D) for linkage analysis
표 Candidate esterase gene selection via RNAseq and genome analysis and the example of following phylogenetic analysis
표 An example of marker development for linkage analysis
표 Comparisons of esterase expression pattern depending on family, tissue (hemolymph and rest of the body) and strain (insecticide susceptible, S and resistant, R strains). IEF, isoelectric focusing gels were stained with coomassie blue staining (A) and substrate specific activity staining (B). 1mM alpha naphthyl acetate was used as a esterase substrate. Native IEF was performed in a vertical electrophoresis unit with the pH 3–7 pre-cast IEF gel. 10ug of proteins were loaded in each well and run 100V for 60min, 200V for 60min and 500V for 30min. Protein concentration was measured by Bradford method. M: IEF marker, N: negative control
표 Alignment of the DNA sequences of CYP337B1, CYP337B2 and 10 different CYP337B3 alleles. CYP337B3v1 and the positions of the six primers designed specifically for amplifying chimeric CYP337B3 by a loop-mediated isothermal amplification (LAMP) reaction. A blue arrowhead points to a crossing-over site. Red arrows indicate the primary priming regions of LAMP (F3 and B3). Purple arrows show the forward internal primer (FIP, a combination of F1c and F2). The backward internal primer (BIP, a combination of B1c and B2) is indicated by yellow arrows. Loop forward (LF) and backward (LB) are indicated by green arrows. CYP337B3v2 (MG744341) K and CYP337B3v7 (MG744342) K were cloned from Korean Helicoverpa armigera. Red letters indicate a different sequence compared to that of CYP337B3v1
표 Genomic DNA used in this study was confirmed by conventional PCR using CYP337B1‐, CYP337B2‐, and CYP337B3‐specific primers. When a CYP337B1 (CYP337B1‐qF and CYP337B1,3‐R)‐ and CYP337B2 (CYP337B2‐qF and CYP337B2‐R)‐specific primer pair was used, each band was detected from genomic DNA from CBW‐S, two CJs and the Bayer susceptible strain (a,b). Alternatively, when a CYP337B3‐specific primer pair (CYP337B3‐qF and CYP337B1,3‐R) was used, all the examined genomic DNAs amplified a strong single band except the two susceptible strains (c). A RPS3‐specific primer pair (RPS3‐F and RPS3‐R) was used as a loading control. The intensities of all bands were comparable
표 The four primers designed for LAMP assay are specific to conventional PCR and LAMP assays. (a) The results of conventional PCR using the F3‐B3 primer pair with the genomic DNA extracted from cotton bollworm susceptible and resistant (CBW‐R) lines. (b–d) LAMP assay using the F3, FIP, BIP, and B3 primers specifically amplify DNA fragments only from CBW‐R genomic DNAs. LAMP reaction results can be confirmed by the naked eye under daylight conditions (b), UV (c), and by separation with 1.0% agarose gel electrophoresis (d). Obviously amplified DNA ladders smeared along the lane were present only in a LAMP assay with CBW‐R genomic DNAs. B3: backward 3 primer; BIP: backward internal primer; CBW‐S: cotton bollworm susceptible; CBW‐R: cotton bollworm resistant; F3: forward 3 primer; FIP: forward internal primer; LAMP: loop‐mediated isothermal amplification; PCR: polymerase chain reaction
표 CBP337B3‐detecting LAMP assay has a wide range of working temperature. To examine a range of working temperatures for this LAMP assay, LAMP mixtures were incubated under various temperatures from 56 to 64°C, at 2°C intervals. Successfully amplified LAMP assays were detected under daylight (a) and UV (b). In addition, amplified DNA ladders were clearly separated by 1.0% agarose gel electrophoresis (c). LAMP: loop‐mediated isothermal amplification
표 The loop primers reduced incubation periods for complete LAMP assays. CYP337B3 LAMP assays were performed using four primers (a‐1 and ‐2), four primers with a LF (b‐1 and ‐2) or a LB (c‐1 and ‐2), and four primers with both loop primers (d‐1 and ‐2) depending on the incubation time 0–30 min), with 5‐min intervals. The addition of one or both loop primers reduced incubation times. LAMP: loop‐mediated isothermal amplification; LB: loop backward; LF: loop forward
표 The detection limit of the LAMP assay using six primers was 10 fg genomic DNA from Helicoverpa armigera. CYP337B3 LAMP assays were performed without genomic DNA inputs or with 1 ng CBW‐S or 1 ng to 1 fg CBW‐R genomic DNA. Reactions were examined by daylight (a), UV (b), and 1% agarose gel separation (c)
표 CYP337B3 loop‐mediated isothermal amplification (LAMP) assay was able to detect pyrethroid resistant strains, field‐collected Helicoverpa armigera by species‐specific pheromone traps and genomic DNAs from CWB‐S, ‐R, or ‐K were used. LAMP reactions were detected when four genomic DNAs extracted from four adult CBWs captured from Daegwallyeong (a) and other field populations, such as Hongcheon (HC), Bongpyeong (BP), and Chungju (CJ), were used (b). The CYP337B3 LAMP assay also detected synthesized CYP337B3v4 (B3v4P), which had one of the most divergent sequences compared to that of CYP337B3v1 (c)
표 Development of loop-mediated isothermal amplification (LAMP) method for insecticide resistant related P450 gene, CYP321 from Helicoverpa armigera. Alignment was performed three DNA sequences of CYP321 allele form insecticide resistant strains such as bA43, bA33 and Kor-R with CYP321A5 from insecticide susceptible strain of Helicoverpa armigera and CYP321A1 from H. zea. CYP321-RF_F3, CYP321-RR_B3 and the positions of the inner primers designed specifically for amplifying CYP321 gene by a LAMP reaction. Reference gene was used CYP321A5 and it amplified CYP321-SF and CYP321-SR1 with same inner primers (A). Gene specific primers were verified by conventional PCR using CYP321 primer set (B) and CYP321A5 primer set (C), respectively. LAMP reactions were confirmed without UV exposure (D) or with UV exposure (E) after adding cybergreen

과제명(ProjectTitle) :	-
연구책임자(Manager) :	-
과제기간(DetailSeriesProject) :	-
총연구비 (DetailSeriesProject) :	-
키워드(keyword) :	-
과제수행기간(LeadAgency) :	-
연구목표(Goal) :	-
연구내용(Abstract) :	-
기대효과(Effect) :	-

내보내기 구분	파일저장 인쇄 메일전송
구성항목	기본정보 상세정보 관리번호, 제목(한글), 저자명(한글), 발행일자, 전자원문, 초록(한글), 초록(영문) 관리번호, 제목(한글), 제목(영문), 저자명(한글), 저자명(영문), 주관연구기관(한글), 주관연구기관(영문), 발행일자, 총페이지수, 주관부처명, 과제시작일, 보고서번호, 과제종료일, 주제분류, 키워드(한글), 전자원문, 키워드(영문), 입수제어번호, 초록(한글), 초록(영문), 목차
저장형식	Text(ASCII format) Excel format
메일정보	받는사람 (필수) @ 보내는사람 (선택) @ 제목 내용 KISTI 검색결과 이메일 서비스
안내	총 건의 자료가 검색되었습니다. 다운받으실 자료의 인덱스를 입력하세요. (1-10,000) 검색결과의 순서대로 최대 10,000건 까지 다운로드가 가능합니다. 데이타가 많을 경우 속도가 느려질 수 있습니다.(최대 2~3분 소요) 다운로드 파일은 UTF-8 형태로 저장됩니다. 파일의 내용이 제대로 보이지 않을실 때는 웹브라우저 상단의 보기 -> 인코딩 -> 자동선택 여부를 확인하십시오. ~ Text(ASCII format) Excel format

연합인증

전사체 분석을 통한 왕담배나방 살충제 저항성 기작 구명
Identification fo insecticide resistance mechanism in Helicoverpa armigera based on transcriptome analysis 원문보기