곤충 유약호르몬의 기능을 교란하는 새로운 곤충 생장 조절 물질의 선별 및 특성 구명

학위논문 (박사)-- 서울대학교 대학원 : 농업생명과학대학 농생명공학부, 2018. 2. 제연호.Mosquitoes are medically important insect pests that transmit various diseases when they feed on humans. The Asian tiger mosquito, Aedes albopictus Skuse (Diptera: Culicidae), is one of the most invasive vectors of various diseases including dengue fever, chikungunya, and zika virus. Chemical insecticides have been commonly used to control mosquitoes. However, due to their toxicity to environments and development of insect resistance, sustained use of chemical insecticides are limited. Insect growth regulators (IGRs) could become an effective alternative to control mosquitoes and other vector transmitting diseases because they are specific to target insects and relatively low toxic to environment. However, recent studies, mosquitoes and other pests also have developed resistance to certain IGR insecticides. Hence, there is an urgent need to develop novel IGR insecticides. In this study, the yeast two-hybrid β-galactosidase assays using the yeast cells transformed with the genes of JH receptor and its partner, Met-FISC of A. aegypti were performed to identify novel juvenile hormone (JH)-related IGR compounds. Among the 2,349 chemical compounds, one JH agonist (JHA) candidate and 17 JH antagonist (JHAN) candidates were screened out and loreclezole hydrochloride and penfluridol were selected based on their high levels of JHA and JHAN activity and insecticidal activity against 3rd instar of A. albopictus larvae, respectively. Loreclezole hydrochloride and penfluridol are well known as anticonvulsant and neuroleptic dug, respectively. However, these compounds showed not only high levels of JH-related IGR activity, but also high levels of insecticidal activity against 3rd instar of A. albopictus larvae compared to those of pyriproxyfen. To find more effective compounds with JH-related IGRs activity for control of mosquitoes, loreclezole hydrochloride and its derivatives were synthesized. Among these derivatives, K21877 demonstrated high level of JHAN activity. Although both loreclezole hydrochloride and K21877 have similar structures, one compound simulated the binding of A. aegypti Met-FISC while the other interfered with the pyriproxyfen-mediated binding of A. aegypti Met-FISC. Both of the JHA and JHAN showed much higher larvicidal activities than that of currently used JHA insecticide, pyriproxyfen. There also showed high embryonic lethality and toxicity against A. albopictus adults, which were due to the modulation of JH-regulated physiological functions such as expression of JH-responsive genes and follicle development. To investigate the transcriptional responses of the A. albopictus treated with loreclezole hydrochloride and K21877, comprehensive transcriptome sequencing was performed and analyzed. These results suggested that JH-interaction of Met could regulate the expression of genes that are related to metabolic process, nucleotide binding process, and translation pathway and loreclezole hydrochloride and K21877 could modulate the JH-regulated gene expression.INTRODUCTION 1 LITERATURE REVIEW 4 1. Juvenile hormone 4 1.1 Historical review 4 1.2 Function 5 2. Insect growth regulators 6 2.1 Juvenile hormone agonists 7 2.2 Juvenile hormone agonists as insect control agents 9 3. Methoprene-tolerant as a JH receptor 10 4. Screening of compounds with JHA and JHAN activity 13 CHAPTER I. Identification of novel juvenile hormone-related insect growth regulators from chemical library 14 ABSTRACT 14 INTRODUCTION 15 MATERIALS AND METHODS 18 1. Insects 18 2. Yeast two-hybrid β-galactosidase assays 18 3. Growth inhibition tests 19 4. Insecticidal activity tests 20 Asian tiger mosquito 20 Diamondback moth 20 Asian corn borer 20 Small brown planthopper 21 Indian mealmoth 21 Fruit fly 21 RESULTS 22 1. Screening of compounds that simulated or interfered with the formation of A. aegypti receptor complex in yeast 22 2. Yeast growth inhibition test 27 3. Screening larvicidal activity against A. albopictus 27 4. Selection of JHA and JHAN compounds from chemical compounds 31 5. Dose-dependent JHA and JHAN activity of loreclezole hydrochloride and penfluridol 32 6. Median lethal concentration (LC50) against 3rd larvae of A. albopictus 32 7. Insecticidal activity of loreclezole hydrochloride and penfluridol against other agricultural pests 36 DISCUSSION 45 CHAPTER Ⅱ. Identification and characterization of loreclezole hydrochloride and its derivatives with juvenile hormone-related insect growth regulator activity against Asian tiger mosquito 48 ABSTRACT 48 INTRODUCTION 50 MATERIALS AND METHODS 52 1. Yeast two-hybrid β-galactosidase assays 52 2. Larvicidal activity tests against Asian tiger mosquito, A. albopictus 52 3. Mosquitocidal activity tests against female adult of A. albopictus 52 4. Embryonic lethality tests 53 5. RNA preparation and quantitative PCR (qPCR) 53 6. Ovary Dissection 54 RESULTS 55 1. Screening of JHA or JHAN from loreclezole hydrochloride derivatives 55 2. Screening larvicidal activity of against A. albopictus 59 3. Median lethal concentrations (LC50) of loreclezole hydrochloride and its derivative against A. albopictus larvae 61 4. Hatching rate of mosquito eggs and mosquitocidal activities against adults treated loreclezole hydrochloride and its derivative 63 5. Expression of the Hairy gene treated loreclezole hydrochloride and its derivative 65 6. Effect of loreclezole hydrochloride and its derivative on the ovary development of female A. albopictus 67 DISCUSSION 70 CHAPTER Ⅲ. The transcriptional responses of Asian tiger mosquito treated with juvenile hormone-related insect growth regulators 75 ABSTRACT 75 INTRODUCTION 77 MATERIALS AND METHODS 79 1. Chemical treatment and RNA extraction 79 2. Illunmina sequencing 79 3. Sequence analysis 80 4. Validation of the sequencing results by qPCR 80 5. Screening of potential target genes for RNAi mediated mosquito control 81 RESULTS 83 1. Overview of the Illumina paired-end sequencing 83 2. Mapping of the short reads to the reference gene set of A. aegypti and analysis of differentially expressed genes 83 3. Gene ontology enrichment analysis 87 4. Analysis of differentially expressed genes related detoxification 96 5. Screening for potential target genes for RNAi mediated mosquito control by qPCR 100 DISCUSSION 103 GENERAL DISCUSSION 114 LITERATURE CITED 118 ABSTRACT IN KOREAN 139Docto

히스테리시스 전동기의 전압원 유한요소 해석

학위논문(석사)--서울대학교 대학원 :전기공학부,2000.Maste

Identification of Plant Compounds that Disrupt the Insect Juvenile Hormone Receptor Complex

학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부, 2015. 7. 제연호.Insects impact human health through vector-borne diseases and cause major economic losses by damaging crops and stored agricultural products. Insect-specific growth regulators represent attractive control agents because of their safety to both humans and the environment. We identified plant compounds that serve as juvenile hormone antagonists (PJHANs). Using the yeast two hybrid system transformed with the mosquito JH receptor as a reporter system, we demonstrate that PJHANs affect the JH receptor, methoprene-tolerant (Met), by disrupting its complex with CYCLE or FISC, formation of which is required for mediating JH activity. We isolated five diterpene secondary metabolites with JH antagonist activity from two plants: Lindera erythrocarpa and Solidago serotina. They are effective at causing mortality of mosquito larvae at relatively low LD50 values. Topical application of two diterpenes caused a reduction in the expression of Met target genes as well as the retardation of follicle development in mosquito ovaries. Hence, the newly discovered PJHANs may lead to the development of a new class of safe and effective pesticides.ABSTRACT I TABLE OF CONTENTS III LIST OF TABLES V LIST OF FIGURES VI INTRODUCTION １ LITERATURE REVIEW ４ 1. Juvenile hormone ４ 2. Methoprene-tolerant as a JH receptor ５ 3. Juvabione and Juvenile hormone mimics from plant ７ 4. Insect growth regulator ７ 1. Juvenile hormone agonists ８ 2. Ecdysone agonists ９ 3. Chitin synthase inhibitor １０ METHODS １２ 1. Yeast two-hybrid binding tests using growth complementation and β-galactosidase assay. １２ 2. Screening of plant extracts. １３ 3. Growth inhibition and anti-yeast activity tests. １４ 4. Extraction, isolation and characterization of plant diterpenes. １４ 5. Larval toxicity tests and LD50 determination. 23 6. In vivo tests of PJHANs. 23 7. RNA preparation and Real-Time RT-PCR. 24 8. Maintenance of mosquitoes and vertebrate approval. 24 RESULTS 25 1. Yeast two-hybrid binding tests using growth complementation and β-galactosidase assays. 25 2. Screening of plant extracts. 27 3. Extraction, isolation and characterization of plant diterpenes 31 4. In vivo tests of PJHANs 35 5. Comparison of antagonist activities of plant diterpenes against JH III and pyriproxyfen. 39 DISCUSSION 41 LITERATURE CITED 44 ABSTRACT IN KOREAN 50 ACKNOWLEDGEMENTS 52Maste