[보고서]꿀벌 병해충 분자 제어 기술 개발

진병래

꿀벌 병해충 분자 제어 기술 개발
Development of molecular control technology of honeybee disease and pests 원문보기

보고서 정보
주관연구기관	동아대학교 Donga University
연구책임자	진병래
참여연구자	이광식 , 김보연 , 박희근 , 등의첩 , 양결 , 하설래 , 박민지 , 제연호 , 김우진 , 이석희 , 김종훈 , 박동환 , 우수동 , 곽원석 , 김동준 , 윤휘건 , 이지훈 , 마지인 , 오종민 , 이우형
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2018-02
과제시작연도	2017
주관부처	농촌진흥청 Rural Development Administration(RDA)
등록번호	TRKO201800043258
과제고유번호	1395049240
사업명	차세대바이오그린21
DB 구축일자	2018-12-15
키워드	꿀벌.병해충.분자제어.벌독.Honeybee.Disease and pests.Molecular control.Bee venom.RNAi.
DOI	https://doi.org/10.23000/TRKO201800043258

초록 ▼

- 토종벌 유래 생리활성 유전자원인 inhibitor cysteine knot, secapin, venom serine protease inhibitor 및 major royal jelly protein 4 등을 확보하고 이 펩타이드들의 꿀벌 질병관련 미생물에 대한 항세균 및 항진균 효과를 확인함
- 꿀벌에 많은 피해를 나타내는 낭충봉아부패병 바이러스(SBV)의 증식을 억제할 수 있는 vp1 유전자를 선발하고 대량생산을 위해 B. thuringiensis 형질전환체를 제작하고 대량생산 조건을 확립하였다. 또한, 봉군에 vp1 dsRNA를 처리하여 낭충봉아부패병 바이러승의 증식 억제 효과를 검정하였다.
- 항노제마 활성을 나타내는 천연물과 dsRNA의 특성을 밝혔으며, 처리조건 및 최적화 기술을 확립하였다.

( 출처: 보고서 요약서 3p )

Abstract ▼

□ Purpose&Contents
- Analysis and usage technology of control effect of honeybee disease and pests using bee venom bioactive substances
1) Identification of genetic resources for bee venom bioactive substances
2) Control effect of bee venom bioactive substances against bee disease and pests
3) Development of utilization technology of bee venom bioactive substances for control of bee disease and pests
4) Screening of anti-viral substances
5) Establishment of mass-production of anti-viral substances
6) Development of treat condition and optimization technology of anti-viral substances
- Development of molecular control technology of honeybee Nosema disease
Bees are a major source of human food resources, and 71% of the world's top 100 crops depend on pollen medicines from bees. Therefore, bees play a very important role in supporting the agricultural ecosystem. In 2006, colony collapse disorder(CCD) of honey bee (Apis mellifera) occurred in the United States. Nosema disease is damaging the damage caused by itself, as well as increasing the susceptibility of bees to other pathogens. Therefore, the prevention and control of Nosema is urgently required worldwide. Currently, only Fumagilin (Fumagilin-B) is used to control Nosema worldwide. However,Fumagilin has a weak control effect against Nosema, it is required to develop a new control agent. Therefore, this study selected effective control substances for Nosema. The effectiveness of the selected control materials was evaluated and an optimal application system was established.

□ Results
- Analysis and usage technology of control effect of honeybee disease and pests using bee venom bioactive substances
1) Molecular cloning of genetic resources of bee-derived bioactive substances - inhibitor cysteine knot, secapin, venom acid phosphatase, venom serine protease inhibitor, major royakl jelly protein 4
2) Inhibitor cysteine knot peptide exhibits anti-fungal effects against entomopathogenic and plant-pathogenic fungi
3) Secapin shows antimicrobial effect against Gram-negative bacteria, Gram-positive bacteria and fungi. Especially, it has excellent antimicrobial effect against P. larvae, one of honeybee disease
4) Major royal jelly protein 4 exhibits antifungal effect against major honeybee pathogens, P. larvae and A. apis
5) dsRNAs dsVP1, dsVP3, and dsRdRp suppress the Sacbrood virus replication in honeybee
6) Construction of B. thuringiensis transformants producing dsVP1 and establishment of mass-production conditions for dsVP1
- Development of molecular control technology of honeybee Nosema disease
In this study, control materials against Nosema were selected using natural materials derived from various entomopathogenic fungi. In addition, Nosema replication related genes were selected and RNAi techniques were applied to them. Based on the above results, dsRNA was selected through inhibition evaluation of Nosema. The mutual synergy between selected natural substances and dsRNA was tested and the effectiveness of the bee suppressant was evaluated by deriving the optimal combination. In addition, the inhibition effect on Nosema was confirmed by treatment with colony and the stability to bees was evaluated.

□ Expected Contribution
- Analysis and usage technology of control effect of honeybee disease and pests using bee venom bioactive substances
1) Development of new management technology using genetic resources to control honeybee disease and pests and this development can contribute to stable productivity improvement of domestic beekeeping industry
2) Beekeeping farmers can use practically useful methods to prevent bee virus and to improve Asian honey species conservation and beekeeping productivity through mass production of anti-viral substances
- Development of molecular control technology of honeybee Nosema disease
For Nosema control, Fumagilin can be substituted by using natural substances and dsRNA which have high control effect.
natural substances and dsRNA can be used to develop new management technology for mite and various bee pests. In economic and industrial terms, effective disease management for bees is possible. Therefore, it is expected to secure competitiveness of the beekeeping industry and increase income.

( 출처: SUMMARY 6p )

목차 Contents

표지 ... 1
제 출 문 ... 2
보고서 요약서 ... 3
국 문 요 약 문 ... 4
Summary ... 6
목차 ... 9
제 1 장 연구 개발 과제의 개요 ... 10
제1절 연구 개발 목적 ... 10
제2절 연구 개발의 필요성 ... 10
제3절 연구 개발 범위 ... 12
제 2 장 연구 수행 내용 및 결과 ... 13
제 3 장 목표달성도 및 관련분야 기여도 ... 143
제1절 : 목표대비 달성도 ... 143
제2절 : 정량적 성과(논문게재, 특허출원, 기타)를 기술 ... 145
제 4 장 연구 결과의 활용 계획 ... 146
제1절 연구 개발 목적 ... 146
제2절 기대성과 ... 146
제 5 장 연구 개발 결과의 보안 등급 ... 148
제 6 장 국가과학기술지식정보서비스에 등록한 연구시설·장비 현황 ... 149
제 7 장 연구개발과제의 대표적 연구실적 ... 150
제 8 장 기타사항 ... 152
제 9 장 참고문헌 ... 154
끝페이지 ... 159

표/그림 (141)

표 지역별 낭충봉아부패병 실태조사 결과 (2010, 농식품부)
표 Oligonucleotide sequences of the primers for dsRNA
표 Oligonucleotide sequences of the primers for qPCR for RNAi pathway genes
표 The primer sequences for cloning of SBV partial genes to synthesize antiviral dsRNAs. The underlined sequences are restriction endonuclease recognition sequences
표 Escherichia coli – Bacillus thuringiensis shuttle vector, pHT1K. Amp r, ampicillin resistant gene; Em r, erythromycin resistant gene; pUC ori, E. coli Replication origin, Bt ori, B. thuringiensis replication origin
표 Phase-contrast microscopy of B. thuringiensis var. israelensis mutant strain 4Q7 (Magnification : X 1,000)
표 Primers used for quantitative PCR of SBV vp1 gene
표 Phase-contrast microscopy of B. thuringiensis var. israelensis mutant strain 4Q7 (Magnification : X 1,000)
표 Hymenopteran ICK amino acid sequences retrived from database searches
표 Transcript expression of the BiICK gene is detected in B. ignitus worker bees. A Northern blot analysis (NB) was performed using the total RNA isolated from the epidermis, fat body, gut, muscle, and venom gland of B. ignitus worker bees. BiICK transcripts are indicated. The ethidium bromide (EtBr)-stained RNA gel shows uniform loading
표 Microbial binding of BiICK occurs in Sf9 insect cells. (A) SDS-PAGE and western blot analysis of purified recombinant BiICK expressed in baculovirus-infected Sf9 insect cells. (B) Western blot analysis of BiICK microbial binding. Live E. coli, B. thuringiensis, P. larvae, A. apis, B. bassiana, or F. graminearum was incubated with recombinant BiICK for 10 min; bound BiICK (P) was separated from free BiICK in the supernatant (S) using centrifugation. The samples were analyzed by western blotting with an anti-His antibody. (C) Immunofluorescence staining of the binding of BiICK to bacterial and fungal cells. Live E. coli, B. thuringiensis, P. larvae, A. apis, B. bassiana, or F. graminearum was incubated with recombinant BiICK for 10 min. BiICK binding (green) to the cell walls of A. apis, B. bassiana, and F. graminearum was visualized using confocal microscopy. The third column is the merged confocal image. Scale bar, 5 mm
표 The antiproliferative activity of BiICK against bacteria and fungi
표 Plasmin inhibition by AcSecapin-1. (A) The expression of recombinant mature AcSecapin-1. Purified, recombinant mature AcSecapin-1 expressed in baculovirus-infected Sf9 insect cells was identified by SDS-PAGE and western blot analysis using anti-His antibodies. (B) AcSecapin-1-mediated human plasmin inhibition assay. Human fibrin was incubated with plasmin or both plasmin and recombinant mature AcSecapin-1 at various time periods (30, 60, 120, or 240 min) and was analyzed using 12% SDS-PAGE. The fibrinogen degradation products (FDPs) are indicated. (C) The inhibitory activity of AcSecapin-1 against human plasmin. Plasmin or both plasmin and recombinant mature AcSecapin-1 was incubated on fibrin plates at 37 °C for 30, 60, 120, 180, or 240 min. (D) Human plasmin-AcSecapin-1 complex formation. Plasmin, recombinant mature AcSecapin-1, or a plasmin-AcSecapin-1 mixture was analyzed by native gel electrophoresis and western blotting using anti-plasmin antibodies or anti-His antibodies. The plasmin, recombinant mature AcSecapin-1, or plasmin-AcSecapin-1 complexes are indicated
표 Inhibitory activity of AcSecapin-1 against elastases. Human or porcine elastase was incubated with recombinant mature AcSecapin-1, and the residual enzyme activity was determined (n = 3)
표 Microbial binding ability of AcSecapin-1. (A) Western blot analysis of AcSecapin-1 microbial binding ability. E. coli, B. thuringiensis, P. larvae, or B. bassiana was incubated with recombinant mature AcSecapin-1 for 10 min. Bound AcSecapin-1 (P) was separated from free AcSecapin-1 in the supernatant (S) using centrifugation. The pellet and supernatant samples were analyzed using western blotting with anti-His antibody. (B) Immunofluorescence staining of the binding of AcSecapin-1 to bacterial and fungal cells. E. coli, B. thuringiensis, P. larvae, or B. bassiana was incubated with recombinant mature AcSecapin-1 for 10 min. Confocal microscopy was performed to visualize AcSecapin-1 binding (green)to the cell walls of E. coli, B. thuringiensis, P. larvae, and B. bassiana. The third column shows merged confocal images. Scale bars, 5 μm or 10 μm
표 The anti-microbial activity of AcSecapin-1 against bacteria and fungi
표 Transcriptional expression of AcSecapin-1 in A. cerana worker bees. (A) Northern blot analysis of AcSecapin-1 expression in A. cerana worker bees. Total RNA was isolated from the epidermis, fat body, gut, muscle, and venom gland of A. cerana worker bees. The AcSecapin-1 transcripts are indicated. (B) Transcriptional expression profiles of the AcSecapin-1 gene in the fat body of A. cerana worker bees injected with E. coli, B. thuringiensis, P. larvae, or B. bassiana (n = 5). PBS-injected A. cerana worker bees were used as controls. Total RNA was isolated from the fat body of worker bees at 1, 3, or 5 h p.i. AcSecapin-1 transcripts were analyzed using northern hybridization. (C) The levels of AcSecapin-1 mRNA were calculated relative to the levels at 0 h before PBS or microbial injection (shown as 100%). The bars represent the means ± SD
표 The alignment of the amino acid sequences for AcVAP and known Hymenoptera venom acid phosphatases. The predicted signal peptide is indicated with a vertical arrow. Amino acids necessary for acid phosphatase activity are boxed. Potential N-glycosylation sites are indicated by crosses. The sources of the aligned sequences are as follows: A. cerana (this study, GenBank accession no. KJ710422), A. mellifera (NM 001013359), A. florea (XM 003696461), Bombus impatiens (XM 003486535), Megachile rotundata (XM 003706060), and Camponotus floridanus (GL444789). The AcVAP sequence was used as the reference for the identity/similarity (Id/Si) values
표 Expression of AcVAP. (A) Expression of AcVAP in A. cerana worker bees. Northern blot analysis was performed with total RNA from the epidermis, fat body, gut, muscle, and venom gland of A. cerana worker bees. The AcVAP transcripts are indicated with an arrow. (B) The expression of recombinant AcVAP. Purified recombinant AcVAP, expressed in baculovirus-infected insect cells, was identified by 10% SDS-PAGE and Western blot analysis using anti-AcVAP antibodies. (C) Detection of AcVAP in the venom gland and the secreted venom of A. cerana worker bees. Protein samples prepared from tissues (epidermis, fat body, gut, muscle, and venom gland) or venom of A. cerana worker bees were analyzed by 10% SDS-PAGE and Western blot analysis using anti-AcVAP antibodies. The AcVAP proteins are indicated with an arrow. (D) Tunicamycin treatment of baculovirus-infected insect cells. Sf9 cells were infected with recombinant AcNPV (AcNPV-AcVAP) expressing AcVAP and cultured without (-) or with (+) tunicamycin (5 μg/mL). At 2 days p.i., total cellular lysates were subjected to 10% SDS-PAGE (left) and Western blot analysis (right) using anti-AcVAP antibodies. The AcVAP proteins are indicated with an arrow
표 Enzymatic properties of recombinant AcVAP. (A) Optimum temperature of recombinant AcVAP. The optimum temperature for AcVAP activity was determined in sodium acetate/acetic acid buffer (0.1 M, pH 4.8) containing 5 mM p-NPP at a temperature range from 25 °C to 80 °C (n = 3). (B) Optimal pH of recombinant AcVAP. The optimal pH for AcVAP activity was determined in sodium acetate/acetic acid buffer (pH 3.6-5.6) and phosphate buffer (pH 5.8-7.2) for 1 h at 45 °C (n = 3)
표 Cloning and recombinant protein expression of AcCBP . (A) Alignment of the predicted amino acid sequences for AcCBP and known bee CBPs. The predicted signal peptide and the conserved peritrophin-A domain are indicated. The six conserved cysteine residues are indicated by circles. The AcCBP sequence was used as a reference for the identity/similarity (Id/Si) values. (B) The expression of recombinant AcCBP. Purified recombinant AcCBP, expressed in baculovirus-infected Sf9 insect cells, was identified by SDS-PAGE and western blot analysis using anti-His antibodies
표 Expression profile of AcCBP in A. cerana worker bees. (A) Expression of AcCBP in A. cerana worker bees. Northern blot analysis was performed with total RNA from the epidermis, fat body, gut (foregut, midgut, and hindgut), muscle, and venom gland of A. cerana worker bees. The AcCBP transcripts are indicated by arrows. (B) Detection of AcCBP in the midgut of A. cerana worker bees. Protein samples prepared from foregut, midgut, and hindgut of A. cerana worker bees were analyzed using 12% SDS-PAGE and western blot analysis using anti-AcCBP antibodies. The AcCBP proteins are indicated with an arrow
표 Localization of AcCBP in A. cerana worker bees. Immunofluorescence staining of the gut sections of A. cerana worker bees was performed by incubation with anti-AcCBP antibodies for 10 min. Confocal microscopy was performed to visualize AcCBP (green) in the PM of A. cerana worker bees. The third column shows merged confocal images. Scale bars, 10 μm
$Chitin-binding ability of recombinant AcCBP. Recombinant AcCBP was incubated with chitin beads, followed by washing and elution. Protein samples from each fraction were analyzed using 12% SDS-PAGE and western blot analysis using anti-AcCBP antibodies. The AcCBP proteins are indicated$ 표 Chitin-binding ability of recombinant AcCBP. Recombinant AcCBP was incubated with chitin beads, followed by washing and elution. Protein samples from each fraction were analyzed using 12% SDS-PAGE and western blot analysis using anti-AcCBP antibodies. The AcCBP proteins are indicated
표 Fungus-binding ability of recombinant AcCBP. (A) Western blot analysis of microbe-binding ability of recombinant AcCBP. Recombinant AcCBP was incubated with E. coli , B. thuringiensis, or B. bassiana for 10 min. Bound AcCBP (Pellet) was separated from free AcCBP in the supernatant via centrifugation. The pellet and supernatant samples were analyzed using western blotting with anti-His antibody. (B) Immunofluorescence staining of the binding of AcCBP to fungal cells but not bacteria. Recombinant AcCBP was incubated with E. coli, B. thuringiensis, or B. bassiana for 10 min. Confocal microscopy was performed to visualize AcCBP binding (green) to the cell walls of E. coli, B. thuringiensis, and B. bassiana. The third column shows merged confocal images. Scale bars, 10 μm
표 The peptide sequences of the synthetic AcSecapin-1 peptides. The two cysteine residues are indicated
표 Cytotoxicity and cell viability in response to AcSecapin-S1. (A) Cytotoxicity of AcSecapin-S1. A cytotoxicity assay was performed using NIH 3T3 cells with AcSecapin-S1 (0.1, 1, 10, 100, 250, or 500 μg per ml of medium) or Triton X-100 (positive control). Bars represent the mean ± SD (n = 3). (B) Cell viability after treatment with AcSecapin-S1. The MTT assay was performed using NIH 3T3 cells with AcSecapin-S1 (0.1, 1, 10, 100, 250, or 500 μg per ml of medium) or Triton X-100 (positive control). The percentage of MTT reduction was determined at 24 and 48 h after AcSecapin-S1 treatment. Bars represent the mean ± SD (n = 3)
표 Caspase-3 activity after treatment with AcSecapin-S1. NIH 3T3 cells were treated with AcSecapin-S1 (10, 100, 250, or 500 μg per ml of medium), and caspase-3 activity was determined at 24 h after AcSecapin-S1 treatment. Bars represent the mean ± SD (n = 3)
표 Release of proinflammatory mediators and cytokines after treatment with AcSecapin-S1. Medium samples from cultures of J774 cells at 5 h after treatment with either AcSecapin-S1 (250 μg per ml of medium) or LPS (100 ng per ml of medium) were evaluated using western blot analysis with an anti-COX-2 antibody (A), anti-IL-1β antibody (B), anti-IL-6 antibody (C), and anti-TNF-α antibody (D). The negative control was untreated J774 cells (control)
표 Thermal stability of AcSecapin-S1. The anti-bacterial activity of AcSecapin-S1 against P. aeruginosa was determined after heating the peptide to various temperatures (25-90 °C) for 3 h. Bars represent the mean ± SD (n = 3)
표 AcVSPI is a low-molecular-weight serine protease inhibitor Api m 6-like peptide. (A) Alignment of the amino acid sequences of AcVSPI, A. mellifera Api m 6, and the A. dorsata Api m 6-like peptide. The predicted signal sequence is underlined in yellow. The conserved cysteine residues are indicated by solid circles. The P1 position is marked with an asterisk. The TIL domain is indicated by a red arrow. The sources of the aligned sequences were A. cerana AcVSPI (this study, GenBank accession no. MF281990), A. mellifera Api m 6 (NP 001035360), and A. dorsata Api m 6-like peptide (XP 006610831). The AcVSPI sequence was used as a reference for the identity/similarity (Id/Si) values. (B) Organization of the AcVSPI gene (GenBank accession no. MF281991). Numbers indicate the position in the genomic sequence. Exons are represented by solid boxes. (C) Expression of AcVSPI in A. cerana worker bees. Total RNA was isolated from the epidermis, fat body, midgut,muscle, and venom gland of A. cerana worker bees. RNA was separated by 1.2% formaldehyde agarose gel electrophoresis, transferred onto a nylon membrane, and hybridized with radiolabeled AcVSPI cDNA (lower panel). The ethidium bromide-stained RNA gel shows uniform loading (upper panel). AcVSPI transcripts are indicated with an arrow
표 AcVSPI functions as an anti-fibrinolytic factor. (A) AcVSPI-mediated human plasmin inhibition assay. The mixture of human fibrin and plasmin was incubated with or without recombinant AcVSPI for 30, 60, 120, or 240 min. AcVSPI-mediated human plasmin inhibition was analyzed using 12% SDS-PAGE. FDPs are indicated. (B) The fibrin plate assay of the inhibitory activity of AcVSPI against human plasmin. Plasmin or the mixture of plasmin and recombinant AcVSPI was incubated on fibrin plates at 37 °C for various periods of time (30, 60, 120, 180, or 240 min)
표 Microbial binding ability of recombinant AcVSPI. (A) Western blot analysis of the binding of AcVSPI to bacterial and fungal cells. E. coli, B. thuringiensis, or B. bassiana was incubated with recombinant AcVSPI for 10 min. Bound AcVSPI (P) of the pellet was separated from free AcVSPI in the supernatant (S) using centrifugation, and the samples were analyzed using Western blotting with anti-Strep-tag II antibody. (B) Immunofluorescence staining of the binding of AcVSPI to bacterial and fungal cells. E. coli, B. thuringiensis, or B. bassiana was incubated with recombinant AcVSPI for 10 min. Confocal microscopy was performed to visualize AcVSPI binding (green) to the cell walls of E. coli, B. thuringiensis, and B. bassiana. A. cerana ICK peptide (Park et al., 2014) was used as a negative control (AcICK). The third column shows merged confocal images. Scale bar, 5 μm
표 SEM images of AcVSPI-induced structural damage to bacterial and fungal cell walls. E. coli, B. thuringiensis, or B. bassiana was treated with (right) or without (left) recombinant AcVSPI. Scale bar, 1 μm
표 The anti-microbial activity of recombinant AcVSPI against bacteria and fungus
표 Alignment of the amino acid sequences of AcMRJP4, AmMRJP4, and the A. florea MRJP 4. The predicted signal sequence is indicated by an arrow. The conserved cysteine residues are indicated by solid circles. The potential N-linked glycosylation sites are marked by asterisks. The AcMRJP4-15 region in the C-terminus of AcMRJP4 is highlighted in yellow. The sources of the aligned sequences were AcMRJP4 (this study, GenBank accession no. MF402924), AmMRJP4 (ADB82660), and A. florea MRJP 4 (XP003695157). The AcMRJP4 sequence was used as a reference to determine the identity/similarity (Id/Si)
표 N-glycosylation of recombinant AcMRJP4 in baculovirus-infected insect cells. Sf9 cells were infected with recombinant AcNPV and cultured without (−, lane 1) or with (+, lane 2) tunicamycin (5 μg/ml). Total cellular lysates were collected 3 days post-infection and then were analyzed using 12% SDS-PAGE (left) and western blotting with the anti-AcMRJP4 antibody (right). AcMRJP4 and its fragments (i.e., AcMRJP4-48 and AcMRJP4-15) are indicated by solid arrows. The non-glycosylated AcMRJP4 and AcMRJP4-48 are indicated by open arrows
표 Proteolytic cleavage of recombinant AcMRJP4. Purified recombinant AcMRJP4 (lane 1) was incubated with mock-infected insect Sf9 cell extracts (lane 2) or wild-type AcNPV-infected insect cell extracts (lane 3) at 27 °C for 3 h. Protein samples were analyzed using 12% SDS-PAGE (left) and western blotting with the anti-AcMRJP4 antibody (right). AcMRJP4 and its fragments (i.e., AcMRJP4-48 and AcMRJP4-15) are indicated
표 Detection of AcMRJP4 from A. cerana RJ. A. cerana RJ (A) and A. mellifera RJ (B) samples were analyzed using 12% SDS-PAGE (left) and western blotting with the anti-AcMRJP4 antibody (right). AcMRJP4 and AmMRJP4 are indicated
표 Microbial binding ability of recombinant AcMRJP4 and AcMRJP4-15. (A, B) Western blot analysis of the binding of AcMRJP4 (A) and AcMRJP4-15 (B) to microbial cell walls. P. larvae, P. aeruginosa, A. apis, and C. albicans were incubated with recombinant AcMRJP4 and AcMRJP4-15 for 10 min. Bound AcMRJP4 and AcMRJP4-15 of the pellet (P) were separated from free AcMRJP4 and AcMRJP4-15 in the supernatant (S) using centrifugation, and the samples were analyzed using western blotting with the anti-His antibody. (C, D) Immunofluorescence staining of the binding of AcMRJP4 (C) and AcMRJP4-15 (D) to microbial cell walls. P. larvae, P. aeruginosa, A. apis, and C. albicans were incubated with recombinant AcMRJP4 and AcMRJP4-15 for 10 min. Confocal microscopy was performed to visualize the binding (green) of AcMRJP4 and AcMRJP4-15 to the microbial cell walls. A. cerana ICK peptide was used as a negative control (AcICK).30 The third column shows merged confocal images. Scale bar, 5 μm
표 SEM images of AcMRJP4-15-induced structural damage in the microbial cell walls. P. larvae, P. aeruginosa, A. apis, and C. albicans were incubated with (right) or without (left) recombinant AcMRJP4-15. Scale bar, 1 mm
표 The relative transcription levels of five genes, Sacbrood virus (SBV), Defensin (Def), Peptidoglycan binding protein (PGBP), Prophenoloxidase (PPO) and Juvenile hormone esterase (JHE) were analyzed by qPCR. The relative transcription levels were demonstrated in fold differences of AcCtrl to AcSBV calculated by 2-ΔΔCT
표 Gene expression pattern of RNAi pathway related genes. Total of six worker bees were collected from SBV infexted hive for qPCR. Y-axis stands for the delta-Ct value bwtween the actin referece gene and target genes. The relative differential transcription levels can be calculated by Log2(delta-Ct). Lower number means less transcription
표 Gene expression pattern of RNAi pathway realted genes of newly infected workers from healthy hive. Total of 4~5 worker bees were pooled for total RNA extraction. Y-axis stands for the delta-Ct value between the actin reference gene and target genes. The relative differential transcription levels can be calculated by Log2(delta-Ct). Lower number means less transcription. ND=Not detected
표 Synthesis of SBV vp1, RdRp, and vp3 dsRNAs by using in vitro transcription system. The molecular sizes of each dsRNA are 595, 457, and 526 bases
표 Stability of dsRNA in honey. Total of 1 ug GFP dsRNA was mixed with 50%, 10%, 1%, and 0.1% of honey in 20 uL of reaction mixture. After 1, 6, and 24 hours of incubation at 30c, 250 ng of dsRNA was examined on 0.8% argarose gel with SYBR green
표 Stability of dsRNA in heat inactivated honey. Total of 1 ug dsRNA was mixed with honey heat inactivated at 95c for 1, and 10 min, and examined on 0.8% agarose gel with SYBR green
표 The relative virus replication levels of dsRNA treated workers. Each sample groups were infected with SBV (109 virus/mL) for 12 hours, and treated with dsRNA for 48 hours followed by total RNA extraction of pooled samples. Y-axis stands for the related transcription level between the actin reference gene and SBV gene. The relative differential transcription levels can be calculated by Ln(delta-Ct). Lower number means less transcription. (N.C; Negative control: feeding 40% sucrose solution for 60 hours, P.C; Positive control: feeding SBV (109 virus/mL) for 12hours, and 40% sucrose solution for 48hours.)
표 The relative virus replication levels of dsRNA treated workers. Each sample groups were infected with SBV (109 virus/mL) for 12 hours, and treated with dsRNA for 48 hours followed by total RNA extraction of pooled samples. Y-axis stands for the natural logarithm related transcription level between the actin reference gene and SBV gene. The relative differential transcription levels can be calculated by Ln(delta-Ct). Lower number means less transcription
표 Nucleotide sequence of Sacbrood virus vp1 gene
표 Construction map of shuttle vector, pHT1K-SBV vp1 cassette, synthesizing SBV vp1 segment dsRNA
표 Construction of shuttle vector expressing SBV vp1 dsRNA
표 Construction analysis of shuttle vectors expressing SBV vp1 dsRNA
표 Construction map of shuttle vector, pHT1K-SBV vp1 cassette, synthesizing SBV vp1 segment dsRNA
표 Construction of shuttle vector expressing EGFP dsRNA
표 Construction analysis of shuttle vectors expressing EGFP dsRNA
표 Construction map of shuttle vector, improved pHT1K-SBV vp1 cassette, synthesizing SBV vp1 segment dsRNA
표 Construction of shuttle vector expressing SBV vp1 dsRNA
표 Construction analysis of shuttle vectors expressing SBV vp1 dsRNA
표 Colony PCR amplification of 4Q7 transformants
표 Verification of 4Q7 transformant’s SBV vp1 fragment
표 Total RNA of 4Q7 transformants
표 Verification of transcription of SBV vp1 gene from 4Q7 transformants
표 Expression profiles of SBV vp1 gene from 4Q7 transformants
표 pHT1K-EGFP vector transformed Bacillus thuringiensis RNA sequencing data
표 Confirmation of self-autolyis condition of 4Q7 transformant
표 The relative virus replication levels of total RNA treated workers. Each sample groups were infected with SBV (109 virus/mL) for 12 hours, and treated with total RNA (1.5ug/ul) from transformed Bacillus thuringiensis for 48 hours followed by total RNA extraction of pooled samples. Y-axis stands for the natural logarithm related transcription level between the actin reference gene and SBV gene. The relative differential transcription levels can be calculated by Ln(delta-Ct). Lower number means less transcription. (N.C; Negative control: feeding 40% sucrose solution for 60 hours, P.C; Positive control: feeding SBV (109 virus/mL) for 12hours, and 40% sucrose solution for 48hours.)
표 The concentration dependent relative virus replication levels of total RNA treated workers. Each sample groups were infected with SBV (109 virus/mL) for 12 hours, and treated with 0.1, 0.5, 1.0, and 1.5 ug/ul of dsRNA containing total RNA from pHT1K-SBV vp1 transformed Bacillus thuringiensis for 48 hours followed by total RNA extraction of pooled samples. Y-axis stands for the natural logarithm related transcription level between the actin reference gene and SBV gene. The relative differential transcription levels can be calculated by Ln(delta-Ct). Lower number means less transcription. (N.C; Negative control: feeding 40% sucrose solution for 60 hours, P.C; Positive control: feeding SBV (109 virus/mL) for 12hours, and 40% sucrose solution for 48hours.)
표 pHT1K-SBV vp1 vector transformed Bacillus thuringiensis total RNA safety test to worker bees. (N.C; Negative control: feeding 40% sucrose solution only.) The mortality of the worker bees fed with various concentration of dsRNA containing total RNA solutions was recorded for ten days
표 N. apis와 N. ceranae에 대한 특이적 Primer
표 후보 유전자 확보를 위한 primer 서열
표 목적 유전자에 대한 2가지 T7 프로모터 합성 유전자
표 유전자 발현 억제 물질 제작을 위한 primer 서열
표 유전자 발현 수준을 확인하기 위한 Primer 서열
표 dsRNA-chitosan particle 제작모식도
표 최종 선발된 균주 정보
표 노제마병 사충으로부터 순수분리한 노제마의 위상차현미경 사진(X400). Scale bar, 10 um
표 분리 Nosema의 동정을 위한 multiplex PCR primers를 이용한 PCR 산물 전기영동 사진. (A) M, 100 bp ladder; lane 1, N. apis, N. bombi, N. ceranae 검출 가능한 multiplex PCR primers를 이용한 PCR 산물; (B) M, 100 bp ladder; lane 1, N. ceranae 검출 primers; lane 2, N. bombi 검출 primers
표 꿀벌 봉군의 유지 모습 (A); 꿀벌 생체를 이용하여 N. ceranae를 계대 배양하는 모습 (B)
표 꿀벌 중장으로부터 분리한 노제마 포자 A, 3 mm bead를 이용한 포자분리; B, 2 mm bead를 이용한 포자 분리; C, 2 mm bead와 효소액을 이용한 포자 분리 Scale bar = 20 um
표 변형된 방법으로 포자를 분리하였을 때의 수율. A, 3 mm bead를 이용한 포자분리; B, 2 mm bead를 이용한 포자 분리; C, 2 mm bead와 효소액을 이용한 포자분리
표 효소 처리시간에 따른 포자 발아율
표 효소 처리시간에 따른 꿀벌 중장으로부터의 분리된 포자 수
표 Filter paper 적용 전,후의 노제마 포자. A, Filter paper 적용 전 노제마 포자 정제; B, Filter paper 적용 후 노제마 포자
표 현탁액 부피에 따른 포자 정제 수율
표 13. 각 처리 과정에서의 포자 수율. A, 중장으로부터 분리 된 포자 수율; B, 정제 후 포자 수율; C, 300 × g 원심분리를 이용한 정제 포자 수율; D, 400 × g 원심분리를 이용한 정제 포자 수율; E, 꿀벌 중장으로부터 최종 정제된 포자 수율
표 꿀벌 중장으로부터 최종 정제 된 노제마 포자
표 항노제마 후보물질 선발을 위한 곰팡이 배양액의 효율적인 생산 및 수거 방법 모식도
표 인위적인 발아 조건에서 의해 polar tube를 형성한 N. ceranae . scale bar: 10 um
표 Polar tube germination asaay를 이용한 항노제마 물질 스크리닝 결과. (A), B. bassiana 243 균주의 항노제마 물질에 의해 발아가 억제된 N. ceranea; (B), Tolypocladium sp. 237 균주의 항노제마 물질에 의해 발아가 억제된 N. ceranea; (C), 무처리구
표 천연 추출물을 이용한 항노제마 활성 비교 (A), High activity - 80% 이상 발아 억제; (B), Middle activity - 60% 이상 발아 억제; (C) Low activity - 60% 이하 발아 억제
표 항노제마 저해율별 곤충병원성 곰팡이의 빈도
표 높은 항노제마 활성을 보인 추출물 균주 비율
표 N . ceranae에 대한 곤충병원성 곰팡이 89개 균주 추출물의 농도별 발아율
표 노제마 유전자 발현 억제 후보 유전자 선별
표 대조군 및 후보 유전자 PCR 산물
표 FNR1과 FNR2를 가지는 재조합 배큘로바이러스 제작을 위한 전이벡터 제작 모식도
표 Bac to Bac system(invitrogen, USA)을 이용한 FNR1과 FNR2를 가지는 재조합 배큘로바이러스 제작모식도
표 Bac to Bac system(invitrogen, USA)을 이용하여 제작한 재조합 배큘로바이러스의 재조합 여부 검정을 위한 PCR 수행 결과
표 재조합 배큘로바이러스와 Sf21 세포를 이용한 FNR1 및 FNR2 발현 여부 검정을 위한 SDS-PAGE(좌), His-tag antibody를 이용한 Western blot 분석(우)
표 Cytochrome P450 환원효소와 Nosema ceranae의 FNR1(A) 및 FNR2(B)의 보존(Conserved domain)영역 분석 결과
표 FNR1 또는 FNR2를 발현하는 재조합 배큘로바이러스에 감염된 sf21세포의 Cytochrome C oxidase 활성 검정
표 bifenthrin (0-100ppm concentrations)처리된 FNR1 또는 FNR2 또는 GFP(Negative control)를 발현하는 sf21세포의 생존률 검정
표 선발되어진 44개 균주
표 10kDa 이하의 배양액 추출물을 이용한 항노제마 활성 (A), 높은 발아 억제를 보인 추출물; (B), 낮은 발아 억제를 보인 추출물
표 선발된 44균주의 노제마 억제 활성 기작 평가
표 미토솜 관련 유전자들의 dsRNA 제작 모식
표 미토솜 관련 유전자들의 dsRNA 전기영동 수행 결과
표 dsRNA의 처리에 따른 포자 증식율
표 dsRNA의 처리에 따른 꿀벌의 생존율
표 실시간 정량 PCR을 이용한 dsRNA의 처리에 따른 Nosema ceranae내 TOM40 (A), FNR1 (B), FNR2 (C), NAR1 (D)의 유전자 발현 억제 검정
표 선발된 유전자의 2가지 dsRNA조합에 따른 포자증식율
표 선발된 유전자의 3가지 dsRNA조합에 따른 포자증식율
표 선발된 유전자의 4가지 dsRNA조합에 따른 포자증식율
표 선발된 유전자의 2가지 dsRNA조합에 따른 꿀벌 생존률
표 선발된 유전자의 3가지 dsRNA조합에 따른 꿀벌 생존률
표 선발된 유전자의 4가지 dsRNA조합에 따른 꿀벌 생존률
표 1%의 추출물에 대한 꿀벌 안전성
표 5% 추출물에 대한 꿀벌 안전성
표 10% 추출물에 대한 꿀벌 안전성
표 배양액 추출물의 벌통 내부조건 안정성
표 5% 추출물에 대한 꿀벌 발병억제 평가 전처리
표 5% 추출물 전처리 포자생산억제 활성
표 1% 추출물에 대한 꿀벌 발병억제 평가 전처리
표 1% 추출물 전처리 포자생산억제 활성
표 5% 추출물에 대한 꿀벌 발병억제 평가 후처리
표 5% 추출물 후처리 포자생산억제 활성
표 1% 추출물에 대한 꿀벌 발병억제 평가 후처리
표 1% 추출물 후처리 포자생산억제 활성
표 P ochonia sp. 60 배양액 추출물에 대한 꿀벌안전성, 발병억제 평가
표 P ochonia sp. 60 배양액 추출물 처리 시기에 따른 꿀벌 생존률
표 P ochonia sp. 60 배양액 추출물 처리 시기에 따른 포자 생산 억제 활성
표 P . marquandii 364 배양액 추출물에 대한 꿀벌안전성, 발병억제 평가
표 P . marquandii 364 배양액 추출물 처리 시기에 따른 꿀벌 생존률
표 P . marquandii 364 배양액 추출물 처리 시기에 따른 포자 생산 억제 활성
표 dsRNA-키토산 나노복합체의 처리에 따른 포자 증식율
표 dsRNA-키토산 나노복합체 처리에 따른 꿀벌 생존률
표 dsRNA-키토산 나노복합체의 제작
표 dsRNA-키토산 나노복합체 섭식을 통한 Nosema ceranae의 포자 증식률
표 dsRNA-키토산 나노복합체 섭식을 통한 Nosema ceranae 감염 꿀벌의 생존률 증대 효과 검정
표 노제마 억제 천연물과 dsRNA의 시너지 효과 검정

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꿀벌 병해충 분자 제어 기술 개발
Development of molecular control technology of honeybee disease and pests 원문보기