[보고서]자생식물에서 천연 항생 펩타이드 개발 및 설계

박윤경

자생식물에서 천연 항생 펩타이드 개발 및 설계
Development of Natural Antibiotic Peptide in Various Natural Plants 원문보기

보고서 정보
주관연구기관	조선대학교 산학협력단
연구책임자	박윤경
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2011-08
과제시작연도	2010
주관부처	교육과학기술부
사업 관리 기관	한국연구재단
등록번호	TRKO201300012550
과제고유번호	1345127239
사업명	중견연구자지원
DB 구축일자	2013-08-26
키워드	자생식물체.천연항균제.다약물 내성균.펩타이드 공학 기술.세포독성.장내 유익균.작용 메카니즘.natural plants antimicrobial peptides.pathogenic strains.Cytotoxic study.human normal cell line.probiotics.food-borne microorganisms.bacterium acnes.therapeutic antibiotics.

초록 ▼

연구의 목적 및 내용
- 각종 자생식물체로부터 병원성 미생물에 대한 항생 작용 펩타이드의 탐색 및 분리
- 탐색 및 분리된 1차 활성이 확인된 자생식물체로부터 항생활성 펩타이드 분리
- 각종 자생식물체에서 유래한 천연형 항균 단백질(펩타이드)을 인체 병원성 미생물에서 작용 메카니즘 탐색
- 천연형 항생제의 단백질(펩타이드) 아미노산 서열 분석
- 식중독 유발 세균 및 여드름 유발 미생물에 대해 항생제 기능성 검증
- 천연 항생펩타이드의 사람의 정상세포 및 장내 유익균에 대한 세포 독성 검사

연구결과
- 자생 식물체 30여종을 이용하여 단백질을 각각 분리 및 정제하여 유해균인 E. coliO17, S. aureus 그리고 곰팡이 C. albicans를 이용하여 항균 및 항진균 실험을 수행하였고, 호박에서 우수한 항균 활성을 나타냄.
- 국내 자생식물에서 항생활성을 가진 heat-stable protein을 분리하였다. 분리한 단백질(펩타이드)를 이용하여 각 종 실험 균주들에 대해 Antifungal activty, Hemolytic effect, Confocal microscopic analysis를 수행하였다.국내 자생식물의 단백질(펩타이드)의 N-teminal 아미노산 서열을 분석하였고, 이를 NCBI database에서 비교 분석함.
- 항생 펩타이드의 항생제 기능을 유발하는 작용 메카니즘을 탐색하기 위해 Confocal laser scanning microscopy, FACScan analysis, CD analysis을 통해 작용기작을 연구하였다. 항생 펩타이드의 NMR 분석 통해 2차 및 3차 구조를 분석 완료 함.
- 분리한 펩타이드의 항균 활성 측정을 위해 식품 유해균인 E.coliO157 및 S. aureus를 사용하여 항균 활성 및 여드름 유발 곰팡이에 대한 항진균 활성을 측정함.
- 분리한 항생펩타이드가 정상의 사람 세포에 대해서 세포독성을 갖는지 여부를 확인하기 위해 LDH test와 장내 유익균인 Lactobacillus에 대해 독성이 나타나는지 여부를 확인함.

연구결과의 활용계획
- 펩타이드 신약 관련 연구의 활성화 및 펩타이드 생산관련 소재의 국가 경쟁력 확보
- 펩타이드 메디컬 소재 개발을 통한 다양한 질병의 치료 및 예방책 개발에 활용
- 펩타이드 친화적인 펩타이드 대량 합성공정기술을 확보
- 다국적 제약회사에 치료제 원천기술을 판매함으로써 국가 경제발전에 활용

Abstract ▼

Purpose&contents
- Screening and the purification of antimicrobial peptides from various natural plants against pathogenic strains
- Study of the mechanism of natural antimicrobial proteins (peptides) derived from various natural plant in human pathogenic microorganisms
- Development of pur

Purpose&contents
- Screening and the purification of antimicrobial peptides from various natural plants against pathogenic strains
- Study of the mechanism of natural antimicrobial proteins (peptides) derived from various natural plant in human pathogenic microorganisms
- Development of purified antimicrobial peptides using peptidomimic techniques

Result
- Screening and purification of antimicrobial peptides from various natural plants against the pathogenic strains
- Screening of antimicrobial activity with extracted proteins
- Purification of antimicrobial peptide will be screened in first year from natural plants
- Mechanism of natural antimicrobial protein (peptides) derived from natural plant in human pathogenic microorganisms
- Amino acid sequence will be analyzed from natural antimicrobial protein(peptide)
- Study of the mechanism of antimicrobial peptide
- Cytotoxic study of antimicrobial peptides in human normal cell lines and probiotics
- Antimicrobial assay will be determined against food-borne microorganisms and propioni bacterium acnes

Expected Contribution
The peptides possessing higher antimicrobial activity without cellular toxicity.
These peptides could be applied in elucidating the details of target cell specificity. We will apply to the development of an antimicrobial peptides as a therapeutic antibiotics.

목차 Contents

중견연구자지원사업(핵심연구) 최종보고서 ... 1
목차 ... 2
연구계획 요약문 ... 3
연구결과 요약문 ... 4
한글요약문 ... 4
SUMMARY ... 5
연구내용 및 결과 ... 6
1. 연구개발과제의 개요 ... 6
2. 국내외 기술개발 현황 ... 10
3. 연구수행 내용 및 결과 ... 13
4. 목표달성도 및 관련분야에의 기여도 ... 48
5. 연구결과의 활용계획 ... 49
6. 연구과정에서 수집한 해외과학기술정보 ... 50
7. 주관연구책임자(공동연구원) 대표적 연구실적 ... 51
8. 참고문헌 ... 51
9. 연구성과 ... 52
10. 기타사항 ... 55
[별첨1] ... 56
대표연구성과 1 ... 56
대표연구성과 2 ... 57
대표연구성과 3 ... 58
대표연구성과 4 ... 59
대표연구성과 5 ... 60

표/그림 (39)

표 Summary for purification of Pr-1 from powders of pumpkin rind

표 Antifungal activity of Pr-1 toward fungal cells

표 Hemolytic effect of the antifungal protein Pr-1

$Purification and antifungal activity of a novel antifungal protein. (A) The unadsorbed fraction with antifungal activity on ion-exchange chromatography was subjected to reverse-phase HPLC on a C18 column.The arrow indicates a protein with antifungal activity. (B)$ 표 Purification and antifungal activity of a novel antifungal protein. (A) The unadsorbed fraction with antifungal activity on ion-exchange chromatography was subjected to reverse-phase HPLC on a C18 column.The arrow indicates a protein with antifungal activity. (B)

표 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Pr-1 was analyzed by SDS-PAGE on a 15% separating gel with Coomassie brilliant blue G-250 stain. Lane M, molecular mass marker proteins obtained from Sigma. Lane 2, proteins extracted from pumpkin rind. Lane 3, heat-stable proteins after heat-treatment, Lane 4, the purified protein from RP-HPLC.

표 Inhibitory activity of purified Pr-1 for fungal growth. Purified Pr-1 was subjected to radial growth inhibition tests with Fusariummoniliformevar. subglutinans (A), B. cinerea (B) and F.solani (C).

표 Localization of rhodamine-labeled Pr-1 (A and B) and SYTOX Green uptake (C and D) on fungal cells. C. albicans cells (A) or F. solani hyphae (B) with 20μM rhodamine-labeled Pr-1 was observed on confocal laser scanning microscopy. The influx of SYTOX Green was visualized using fluorescence microscopy in the presence (C) or absence (D) of Pr-2. Panel 1 shows the bright field image, while panel 2 is a fluorescence image of the same cells shown in the bright field micrograph. Panel 3 shows the merged images.

표 Summary of the purification of Pr-2 from pumpkin rind powders

표 Inhibitory effects of the antifungal protein, Pr-2, against fungal and bacterial cells

표 Proteolytic digestion of crude extracts with trypsin. A, Crude extracts were digested with 0.25 mg/ml trypsin at 37 °C for 4hr, and then the sample was subjected to SDS-PAGE. Lane 1: without trypsin and lane 2: with trypsin. B,digested crude extracts were assayed toward Candida albicans. 1: Control (no extracts and trypsin), 2: only extracts (no trypsin), 3: digested extracts (with trypsin), 4: trypsin only (no extracts).

$Chromatographical purification of antifungal proteins (Pr-2). A and B, Pr-2, antifungal protein, was purified using cation exchange chromatography on CM-Sepharose (A), reverse-phase HPLC on a C18 column (B). Extraction of the soluble proteins and chromatographic procedures are described in the Materials & Methods section. (I): Unadsorbed fraction, (II-IV): fraction eluted with 0.1 M (II), 0.3 M (III) and 1 M (IV) NaCl. Fraction (II) containing antifungal activity was collected and purified on a C18 column (B).$ 표 Chromatographical purification of antifungal proteins (Pr-2). A and B, Pr-2, antifungal protein, was purified using cation exchange chromatography on CM-Sepharose (A), reverse-phase HPLC on a C18 column (B). Extraction of the soluble proteins and chromatographic procedures are described in the Materials & Methods section. (I): Unadsorbed fraction, (II-IV): fraction eluted with 0.1 M (II), 0.3 M (III) and 1 M (IV) NaCl. Fraction (II) containing antifungal activity was collected and purified on a C18 column (B).

$Tricine-SDS-PAGE, molecular massand growth inhibition activity of purified Pr-2. A, Pr-2 was analyzed by Tricine-SDS-PAGE on a 16.5% separating gel with Coomassie brilliant blue G-250 staining. Lane M: Molecular mass marker proteins from Sigma, Lane 2: the protein purified by RP-HPLC. B, The molecular mass of the purified Pr-2 was examined using MALDI-TOF/MS. C, After collection of the major peak fraction from HPLC, antifungal activities were assayed against F. solani without (1) or with Pr-2 protein (2).$ 표 Tricine-SDS-PAGE, molecular massand growth inhibition activity of purified Pr-2. A, Pr-2 was analyzed by Tricine-SDS-PAGE on a 16.5% separating gel with Coomassie brilliant blue G-250 staining. Lane M: Molecular mass marker proteins from Sigma, Lane 2: the protein purified by RP-HPLC. B, The molecular mass of the purified Pr-2 was examined using MALDI-TOF/MS. C, After collection of the major peak fraction from HPLC, antifungal activities were assayed against F. solani without (1) or with Pr-2 protein (2).

표 Inhibitory activity of purified Pr-2 for fungal growth (A) and human red blood cells (B). A, Purified Pr-2 was subjected to radial growth inhibition tests using Fusarium solani (1), B. cinerea (2), Trichoderma harzianum (3) and Colletotrichum coccodes (4). Paper disks were loaded with buffer alone as a negative control (disk C; 25 mM Hepes buffer, pH 7.2) or with 150 μg (disk 1) or 300 μg (disk 2) of purified Pr-2. B, The dose response of hemolytic activity toward 8% hRBCs with Pr-2 (▲) and melittin (●) was evaluated.

표 Localization of rhodamine-labeled Pr-2 in fungal cells. Fungal cells with rhodamine-labeled Pr-2 were visualized on confocal laser scanning microscopy. Conidia of C. albicans (A) and hyphae of F. oxysporum (B) were incubated with 20 μM rhodamine-labeled Pr-2 for 30 min. A representative fluorescence image of fungal cells with the protein is shown in panel 1, and a bright field image of the same cells is presented in panel 2. The merged image of the two panels is shown in panel 3.

표 Permeable activity of Pr-2 in fungal cells. A, Influx of the vital dye, SYTOX Green, into C. albicans was measured at an excitation 484 nm and emission 520 nm after the addition of Pr-2 (■) and BSA (□). The fluorescent intensity with 0.1% Triton X-100 (final concentration) was calculated as 100% permeabilization. B and C, visualization of SYTOX Green uptake by F.oxysporum treated with BSA (B) or Pr-2 (C) at a concentration of 20 μM was observed using fluorescence microscopy. Panel 1 and 2 show the fluorescence image and bright field image, respectively.

표 Scheme for the purification of antimicrobial peptides from potato tubers.

표 Inhibition of chymotrypsin, trypsin and papain by Potide-G. Fluorescently labeled casein was incubated at room temperature with 25 μg of the indicated enzyme for 60 min, with or without the indicated concentration of potide-G, after which the fluorescence was measured. Modified with permission from REF. 60.(2006) Biochem Biophys Res Commun

표 Antifungal activity of potide-G on agar containg C. albicans. After peptide was untreated (A) or treated (B, 5 μg) on paper discs, the plated was incubated for 24 hr at 37 °C.

표 Antibacterial assay of PT-1 peptide in the absence (A) or presence (B) of DTT against S. aureus. After reducing the intramolecular disulfide bonds of peptide with DTT, substance was mixed with bacterial cell suspension.

표 Lists and Description of Primers

표 Potato Regeneration Medium

표 Comparison of putative proteins encoded for by G-Ry, Y-1 (16), and W-Ry.

표 Strategy for construction: (A) two fragments of the Ry gene (left, 1.3 kb; right, 2.2 kb) were amplified by PCR; (B) both products were assembled on the SphI linker site. pBI121 contains NPT (KanR) and CaMV promoter. In B, the full-length Ry gene was digested and cloned on the XmaI site of the pBI121 binary vector.

표 Regeneration of ’

표 Comparative analysis of Ry gene and CP gene by RT-PCR: (A) comparative band intensities of the Ry gene were detected via RT-PCR using specific primers (Table 1) (lanes 1 and 3, ’

표 Antiviral activity of ’

표 Phenotypic responses of PVYO infection. Several local lesions and leaf spots developed on most of the leaf lamina of infected ’

표 Sequencing primer. cDNA sequencing used WF and WR serise primer, and genomic DNA sequencing used WiF and WiR serise primer.

표 W-Ry cloning primer

표 G-Ry and W-Ry Point-mutation primer.

표 The site-direct mutagenesis of W-Ry.

표 Mutated W-Ry construction.

표 Strategy for mutation of the MHD motif in W-Ry and G-Ry.

표 WF4-3, mutated WR31-9 and pEGAD digested with XmaⅠ.

표 pEGAD and mW-Ry were digestedwith XmaⅠ. The two fragments were directly cloned on the XmaⅠ restriction site of the pEGAD binary vector. The two fragment, 1.3 kb and 2.0 kb The digested mW-Ry fragment (3.6 kb). M, size marker.

표 감자잎에서 total RNA를 뽑아 cDNA를 만든후 CP primer를 사용하여 확인하였다. 사용된 CP primer는 Forward primer (CPF2）: 5'-GACACATACTGTGCCGAGAATCAAGGCTA-3'과 Reverse primer (CPR2) : 5'-CATTCATCACAGTTGGCATCTCAGT-3' 를 이용하였다. 실험7일 째에 확인된 결과이다. 감염되어진 Winter(S) PVY가 CP가 확인되지 않는 것은 형질전환으로 인해 저항성이 증가 되어진 것으로 보여 진다.

표 형질전환된 감자의 PVY 감염여부 확인. OD405 에서 PVY에 감염된 잎과 감염되지 않은 잎을 ELISA 방법을 통하여 확인하였다. 감염되어진 Winter(S) PVY는 감염되지 않은 것과 비교해 보았을 때 거의 비슷한 수준으로 나오며, 다른 감염된 감자와 비교했을 때 CP양에 차이가 나는 것으로 보아 PVY에 대한 저항성이 향상 되었음을 보인다.

표 Golden valley의 Ry gene이 들어간 형질전환된 Winter valley에서 PVY에 의하여 감염이 되었을 때 감염이 되지 않았을 때와 비교할 수 있었다.PVY감염 여부에 따른 형질전환 감자의 표현형. PVY에 감염된 감자는 검은 반점 및 황백화, 잎 끝이 말려 들어가는 특징이 있으며, 그 중 가장 주요한 검은 반점이 두드러지게 잘 나타나고 있으며, 형질전환된 감자에서는 그 특징이 보이지 않는다. 즉, Winter(S)는 저항성이 향상되었음을 확인할 수 있다.

표 감염되지 않은 감자에서는 PVY의 CP밴드가 나오지 않았고, 감염된 감자에서 30kDa가 약간 못되는 부위에서 CP밴드가 확인이 되었다. 형질전환된 infected Winter(S)는 CP밴드가 확인되지 않은 것으로 보아 Ry gene이 제대로 활성을 가지고 있다는 것을 확인하였다.

표/그림 (39)

표 Summary for purification of Pr-1 from powders of pumpkin rind

표 Antifungal activity of Pr-1 toward fungal cells

표 Hemolytic effect of the antifungal protein Pr-1

$Purification and antifungal activity of a novel antifungal protein. (A) The unadsorbed fraction with antifungal activity on ion-exchange chromatography was subjected to reverse-phase HPLC on a C18 column.The arrow indicates a protein with antifungal activity. (B)$ 표 Purification and antifungal activity of a novel antifungal protein. (A) The unadsorbed fraction with antifungal activity on ion-exchange chromatography was subjected to reverse-phase HPLC on a C18 column.The arrow indicates a protein with antifungal activity. (B)

표 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Pr-1 was analyzed by SDS-PAGE on a 15% separating gel with Coomassie brilliant blue G-250 stain. Lane M, molecular mass marker proteins obtained from Sigma. Lane 2, proteins extracted from pumpkin rind. Lane 3, heat-stable proteins after heat-treatment, Lane 4, the purified protein from RP-HPLC.

표 Inhibitory activity of purified Pr-1 for fungal growth. Purified Pr-1 was subjected to radial growth inhibition tests with Fusariummoniliformevar. subglutinans (A), B. cinerea (B) and F.solani (C).

표 Localization of rhodamine-labeled Pr-1 (A and B) and SYTOX Green uptake (C and D) on fungal cells. C. albicans cells (A) or F. solani hyphae (B) with 20μM rhodamine-labeled Pr-1 was observed on confocal laser scanning microscopy. The influx of SYTOX Green was visualized using fluorescence microscopy in the presence (C) or absence (D) of Pr-2. Panel 1 shows the bright field image, while panel 2 is a fluorescence image of the same cells shown in the bright field micrograph. Panel 3 shows the merged images.

표 Summary of the purification of Pr-2 from pumpkin rind powders

표 Inhibitory effects of the antifungal protein, Pr-2, against fungal and bacterial cells

표 Proteolytic digestion of crude extracts with trypsin. A, Crude extracts were digested with 0.25 mg/ml trypsin at 37 °C for 4hr, and then the sample was subjected to SDS-PAGE. Lane 1: without trypsin and lane 2: with trypsin. B,digested crude extracts were assayed toward Candida albicans. 1: Control (no extracts and trypsin), 2: only extracts (no trypsin), 3: digested extracts (with trypsin), 4: trypsin only (no extracts).

$Chromatographical purification of antifungal proteins (Pr-2). A and B, Pr-2, antifungal protein, was purified using cation exchange chromatography on CM-Sepharose (A), reverse-phase HPLC on a C18 column (B). Extraction of the soluble proteins and chromatographic procedures are described in the Materials & Methods section. (I): Unadsorbed fraction, (II-IV): fraction eluted with 0.1 M (II), 0.3 M (III) and 1 M (IV) NaCl. Fraction (II) containing antifungal activity was collected and purified on a C18 column (B).$ 표 Chromatographical purification of antifungal proteins (Pr-2). A and B, Pr-2, antifungal protein, was purified using cation exchange chromatography on CM-Sepharose (A), reverse-phase HPLC on a C18 column (B). Extraction of the soluble proteins and chromatographic procedures are described in the Materials & Methods section. (I): Unadsorbed fraction, (II-IV): fraction eluted with 0.1 M (II), 0.3 M (III) and 1 M (IV) NaCl. Fraction (II) containing antifungal activity was collected and purified on a C18 column (B).

$Tricine-SDS-PAGE, molecular massand growth inhibition activity of purified Pr-2. A, Pr-2 was analyzed by Tricine-SDS-PAGE on a 16.5% separating gel with Coomassie brilliant blue G-250 staining. Lane M: Molecular mass marker proteins from Sigma, Lane 2: the protein purified by RP-HPLC. B, The molecular mass of the purified Pr-2 was examined using MALDI-TOF/MS. C, After collection of the major peak fraction from HPLC, antifungal activities were assayed against F. solani without (1) or with Pr-2 protein (2).$ 표 Tricine-SDS-PAGE, molecular massand growth inhibition activity of purified Pr-2. A, Pr-2 was analyzed by Tricine-SDS-PAGE on a 16.5% separating gel with Coomassie brilliant blue G-250 staining. Lane M: Molecular mass marker proteins from Sigma, Lane 2: the protein purified by RP-HPLC. B, The molecular mass of the purified Pr-2 was examined using MALDI-TOF/MS. C, After collection of the major peak fraction from HPLC, antifungal activities were assayed against F. solani without (1) or with Pr-2 protein (2).

표 Inhibitory activity of purified Pr-2 for fungal growth (A) and human red blood cells (B). A, Purified Pr-2 was subjected to radial growth inhibition tests using Fusarium solani (1), B. cinerea (2), Trichoderma harzianum (3) and Colletotrichum coccodes (4). Paper disks were loaded with buffer alone as a negative control (disk C; 25 mM Hepes buffer, pH 7.2) or with 150 μg (disk 1) or 300 μg (disk 2) of purified Pr-2. B, The dose response of hemolytic activity toward 8% hRBCs with Pr-2 (▲) and melittin (●) was evaluated.

표 Localization of rhodamine-labeled Pr-2 in fungal cells. Fungal cells with rhodamine-labeled Pr-2 were visualized on confocal laser scanning microscopy. Conidia of C. albicans (A) and hyphae of F. oxysporum (B) were incubated with 20 μM rhodamine-labeled Pr-2 for 30 min. A representative fluorescence image of fungal cells with the protein is shown in panel 1, and a bright field image of the same cells is presented in panel 2. The merged image of the two panels is shown in panel 3.

표 Permeable activity of Pr-2 in fungal cells. A, Influx of the vital dye, SYTOX Green, into C. albicans was measured at an excitation 484 nm and emission 520 nm after the addition of Pr-2 (■) and BSA (□). The fluorescent intensity with 0.1% Triton X-100 (final concentration) was calculated as 100% permeabilization. B and C, visualization of SYTOX Green uptake by F.oxysporum treated with BSA (B) or Pr-2 (C) at a concentration of 20 μM was observed using fluorescence microscopy. Panel 1 and 2 show the fluorescence image and bright field image, respectively.

표 Scheme for the purification of antimicrobial peptides from potato tubers.

표 Inhibition of chymotrypsin, trypsin and papain by Potide-G. Fluorescently labeled casein was incubated at room temperature with 25 μg of the indicated enzyme for 60 min, with or without the indicated concentration of potide-G, after which the fluorescence was measured. Modified with permission from REF. 60.(2006) Biochem Biophys Res Commun

표 Antifungal activity of potide-G on agar containg C. albicans. After peptide was untreated (A) or treated (B, 5 μg) on paper discs, the plated was incubated for 24 hr at 37 °C.

표 Antibacterial assay of PT-1 peptide in the absence (A) or presence (B) of DTT against S. aureus. After reducing the intramolecular disulfide bonds of peptide with DTT, substance was mixed with bacterial cell suspension.

표 Lists and Description of Primers

표 Potato Regeneration Medium

표 Comparison of putative proteins encoded for by G-Ry, Y-1 (16), and W-Ry.

표 Strategy for construction: (A) two fragments of the Ry gene (left, 1.3 kb; right, 2.2 kb) were amplified by PCR; (B) both products were assembled on the SphI linker site. pBI121 contains NPT (KanR) and CaMV promoter. In B, the full-length Ry gene was digested and cloned on the XmaI site of the pBI121 binary vector.

표 Regeneration of ’

표 Comparative analysis of Ry gene and CP gene by RT-PCR: (A) comparative band intensities of the Ry gene were detected via RT-PCR using specific primers (Table 1) (lanes 1 and 3, ’

표 Antiviral activity of ’

표 Phenotypic responses of PVYO infection. Several local lesions and leaf spots developed on most of the leaf lamina of infected ’

표 Sequencing primer. cDNA sequencing used WF and WR serise primer, and genomic DNA sequencing used WiF and WiR serise primer.

표 W-Ry cloning primer

표 G-Ry and W-Ry Point-mutation primer.

표 The site-direct mutagenesis of W-Ry.

표 Mutated W-Ry construction.

표 Strategy for mutation of the MHD motif in W-Ry and G-Ry.

표 WF4-3, mutated WR31-9 and pEGAD digested with XmaⅠ.

표 pEGAD and mW-Ry were digestedwith XmaⅠ. The two fragments were directly cloned on the XmaⅠ restriction site of the pEGAD binary vector. The two fragment, 1.3 kb and 2.0 kb The digested mW-Ry fragment (3.6 kb). M, size marker.

표 감자잎에서 total RNA를 뽑아 cDNA를 만든후 CP primer를 사용하여 확인하였다. 사용된 CP primer는 Forward primer (CPF2）: 5'-GACACATACTGTGCCGAGAATCAAGGCTA-3'과 Reverse primer (CPR2) : 5'-CATTCATCACAGTTGGCATCTCAGT-3' 를 이용하였다. 실험7일 째에 확인된 결과이다. 감염되어진 Winter(S) PVY가 CP가 확인되지 않는 것은 형질전환으로 인해 저항성이 증가 되어진 것으로 보여 진다.

표 형질전환된 감자의 PVY 감염여부 확인. OD405 에서 PVY에 감염된 잎과 감염되지 않은 잎을 ELISA 방법을 통하여 확인하였다. 감염되어진 Winter(S) PVY는 감염되지 않은 것과 비교해 보았을 때 거의 비슷한 수준으로 나오며, 다른 감염된 감자와 비교했을 때 CP양에 차이가 나는 것으로 보아 PVY에 대한 저항성이 향상 되었음을 보인다.

표 Golden valley의 Ry gene이 들어간 형질전환된 Winter valley에서 PVY에 의하여 감염이 되었을 때 감염이 되지 않았을 때와 비교할 수 있었다.PVY감염 여부에 따른 형질전환 감자의 표현형. PVY에 감염된 감자는 검은 반점 및 황백화, 잎 끝이 말려 들어가는 특징이 있으며, 그 중 가장 주요한 검은 반점이 두드러지게 잘 나타나고 있으며, 형질전환된 감자에서는 그 특징이 보이지 않는다. 즉, Winter(S)는 저항성이 향상되었음을 확인할 수 있다.

표 감염되지 않은 감자에서는 PVY의 CP밴드가 나오지 않았고, 감염된 감자에서 30kDa가 약간 못되는 부위에서 CP밴드가 확인이 되었다. 형질전환된 infected Winter(S)는 CP밴드가 확인되지 않은 것으로 보아 Ry gene이 제대로 활성을 가지고 있다는 것을 확인하였다.

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