Cryphonectria Parasitica에서 CpPK1 기질 단백질의 동정 = Identification of CpPK1 substrates protein in Cryphonectria Parasitica

다국어 초록 (Multilingual Abstract)

The cppk1 gene encodes the Ser/Thr protein kinase of Cryphonectria parasitica and is trascriptionally up-regulated by the presence of hypovirus CHV1-EP713. The cppk1 is important for coordinating growth with development and maintaining cell wall integrity. In ordet to identify CpPK1 substrates, we used yeast two hybrid system and in order to identify CpPk1-dependent substrates, we conducted kinase-assisted two dimensional (2-D) polyacrylamide gel elestrophresis using partially purified CpPk1 and protein substrate. Among 70 fractions of cell-free extracts by HPLC using gel-filtration column, number 10, 12, 14, 16, 18 fractions have shown the presence of the CpPK1 protein by Western blot analysis. In order for the CpPK1-dependent, substrates, fractions which were expected to contain the putative substrate of CpPK1 was obtained by HPLC using ion-exchange column. A kinase assay using CpPK1 fration revealed CpPK1-dependently phosphorylated protein fractions containing putative substrates with estimated masses of 70kDa, 50kDa, and 40kDa. In order to identify these two proteins, we conducted kinase-assisted two dimensional (2-D) polyacrylamide gel electrophoresis using partially purified CpPK1 and protein substrate and then the corresponding radioactive protein spots were further analyzed by using MALDI- and Q-TOF mass spectrophotometry.

목차 (Table of Contents)

• ABSTRACT = 1
• Ⅰ. 서론 = 2
• Ⅱ. 실험재료 및 실험 방법 = 4
• 1. Yeast two hybrid system = 4
• (1) 균주 및 균주 배양 방법 = 4
• (2) 사용한 vecter의 제조 = 5
• 2. Kinase assay 및 2-D polyacrylamide gel electrophoresis = 5
• (1) 곰팡이 균주 및 배양 방법 = 5
• (2) C. Parasitica cell-extract 추출 및 HPLC를 이용한 CpPK1의 정제 = 6
• (3) CpPK1-dependent substrates protein의 확인 = 6
• (4) Kinase assay = 7
• (5) 2-D polyacrylamide gel electrophoresis = 7
• Ⅲ. 결과 = 9
• 1. Yeast two hybrid system을 이용한 CpPK1 substrates protein의 동정 = 9
• (1) Bait vector와 prey vector의 제조 및cloning = 9
• (2) Yeast transformant의 선별 = 9
• 2. Proteomic 분석을 통한 CpPK1-dependent substrates protein의 동정 = 10
• (1) HPLC 를 이용한 CpPK1 protein의 부분 정제 = 10
• (2) Western blot analysis = 10
• (3) HPLC 를 이용한 CpPK1-dependent substrates protein의 부분 정제 = 10
• (4) Kinase assay를 통해 CpPK1-dependent substrate protein으로 추정되는 protein 확인 = 11
• (5) Two dimensional polyacrylamide gel electrophoresis = 11
• Ⅳ. 고찰 = 12
• Ⅴ. Figures = 14
• Ⅵ. 참고 문헌 = 23