Aspergillus nidulans의 citrate synthase 유전자의 발현조절 분석 = Analysis of expression and regulation of citrate synthase gene in Aspergillus nidulans

다국어 초록 (Multilingual Abstract)

cDNA of citrate synthase gene(citA) was isolated from Aspergillus nidulans. The nucleotide sequence of a 1.7 kb fragment of the citA cDNA was determined.
citA promoter of A.nidulans investigated. The nucleotide sequence of a 1.45 kb fragment of the citA promoter was determined. To identify the region responsible for high level expression and regulation within citA promoter, a series of deletion promoters fused to the GFP(Green Fluorescent Protein) gene was constructed and introduced into the A.nidulans trpC locus by homologus recombination.
Two classes of transformants in which pTGmT5C△904(the number after the △ symbol indicates the length of the deletion promoter) and the plasmid containing promoter with MTS(mitochondrial targeting signal), pTGmT5C_MTS were integrated into the trpC locus respectively were obtained. The data of western blot revealed that the R3(RCY↓S) motif functioned as mitochondrial targeting sequence. Expression of GFP in the transformants grown under the catabolite carbon sources(glucose) and the noncatabolite carbon source(glycerol, potassium acetate) was observed. The pTGmT5C_MTS transformant exhibited quite strong fluorescence of GFP in the medium containing glycerol or potassium acetate as a sole carbon source, but showed severely weakened GFP flurescence in the medium supplimented with glutamate. The pTGmT5C△904 transformant, on the contrary, showed similar strength GFP fluorescence in either medium.
These results supports that the expression of citA gene is strongly repressed by glutamate, a plausible catabolite repressor for genes that play major roles in the central catabolic and biosynthetic pathways, and that the putative CREA-binding site located at position -1116 and -913 bp functions as a main regulatory motif for the repression caused by glutamate.

목차 (Table of Contents)

• 목차
• I. 서론 = 1
• II. 재료 및 방법 = 4
• 1. 균주 및 플라스미드 = 4
• 2. 배지 및 배양 = 4
• 3. Plaque hybridization = 4
• 4. 생체 내에서의 phagemid excision = 6
• 5. 염기서열 분석 = 6
• 5.1. primer = 6
• 5.2. 염기서열 분석 = 6
• 6. Deletion = 7
• 7. Polymerase chain reaction = 8
• 8. Aspergillus의 형질전환 = 8
• 9. Aspergillus nidulans의 genomic DNA 준비 = 9
• 10. Genomic Southern Hybridization = 10
• 11. 형질 전환체에서의 GFP의 발현분석 = 11
• 11.1. SDS-PAGE = 11
• 11.2. Western Blotting = 11
• 11.3. GFP(Green Fluorescent Protein) 관찰 = 12
• III. 결과 및 고찰 = 17
• 1. citA cDNA cloning = 17
• 1.1. Plaque hybridization = 17
• 1.2. citA cDNA 염기서열 분석 = 17
• 2. citA promoter의 염기서열 분석 = 21
• 3. Promoter분석 vector제조 = 21
• 4. citA promoter와 GFP의 융합유전자 제조 = 24
• 4.1. PCR을 이용한 citA promoter 부위의 증폭 및 promoter분석 vector에 cloning = 24
• 4.2. Mutant citA promoter와 GFP의 융합유전자 제조 = 24
• 5. Aspergillus 형질전환 및 형질전환체 분석 = 27
• 5.1. Aspergillus 형질전환 = 27
• 5.2. Southern hybridization = 27
• 5.3. Western blot = 27
• 5.4. GFP 발현관찰 = 28
• IV. 결론 = 36
• V. 참고문헌 = 37
• ABSTRACT = 40