Abstract
本篇論文研究大腸桿菌(Escherichia coli)的離氨酸生合成途徑中dapA與lysC兩個基因。 關於dapA基因之研究首先由大腸桿菌菌株DH1染色體選殖此基因。其步驟為構築迷你基因庫,由此迷你基因庫藉聚合連鎖反應 (polymerase chain reaction, PCR)放大dapA基因,將PCR生成物嵌入載體pUC18,最後利用限制圖譜(restriction pattern)及核甘酸序定法確定選殖正確基因。 質體pCR13為攜帶dapA基因之多套型質體。將其殖入大腸桿菌菌株AT998及菌株DH1,由dapA基因表現所形成之合成酵素(dihydrodipicolinate synthase)可藉菌體之粗抽出液進行電泳分析及酵素活性分析觀察之。電泳分析顯示含有質體pCR13之大腸桿菌菌株具有一個分子質量約35kDa之色帶,此色帶推測為合成酵素(dihydrodipicolinatesynthase) 。電泳分析及酵素活性分析顯示pCR13所攜帶之dapA基因之表現能力隨寄主菌株而異。表現能力在DH1中較在AT998中為強。電泳分析及酵素活性分析亦顯示增加dapA基因之套數會提高合成酵素(dihydrodipicolinate synthase)量及活性。外加離氨酸會抑制合成酵素(dihydrodipicolinate synthase)活性。 關於lysC基因之研究,首先構築lysC-lac'Z融合質體,以研究lysC基因之調控區。藉著刪除變異作用(deletion mutation),證實有另一個啟動子(promoter) P2存在。由啟動子P2開始進行之轉錄作用受離氨酸調控,其調控機制作用在啟動子P1之上游。啟動子P1亦受離氨酸調控,其調控機制作用在其下游之未轉譯區(untranslated region)。啟動子P2轉錄作用之啟始點位於啟動子P1轉錄作用啟始點之上方85bp處。lysC基因之未轉譯區利用核酸分解酵素(nuclease Bal31)刪除以及特定點嵌入變異法分析,結果顯示此調控機制似乎不是傳統的抑制子－操縱子結合作用(repressor-operator binding)。The dapA gene of Escherichia coli encodingdihydrodipicolinate synthase (DHDPS), the first enzyme of thebiosynthetic branch leading to diaminopimelate and lysine, wascloned in this study. We amplified the dapA gene by PCR(polymerase chain reaction) from a minilibrary of E. coli DH1chromosome, inserted the PCR product into vector pUC18, andconfirmed the insert first by restriction patterns and finallyby DNA sequence analysis. Plasmid pCR13, the multicopy plasmidcarrying the dapA gene, wasintroduced into both E. coli AT998and DH1. Expression of dihydrodipicolinate synthase was detectedfrom SDS-PAGE analysis and theenzyme activity of the crude cellextract. SDS-PAGE analysis revealed aspecific band of about 35kDa present on E. coli strains harboring pCR13.SDS-PAGE analysisand enzyme assay showed that expression of the dapA geneon pCR13varied with the host strains. The expression level was higher inDH1 than in AT998. SDS-PAGE analysis and enzyme assay alsoindicated thatincrease of the copy number of the dapA gene ledto increase of the DHDPS activity. Moreover, DHDPS activity wasinhibited 80 to 84 % by 20 mM lysinein vitro. A lysC-lac¢Zfusion plasmid was constructed to study the regulatoryregion ofthe lysC gene. Analysis by deletion mutations confirmedtheexistence of an alternative promoter, P2, located upstream ofthe previouslyidentified promoter, P1. The transcription startsite of promoter P2 was located 85 base pairs upstream thetranscription start site of promoter P1.Both promoters areregulated by lysine. The untranslated region of the lysCgene wasanalyzed by Bal 31 nested deletion and specific-site insertionmutations. It seemed that the regulation mechanism might not beconventionalrepressor-operator binding.