微生物hydrolase和PHB synthase之基因選殖與探討

Cloning and studying genes encoded hydrolase and PHB synthase from microorganisms

鍾婷(Ting Chung)

指導教授 ： 簡志青

元智大學/工程學院/生物科技與工程研究所/碩士(2008年)

https://doi.org/10.6838/YZU.2008.00102

摘要

本論文主要分為兩大部份；第一部份是選殖Cupriavidus taiwanensis 184的PHB合成基因(phbC基因)序列。C. taiwanensis 184是從台灣土壤中分離出來的根瘤菌，可在菌體內累積大量聚羥基丁酸（poly-hydroxbutyrate，PHB）。在實驗室醱酵培養時，C. taiwanensis 184 之PHB累積量可達其菌體乾重的80%以上。然而，C. taiwanensis 184 參與PHB合成之相關基因與機制仍未被探討。在PHB的相關合成基因中，Cupriavidus necator H16 (早期分類為Ralstonia eutropha H16)已被廣泛研究，並探討其應用在PHB合成菌上，如利用基因轉殖大腸桿菌來生產PHB。因此利用Cupriavidus necator H16的PHB合成基因(phbC基因)序列來設計primer，欲選殖出Cupriavidus taiwanensis 184的PHB合成基因(phbC基因)序列，分別以PCR、南方墨點法和轉位子突變的實驗來選殖C. taiwanensis 184的PHB合成基因，現在已順利獲得部分PHB合成基因(phbC基因)序列，並與Cupriavidus necator H16的PHB合成基因(phbC基因)序列比對後有高達91~92％的同源性。
第二部份是選殖Acinetobacter sp. TMD1中的hydrolase基因，Acinetobacter spp.在文獻中指出是一種會在細菌體內累積PHA的菌屬，因此推測Acinetobacter sp. TMD1的hydrolase基因可能與PHA分解基因(phaZ基因)有關，所以將此DNA片段選殖至pBluescript II SK（+/-）載體上，並送入大腸桿菌XL-1 Blue中，再使用含葡萄糖及Nile Red 的培養基，篩選菌體內含有大量脂質堆積物之菌株。

關鍵字

Cupriavidus taiwanensis 184 ； Cupriavidus necator H16 ； 聚羥基丁酸(poly-hydroxbutyrate，PHB) ； Acinetobacter sp. TMD1

並列摘要

Polyhydroxybutyrate (PHB) is polyester accumulated by various bacteria under unbalanced growth conditions when the carbon substrate is in excess to other nutrients such as nitrogen, sulfur, phosphorus or oxygen. Of the variety of bacteria that synthesize PHB, Cupriavidus necator H16 (formally known as Ralstonia eutropha H16) is the most intensively studied, and the genes involved in PHA metabolism in this bacterium have been well characterized. PHB operon of C. necator H16 has been cloned and introduced into Escherichia coli as PHB-producing recombinant strain. Cupriavidus taiwanensis 184, an indigenous strain isolated from soil of Taiwan, also accumulate significant amount of PHB inside the cells. However, the genes and mechanisms related to PHB metabolism in the bacterium is not yet explored. In this study, we aim to identify and clone the genes that related to PHB metabolism in C. taiwanensis 184.

並列關鍵字

Cupriavidus taiwanensis 184 ； Cupriavidus necator H16 ； 聚羥基丁酸(poly-hydroxbutyrate，PHB)

參考文獻

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