- 學位論文
Stenotrophomonas sp. CD02抗重金屬鎘相關基因之選殖與探討
Cloning and studying the role of genes related to cadmium resistance of Stenotrophomonas sp. CD02
摘要
由被重金屬鎘污染的土壤環境中分離出來的Stenotrophomonas sp. CD02，可以抵抗高濃度（最高到4 mM）的重金屬鎘以及其他重金屬汙染，例如鉻、鉛、鎳、銅跟鋅。此菌株經實驗證實顯示當其生長到穩定期時，可以從環境中移除重金屬鎘。本實驗藉由Stenotrophomonas sp. CD02的基因體DNA建構一fosmid基因庫，來探討與抗重金屬有關的基因。利用體外轉位子插入突變法，得到23株轉殖菌株與抗重金屬鎘的表現有關，並分析、定序其相關基因。經定序及BLAST同源性比對，得到與Enterobacter sp. 638的heavy metal translocating P-type ATPase基因有很高的同源性。利用PCR技術將Stenotrophomonas sp. CD02體內的heavy metal translocating P-type ATPase分離出來，與大量表現載體（over-expression vector）接合之後送入經體外轉位子插入突變之後的轉殖菌株F35。利用生長曲線的測試，觀察其抗重金屬鎘的能力。
並列摘要
Stenotrophomonas sp. CD02 was isolated from soil contaminated with the heavy metal cadmium. The bacterium can resist high concentration of Cd 2+ (up to 4 mM) and other heavy metals such as chromium, lead, nickel, copper and zinc. This bacterium showed the ability to remove cadmium ions from medium when it reached stationary growth phase. A fosmid library was constructed for studying the genes related to heavy metal resistance in this bacterium. Using in vitro transposon mutagenesis techniques, obtain 23 clones that contain genes involved in Cd resistance were selected and the sequences were determined. The identified genes include genes encode homologues of heavy metal translocating P-type ATPase of Enterobacter sp. 638. Isolating heavy metal translocating P-type ATPase of Stenotrophomonas sp. CD02 by PCR technique, then ligate with over-expression vector transform to clone F35. To investgate the ability of heavy metal cadmium resistant by measuring the growth curve.