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標題: 嗜鹽性甲烷古生菌抗鹽基因的轉殖與分析
Cloning and analysis of salt resistant genes from halophilic methanogenic archaea Methanohalophilus portucalensis FDF1
作者: 賴麗娟
Lai, Li-Jane
關鍵字: compatible
Methanohalophilus portucalensis
salt-resistant recombinant
出版社: 植物學系
摘要: 在高鹽生態環境中,生物會以改變細胞結構及組成、調節細胞體積及累積 相容質等方式存活於高滲透壓力下。為瞭解嗜鹽生物抗高鹽的分子機制, 我們以ZAP express vector構築嗜鹽性甲烷古生菌Methanohalophilus portucalensis FDF1 的基因庫,並利用mass excision方式,將攜帶高鹽 古生菌DNA的噬菌體轉化成以質體(pBK- CMV vector)形式存於E. coli strain XLOLR寄主中,並於含2 M NaCl的LA-kanamycin篩選到166株抗鹽 轉殖株。進一步分析抗鹽轉殖株FII0065,發現此能生長於2 M NaCl的抗 鹽轉殖株之全細胞蛋白質具有四個高產量且類似嗜鹽性甲烷古生菌的蛋白 產物(69 kDa, 44 kDa, 30 kDa及21 kDa),且此四個polypeptides的N-端 氨基酸序列顯示,其和E. coli蛋白產物較不相關反而與真核生物及古生 菌類蛋白產物較為相關。但是此能生長於含2 M NaCl LA-kanamycin的抗 鹽轉殖株FII0065,卻無法生長於LA-kanamycin中,並且無法萃取到原先 殖入的pBK-CMV質體。因此推測嗜鹽古生菌FDF1的DNA片段已鑲入寄主染色 體中。為了找出鑲於寄主染色體內的古生菌抗鹽基因,我們選用30 kDa蛋 白之N-端氨基酸序列,以甲烷菌常用之基因密碼,利用DNA合成儀合成一 段30個鹼基之oligonucleotides。以此段oligonucleotides及T7為引子, 利用PCR技術合成出兩段嗜鹽甲烷古生菌FDF1的DNA片段:1.2 kb和1 kb。 再以1.2 kb DNA 作為探針自嗜鹽甲烷古生菌FDF1釣到一段約6.5 kb的DNA ,進而用此段DNA進行選殖,篩選到兩株分別含4.5及6.5 kb古生菌DNA的 轉殖株:FPSII14和 FPSII15。E. coli FPSII14和 FPSII15能生長於含1 M鹽濃度,但卻無法生長至2 M NaCl的環境。SDS-PAGE分析顯示,這兩株 轉殖株均具有30 kDa的polypeptides,且生長鹽度提高,蛋白產量提高, 顯示這是一個受滲透壓調節的蛋白。
The molecular mechanism of how life adapt/survive in the high salt environment is still remain unsolved. The halophilic methanogenic archaea-Methanohalophilus portucalensis strain FDF1 can grow optimally in the salt range of 1.2 to 2.9 M and transport glycine betaine or de novo biosynthesis (-glutamine, N(-acetyl-(-lysine and glycine betaine as compatible solutes in response to the changing osmotic stress. The genomic library of Mh. portucalensis FDF1 was constructed and 166 strains of E. coli XLOLR recombinant clones that can grow of 2 M NaCl were selected for further investigation in salt resistant genes.Salt resistant clone-FII0065 can grow in LA medium containing 2 M NaCl and the total protein profile in SDS-PAGE contains four significant additional polypeptides of 69 kDa, 44 kDa, 30 kDa and 21 kDa. These four polypeptides showed higher similarity with halophilic methanogen than the E. coli in both total cell protein profile and N-terminal amino acid compareness. However, plasmid vector with insert archaeal gene can(t locate in FII0065 and FII0065 can(t grow in LA-kanamycin plate. It was suspicious that they were integrated into the host genome in part. The N- terminal amino acid sequence of 30 kDa polypeptide was used to deduce the 30 base oligonucleotides and T7 primer, 1.2 kb and 1.0 kb DNA fragements were amplified by PCR with Mh. portucalensis FDF1 DNA as template. A 6.5 kb DNA fragment from Mh. portucalensis FDF1 were hybridized with 1.2 kb probe through Southern analysis and further recovered and transformed to E. coli DH5(. Two transformants FPSII14 and FPSII15 were selected and both can grow in the medium contain 1 M NaCl but not in 2.0 M NaCl. Moreover, the total protein profile of these two transformants both showed the existence of 30 kDa polypeptides and the expression of this polypeptides were salt induced.
Appears in Collections:生命科學系所



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