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標題: 極端高鹽太古生物二級相容質運輸蛋白基因的選殖與特性分析
Cloning, Expression, and Functional Characterization of two Possible Betaine-Choline-Carnitine Transporter Genes from an Extreme Halophilic Archaeon Haloterrigena sp. H13
作者: 張柏迪
Chang, Po-Ti
關鍵字: Osmolyte
secondary compatible solute transporter
出版社: 生命科學系所
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摘要: 微生物在面臨高滲透壓逆境時會在胞內累積鉀離子或有機相容質(compatible solutes)來平衡胞內外的滲透壓。BCCT (Betaine-choline-carnitine transporter) family 運輸蛋白是生物細胞當中普遍存在的二級相容質運輸系統 (secondary transporter),可藉由氫離子/鈉離子濃度梯度差(proton/ sodium gradient force)作為驅動能源,由胞外攝取相容質至胞內累積。極端高鹽太古生物生長在高鹽環境中,主要以累積大量鉀離子於胞內來因應外界的滲透壓力,細胞蛋白中也含有較高比例的酸性胺基酸藉以在大量鉀離子濃度下維持蛋白構型。到目前為止,並無文獻證實極端高鹽太古生物具有可運輸相容質的二級運輸蛋白。極端高鹽太古生物Haloterrigena sp. H13的比較基因體分析發現具有兩套二級相容質運輸蛋白的基因betLH13與opuDH13,但本實驗並未測出Haloterrigena sp. H13 在最適鹽度生長狀態下的glycine betaine與choline chloride的運輸能力。藉由南方墨點法及菌落雜合,篩選出帶有betLH13與opuDH13基因全長的菌落。由定序及序列分析比較,BetLH13與Halorubrum lacusprofundi的BetT有75%的胺基酸相同度,而OpuDH13與Halobacterium sp. NRC-1的OpuD有69%的胺基酸相同度。在序列分析上,betLH13與opuDH13的GC含量高,且蛋白的等電點較低,具普遍極端高鹽菌的蛋白特色。BetLH13與OpuDH13的二級結構都具有BCCT family運輸蛋白的特性,然而BetLH13在BCCT保留性區域的四個色胺酸保留位上,其中一個色胺酸由丙胺酸所取代。但是將betLH13表現於glycine betaine運輸突變株E. coli MKH13,測試BetLH13 的glycine betaine運輸活性,在0 M NaCl與0.5 M NaCl的條件下都偵測不到glycine betaine攝取活性。進一步再確認與分析betLH13在E. coli MKH13的基因序列、轉錄與轉譯,發現betLH13序列第1173的位置上少了一個鹼基,使得序列於第1181~1183形成一個終止密碼,導致所表現出的蛋白分子量僅只有約48 kD,而並非原先預設的62.46 kD。因此需要重新構築具有正確序列的betLH13於大腸桿菌上再分析glycine betaine運輸活性。以RT-PCR初步分析betLH13與opuDH13 mRNA的表現量,發現在高滲透壓(5 M NaCl)與高溫(55°C)的逆境下,這兩個基因的表現量皆為最適生長環境(45°C 4 M NaCl)下的2.7倍;在低滲透壓(3 M NaCl)與低溫(20°C)的逆境下之基因表現量個別為最適生長下的2.2與1.4倍。研究結果顯示betLH13與opuDH13基因的表現和極端高鹽太古生物因應環境的滲透壓與高溫逆境有關。
Microorgansims could accumulate organic compatible solutes (osmolytes) to encounter the osmotic stress. BCCT family transporters can transport glycine betaine, choline and carnitine from extracellular environment by proton/ sodium motive force as energy source. Extreme halophile lives in the highly saline environment and can internalize large amount of potassium to balance the osmolality. Due to the high concentration of K+ inside the cells, there are more acidic amino acids used in proteins for maintaining the proper protein structure. There are no direct evidences so far to show that extreme halophile could uptake osmolyte from environments. There are two BCCT secondary transporter genes, betLH13 and opuDH13, have been identified by Ding and Lai through comparative genomics of Haloterrigena sp. H13. In this study, the full length of betLH13 and opuDH13 were cloned and sequenced. The betLH13 and opuDH13 share 75% identity of Halorubrum lacusprofunid BetT and 69% identity Halobacterium sp. NRC-1 OpuD, respectively. From sequence analysis, they both have high GC content and low pI. The secondary structure prediction shows that both of BeLH13 and OpuDH13 have the characteristic signature of BCCT, however BetLH13 replace one of four tryptophans with an alanine. The betLH13 was further transformed into the glycine betaine transport-negative mutant E. coli MKH13 for glycine betaine transport assay. No glycine betaine uptake activity was detected whether cell grown at 0 M NaCl or 0.5 M NaCl. Reconfirmed the betLH13 sequence, transcription and translation in E. coli MKH13 and found that single base deletion occurred at the betLH13 sequence and formed a early stop codon which generated a 48 KD BetLH13 instead of 63 KD. Additionally, 14C labeled glycine betaine was prepared, purified and quantitated from the 14C choline oxidation and tested the glycine betaine transport activity of Haloterrigena sp. H13 under optimal growth condition with 45C 4 M NaCl. No transport activity was detected. However, the RT-PCR analysis the expression of betLH13 and opuDH13 at Haloterrigena sp. H13 under hyperosmotic stress (5 M NaCl) and high temperature stress (55C) increased 2.7 fold than the culture at optimal growth condition. And under the hypoosmotic stress (3 M NaCl) and low temperature stress (20C), the expression of the betLH13 and opuDH13 increased 2.2 and 1.4 fold, respectively. The primarily result showed here indicating that the expression of BCCT transports BeLH13 and OpuDH13 associated with the adaptation of osmotic and temperature stresses of the extreme halophiles.
其他識別: U0005-2508200917353300
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