| Summary: | 碩士 === 國立中興大學 === 生命科學系所 === 97 === 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 45°C 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 (55°C) increased 2.7 fold than the culture at optimal growth condition. And under the hypoosmotic stress (3 M NaCl) and low temperature stress (20°C), 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.
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