Academic literature on the topic 'Strawberry; Antifreeze protein gene'

A variety of antifreeze proteins is produced by marine teleosts inhabiting polar regions to ensure protection from internal ice formation at subzero temperatures. Combining evidence from paleoclimatology, teleostian evolution, and studies of antifreeze gene organization, the case is made for Cenozoic cooling as the force driving antifreeze evolution in marine teleosts. The distribution of antifreeze types amongst teleost suborders, families, genera, and species correlates with Cenozoic glaciation in the Southern Hemisphere preceding that in the Northern Hemisphere by approximately 25 million yr. The genomic organization of antifreeze genes suggests recent and extensive amplification, an event compatible with their proposed stress-induced origin. Also, a realignment of the nototheniids with the Gadiformes based on antifreeze protein data is suggested.

Many marine teleosts have adapted to ice-laden seawater by evolving antifreeze proteins and glycoproteins. These proteins are synthesized in the liver for export to the blood where they circulate at levels of up to 20 mg/mL. There are at least four distinct antifreeze protein classes differing in carbohydrate content, amino acid composition, protein sequence, and secondary structure. In addition to antifreeze structural diversity, fish species differ considerably with respect to mechanisms controlling seasonal regulation of plasma antifreeze concentrations. Some species synthesize antifreeze proteins immediately before the onset of freezing conditions, some synthesize them in response to such conditions, whereas others possess high concentrations all year. Endogenous rhythms, water temperature, photoperiod, and pituitary hormones have all been implicated as regulators of plasma antifreeze protein levels. The structural diversity of antifreeze proteins and their occurrence in a wide range of fish species suggest that they evolved separately and recently during Cenozoic glaciation. Invariably, the genes coding for these antifreeze proteins are amplified, sometimes as long tandem arrays, suggesting intense selective pressure to produce large amounts of protein. The distribution of antifreeze gene types among fish species suggests that they could serve as important tools for studying phylogenetic relationships.

Antifreeze proteins inhibit ice growth and are crucial for the survival of supercooled fish living in icy seawater. Of the four antifreeze protein types found in fishes, the globular type III from eelpouts is the one restricted to a single infraorder (Zoarcales), which is the only clade know to have antifreeze protein-producing species at both poles. Our analysis of over 60 unique antifreeze protein gene sequences from several Zoarcales species indicates this gene family arose around 18 Ma ago, in the Northern Hemisphere, supporting recent data suggesting that the Arctic Seas were ice-laden earlier than originally thought. The Antarctic was subject to widespread glaciation over 30 Ma and the Notothenioid fishes that produce an unrelated antifreeze glycoprotein extensively exploited the adjoining seas. We show that species from one Zoarcales family only encroached on this niche in the last few Ma, entering an environment already dominated by ice-resistant fishes, long after the onset of glaciation. As eelpouts are one of the dominant benthic fish groups of the deep ocean, they likely migrated from the north to Antarctica via the cold depths, losing all but the fully active isoform gene along the way. In contrast, northern species have retained both the fully active (QAE) and partially active (SP) isoforms for at least 15 Ma, which suggests that the combination of isoforms is functionally advantageous.

Strawberry (Fragaria ananassa) is an economically important soft fruit that is highly valued as a fresh product and flavouring. During ripening, strawberry fruits undergo a number of physiological changes affecting colour, texture and flavour. An understanding of these changes at the biochemical and molecular level will be important in developing strategies for enhancing the quality attributes of this fruit. A cDNA encoding a ripening- enhanced acyl carrier protein (RE-ACP) was previously isolated from strawberry fruit. AC? is an essential component of fatty acid synthesis in both plants and animals. The aims of this thesis were to isolate and characterise this and other members of the ACP multigene family expressed in strawberry fruit. Six closely related putative AC? cDNA isoforms were identified from strawberry. Two of these were obtained by screening a cDNA library from ripe fruit and three were obtained by a technique known as candidate fragment length polymorphism (CFLP) that utilised ACP gene-specific primers for AFLP-cDNA display. Northern analysis was not able to differentiate their expression but ACP was highly up-regulated in ripening fruit whereas low levels of expression were detected in other strawberry tissues, including achenes (seeds), expanding leaves and flowers. The RE-ACP was over-expressed in E. coli and the recombinant protein partially purified. The over-expressed protein had a M(_r) of 20kDa on SDS-PAGE and appeared to form a dimer. A genomic library was constructed from F. ananassa from which two different genomic clones closely related to RE-ACP were obtained. Promoter analysis indicated the presence of regulatory elements. The characterization of putative ACP cDNA and genomic clones, including the 5' upstream regions, is described and their possible role in strawberry fruit is discussed. Key words: Strawberry, fruit, ripening, gene expression, genomic, cDNA, fatty acid, acyl carrier protein, aroma, promoter.

Antifreeze proteins (AFPs) protect marine teleosts from freezing in icy seawater by binding to nascent ice crystals and preventing their growth. It has been suggested that the gene dosage for AFPs in fish reflects the degree of exposure to harsh winter climates. The starry flounder, _Platichthys stellatus_, has been chosen to examine this relationship because it inhabits a range of the Pacific coast from California to the Arctic. This flatfish is presumed to produce type I AFP, which is an alanine-rich, amphipathic alpha-helix. Genomic DNA from four starry flounder was Southern blotted and probed with a cDNA of a winter flounder liver AFP. The hybridization signal was consistent with a gene family of approximately 40 copies. Blots of DNA from other starry flounder indicate that California fish have far fewer gene copies whereas Alaska fish have far more. This analysis is complicated by the fact that there are three different type I AFP isoforms. The first is expressed in the liver and secreted into circulation, the second is a larger hyperactive dimer also thought to be expressed in the liver, and the third is expressed in peripheral tissues. To evaluate the contribution of these latter two isoforms to the overall gene signal on Southern blots, hybridization probes for the three isoforms were isolated from starry flounder DNA by genomic cloning. Two clones revealed linkage of genes for different isoforms, and this was confirmed by genomic Southern blotting, where hybridization patterns indicated that the majority of genes were present in tandem repeats. The sequence and diversity of all three isoforms was sampled in the starry flounder genome by PCR. All coding sequences derived for the skin and liver isoforms were consistent with the proposed structure-function relationships for this AFP, where the flat hydrophobic side of the helix is conserved for ice binding. There was greater sequence diversity in the skin and hyperactive isoforms than in the liver isoform, suggesting that the latter evolved recently from one of the other two. The genomic PCR primers are currently being used to sample isoform diversity in related right-eyed flounders to test this hypothesis.
Thesis (Master, Biochemistry) -- Queen's University, 2009-01-30 13:38:08.346

碩士
國立中興大學
食品科學系
89
I.Part I. Improvement of the electrotransformation efficiency of threonine-treated Bacillus subtilis Bacillus subtilis is an industrial important bacterium, which has been used for the manufacture of a variety of enzymes, antibiotics and fine biochemicals. For strain improvement and genetic manipulation, transformation of this bacterium is an important step. There are several methods to introduce plasmid DNA into Bacillus subtilis, such as competent cell transformation, protoplast transformation, and electrotransformation. Among these methods, electro-transformation is the most attractive approach for its simplicity and easiness. However, the transformation efficiency by electrotransformation is generally low. For this reason, many researchers are attempting to improve the transformation efficiency of electrotransformation. In this study, we tried to improve the electrotransformation efficiency of Bacillus subtilis DB104. The cell wall of Bacillus subtilis DB104 is weakened by supplement of threonine in culture medium. We examined the effect of electrical parameters, cell concentration, the concentration of plasmid DNA, plasmid purity, plasmid source, electroporation buffer, recovery medium and recovery time on the electrotransformation efficiency of threonine-treated Bacillus subtilis. The results showed: (1)Efficiencies of transformation increased with applied voltage to an optimum of 1.77×104 transformants /μg at a field strength of 8.75 kV/cm and resistance of 500Ω. (2)The transformation efficiency increased with the increases of cell concentrations. (3)Plasmid concentration did not influence transformation efficiency, but the plasmid purity and source did. (4)The best electrotransformation buffer is SHMPYT(0.25M sucrose, 1mM Hepes, 1mM MgCl2, 20%(v/v) PEG (polyethylene glycol) 6000, 0.125% yeast extract, 0.25% tryptone). (5)The optimal recovery time is 2 hours. (6) The optimal medium is 2LB. The maximum transformation efficiency of Bacillus subtilis DB104 was 2.25×105 transformants/μg. It is much higher than the transformation efficiency of original procedure (7.22×103 transformants/μg). Transferred plasmid DNAs isolated from transformants were the same as those of intact plasmids. Therefore, it is clear that the transferred DNAs did not undergo significant rearrangement or deletion. Using this procedure, ligation mixture can be directly transformed into Bacillus subtilis DB104, allowing direct molecular cloning of DNA into this organism. II.Design and synthesis of type I antifreeze protein gene and expression in Bacillus subtilis and Escherichia coli. Abstract Antifreeze proteins (AFPs) are a group of proteins that can depress freezing point, inhibit ice recrystallization and modify the morphology of ice crystal. They are found in a wide range of organisms living in cold ambient conditions, including bacteria, fungi, plants, invertebrates and fish. Among these proteins, fish antifreeze proteins have been studied extensively. To date, five distinct types of fish AFPs have been described: antifreeze glycoproteins (AFGP), type I antifreeze proteins (AFP I), type II antifreeze proteins (AFP II), type III antifreeze proteins (AFP III) and type IV antifreeze proteins (AFP IV). In order to study their structure-function relationship or to apply them in industry, it is essential to gain a great quantity of these proteins. Three methods were developed to achieve this purpose: chemical synthesis, genetic engineering and isolation from fish bloods. Among these methods, genetic engineering is the most attractive one for scientists. In this study, we designed and constructed a synthetic gene for recombinant antifreeze protein( rAFP ). The protein sequence of rAFP was designed to include four copies of the 11 amino acid antifreeze motif (Thr-X2-polar amino acid-X7) and was reverted into nucleotide sequence by Bacillus subtilis preferred codon usage. Using overlap extension polymerase chain Reaction technique to synthesize rAFP gene. Then, the PCR product was cloned into the Bacillus subtilis expression vector or Escherichia coli expression vector, and was transformed into the host strains by electroporation. In B. subtilis DB430, the expression level of antifreeze protein is very low. In E. coli host cells, the rAFP can be expressed after isopropyl-β-D-thiogalactopyranoside (IPTG) induction. rAFP fused to Subtilisin YaB signal peptide can be exported to periplasmic space, even secreted to medium by signal peptide cleavage through secretion machinery. Otherwise rAFP fused to OmpT signal peptide accumulated as insoluble inclusion bodies in the E. coli host. Expressed rAFPs can be purified by using Ni-NTA affinity chromatography. The N-terminal amino acid seqence of purified protein confirmed the identity of the expressed and purified protein as rAFP. All puried rAFPs can decrease the size of ice crystal.