• The Plant Pathology Journal
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• 제20권2호
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• pp.158-163
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• 2004

Chlorella is a eukaryotic microalgae which shares metabolic pathways with higher plants. These charac-teristics make chlorella a potential candidate for eukaryotic overexpression systems. Recently, a foreign flounder growth hormone gene was stably introduced and expressed in transformed Chlorella ellipsoidea by using a modified plant transformation vector that contains cauliflower mosaic virus (CaMV) 35S pro-moter and the phleomycin resistant Sh ble gene as a selection marker. In this study, this same vector was modified by incorporating a promoter and a 3' UTR region of the 33kDa peptide gene from a chlorella virus that was isolated in our laboratory. The 33kDa gene promoter was used to replace the 35S promoter and the 3' UTR was introduced to separate the target gene and downstream Sh ble gene. Three different chlorella transformation vectors containing human erythropoietin (EPO) gene were constructed. The mp335EPO vector consists of a promoter from the 33kDa peptide gene, whereas the mp3353EPO vector contains the same promoter from the 33kDa peptide gene and its 3' UTR. The mp35S33pEPO vector contains the 35S promoter and the 3' UTR from the 33 kDa peptide gene. There was no significant difference in the expression levels of EPO protein in chlorella cells transformed with either of three of the transformation vectors. These data indicate that the promoters from the chlorella virus are comparable to the most common CaMV 35S promoter. Furthermore, these data suggest that other promoters from this virus can be used in future construction of chlorella transformation system for higher expression of target proteins.

• 미생물학회지
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• 제7권4호
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• pp.143-152
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• 1969

This investigation was proceeded to define the effects of gibberellic acid on the growth of Chlorella by determining the contents of chlorphyll and changes in various components in Chlorella cells according to the concentration of treated GA. The growth of Chlorella was accelerated with telative low concentrations of GA(10, 40 ppm) and was restrained with relative high concentrations of GA(70, 100, 200 ppm). The synthetic ability of chlorophyll of GA was inhibited generally in proportion to the concentration of treated-GA and the higher the concentration of GA was applied, the longer time was required in the restoration. The contents of RNA, protein and soluble carbohydrate were increased PCA-soluble amino acid and polysacharide were decreased in those cell components between the accelerated and restrained group. Consequently, the effect on accelerated growth in relative low concentrations of GA is considered to be caused by the powerful effet on expansion growth of GA. It is presumed that the effect of restrained growth in relative high concentrations of GA is due to the inhibitory effect on the chlorophyll synthesis.

• 미생물학회지
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• 제20권1호
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• pp.27-40
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• 1982

The effects of heavy metals on the growth rate and phosphate metabolism of Chlorella elliposidea cells were investigated. Chlorella cells were cultured in the media treated with Hg(0.3, 0.7, 0.9 ppm), Cd(1, 5, 15ppm), and Zn(1, 5, 50ppm) for 6days. Aliquots cells were taken out at the inoculation and at intervals during the culture, and measured packed cell vlolume and optical density. The inhibitions of heavy metals on the growth rate and chlorophyll contents were traced. Also after 6 days culture, the amounts of inorganic phosphate and organic compounds of various fractions in Chlorella cells were observed. The turbid effects of heavy metals on the growth rate and chlorphyll contents of Chlorella cells were in order of Hg>Cd>Zn. Because heavy metals depressed the biosynthesis of inorganic polyphosphates and nucleic acids and turn over of inorganic phosphates, the amounts of various phosphate compounds were decreased. The inhibitory effect of photosynthesis by heavy metals resulted in lower contents of carbohydrate. Due to the turbidity of biosynthesis of amino acids by heavy metals, contents of protein were reduced in comparison with those of control. It is suggested conciusively that the minimum concentrations affected by heavy metals on the growth rate and phosphate metabolism of Chlorella cells were 0.7 ppm Hg, 15ppm Cd, 50ppm Zn.

• 한국해양바이오학회지
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• 제16권1호
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• pp.26-35
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• 2024

Chlorella has various biotechnological applications, including in the biomedical and pharmaceutical industries, because of its advantages, including rich nutrients, fast growth rate, easy cultivation, and high biomass. We used the Agrobacterium-mediated transformation method to express human GM-CSF and EGF proteins, which are widely used in regenerative medicine, cosmetics, and pharmaceutical materials in Chlorella. The codon-optimized hGM-CSF and hEGF genes were cloned into plant binary vectors and transformed into Chlorella vulgaris using the Agrobacterium-mediated coculture transformation method. After transformation, genomic DNA PCR was performed for each C. vulgaris line that was stably subcultured on an antibiotic-resistant solid medium to confirm the insertion of hGM-CSF and hEGF into the chromosome. Furthermore, PT-PCR and protein expression of hGM-CSF and hEGF in each transformed C. vulgaris were significantly increased compared to the untransformed Chlorella. This study suggests that high-value proteins, including hGM-CSF and hEGF, which are foreign genes of C. vulgaris, can be stably expressed through the Agrobacterium-mediated Chlorella transformation system.

• Journal of Microbiology and Biotechnology
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• 제16권6호
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• pp.952-960
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• 2006

As a unicellular green alga that possesses many of the metabolic pathways present in higher plants, Chlorelia offers many advantages for expression of heterologous proteins. Since strong and constitutive promoters are necessary for efficient expression in heterologous expression systems, the development of such promoters for use in the Chlorella system was the aim of this study. Proteins encoded by the early genes of algal viruses are expressed before viral replication, probably by the host transcriptional machinery, and the promoters of these genes might be useful for heterologous expression in Chlorella. In this study, putative promoter regions of DNA polymerase, ATP-dependent DNA ligase, and chitinase genes were amplified from eight Korean Chlorella virus isolates by using primer sets designed based on the sequence of the genome of PBCV-1, the prototype of the Phycodnaviridae. These putative promoter regions were found to contain several cis-acting elements for transcription factors, including the TATA, CAAT, NTBBF1, GATA, and CCAAT boxes. The amplified promoter regions were placed into Chlorella transformation vectors containing a green fluorescence protein (GFP) reporter gene and the Sh ble gene for phleomycin resistance. C. vulgaris protoplasts were transformed and then selected with phleomycin. The GFP fluorescence intensities of cells transformed with chitinase, DNA polymerase, and DNA ligase gene promoter-GFP fusion constructs were 101.5, 100.8, and 95.8%, respectively, of that of CaMV 35S-GFP-transformed Chlorella cells. These results demonstrate that these viral promoters are active in transformed Chlorella.

• 미생물학회지
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• 제4권1호
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• pp.14-20
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• 1966

1. Uniformly $^{32}P$-labeled Chlorella cells were further grown in a standard "cold" medium and aliquots of the algal cells were taken out at the beginning of, and at intervals during the culture, and subjected to analyze the contents of $^{32}$ P and total P in various fractions of the cell constituents. 2. When the $^{32}P$--labeled algae were grown in a normal "cold" medium, the P-contents in the fractions of DNA and protein increased. In the meantime the $^{32}P$- in acid-insoluble polyphosphate fraction decreased considerably, while that in RNA-polyphosphate complex significantly increased. 3. It was inferred that, under the experimental conditions of the present study, the phosphorus in polyphosphate seems to be transferred to RNA polyposphate complex and the phosphorus used in the synthesis of DNA and protein was, directly or indirectly, taken from those fractions above.ose fractions above.

• 미생물학회지
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• 제5권2호
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• pp.61-68
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• 1967

In the process of the incorporation of orthophsphate into protein and other cell constituents, the role of inorganic polyphosphate and RNA-polyphosphate complex and the correlation between them were pursued by analyzing the contents of $^{32}P$ and total P in various fractions of Chlorella cells, which had been uniformly labeled with $^{32}P$ before the inoculation in a normal "cold" medium or P-free medium during the culture. The effects of ionizing radiation and various micronutritional-element deficiencies on the phosphate incorporation into, and biosynthesis of, protein and other introgenus compounds in the cells were also observed. When the uniformly $^{32}P$-labeled algae were grown in a normal "cold" medium the contents of $^{32}$ P in the fractions of protein, DNA and RNA-polyphosphate complex increased, but those in the fraction of acid-insoluble polyphosphate decreased. On the other hand, amount of $^{32}P$in the fraction of RNA was almost unchanged in spite of rapid increase of the total P. In the growing period of $^{32}P$-labeled algae in a P-free medium, amounts of $^{32}P$ in the fractions of DNA, protein and lipid increased, while those in the fractions of RNA-polyphosphate and inorganic polyphosphates decreased. When the algal cells were irradiated with about 70, 000r of gamma-rays before the inoculation in the medium, amounts of phosphate in the fractions of DNA, RNA, nucleotides and protein decreased during the culture, compared with those of the control. However, the phosphate content in the fraction of acid-insoluble polyphosphate of the irradiated cells increased than those of the control. In the growing period of the algae in a Mo-free, medium, amounts of acid-soluble total phosphate and nucleotides of the cells increased, while the amounts of residual protein and RNA decresed compared with those of the normal cells. Amounts of alkali-labile protein and phospholipid of the cells grown in a B-free medium decreased, whereas amount of phosphate in acid-soluble fraction increased compared with the control. In general, the contents of protein and RNA in each microelement deficient cells decreased more or less, compared with those in the normal cells.in the normal cells.

• Fisheries and Aquatic Sciences
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• 제22권1호
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• pp.2.1-2.9
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• 2019

In the present study, the protective effects of Hizikia fusiforme and Chlorella sp. extracts on lead acetate-induced hepatotoxicity were investigated. Hepatic damage was induced in rats by intraperitoneal (i.p.) injection of lead acetate and the protective effects of H. fusiforme (HZK) and Chlorella sp. (CHL) extracts on lead acetate-induced hepatic damage in rat liver were examined. The results revealed significantly increased glutamic oxaloacetate and glutamic pyruvic transaminase levels in the group treated with lead acetate only (Pb group); oral administration of HZK and CHL extracts tended to decrease the enzyme levels similar to those observed in the control group. Regarding antioxidant enzymes, superoxide dismutase activity was increased in the Pb group and decreased in a concentration-dependent manner in the HZK- and CHL-treated groups. Glutathione levels were increased in a concentration-dependent manner in the HZK- and CHL-treated groups. There was no significant difference in catalase activity. Western blot analysis showed inflammation-related protein expression in mitogen-activated protein kinase and Nrf2 pathways was affected in the HZK- and CHL-treated groups. Therefore, HZK and CHL extracts exerted antioxidant and anti-inflammatory effects against lead acetate-induced hepatotoxicity. Development of functional health foods containing HZK and CHL extracts, which have hepatoprotective effects against inhaled lead acetate, should be considered.

• 한국해양바이오학회지
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• 제16권1호
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• pp.45-54
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• 2024

Microalgae have great potential in the biomedical and pharmaceutical industries as a new type of bioreactor that can produce proteins for specific purposes, including recombinant proteins, pharmaceuticals, and industrial enzymes. Despite the production advantages and importance of microalgae-based expression systems, studies on secretion efficiency are limited. In this study, for stable expression and efficient secretion of the heterologous protein (human SCF and human INFγ) in Chlorella vulgaris, we constructed SP:hSCF:His and SP:hINFγ:His plant binary vectors using the signal peptide (SP) of Chlamydomonas reinhardtii, and we obtained stable transformants through the effective agrobacterium-mediated transformation of these vectors. Transformants with accurately inserted hSCF and hINFγ demonstrated stably increased mRNA and protein expression using RT-PCR and western blotting under the same culture conditions. Following the analysis of the proteins secreted into the culture medium using ELISA, it was confirmed that hINFγ was effectively produced in the transformed C. vulgaris culture medium. The overall findings indicate that the combination of heterologous protein and SP may be crucial for ensuring the expression and secretion of recombinant proteins in Chlorella culture systems.

• 한국유기농업학회지
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• 제26권3호
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• pp.393-408
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• 2018

본 연구는 클로렐라 건조분말을 산란계의 사료에 첨가해서 계란의 품질과 난황의 지방산 조성에 미치는 영향에 대해서 분석하였다. 시험에 사용한 유기 산란계 사료의 수분함량은 약 12.8%, 회분함량은 10.8%, 조단백질함량은 18.0%, 조지방 함량은 2.5%이었다. 사료에 첨가한 클로렐라분말의 수분함량은 약 1.54%, 회분함량은 6.53%, 조단백질은 54.56%, 조지방 함량은 2.45%이었다. 계란 난각의 색깔은 클로렐라를 급여한 후, 시간의 경과에 따라 대조구에 비해 유의적으로 진해졌다. 파각강도는 클로렐라를 급여한 후, 10일까지 증가하였으며 대조구에 비해 유의적으로 강하였다. 계란 난각의 두께도 클로렐라를 급여한 것이 대조구에 비해 유의적으로 두꺼웠다. 클로렐라 급여한 계란 난백의 높이는 대조구에 비해 높았다. 계란 난백의 품질 기준이 되는 호유닛은 클로렐라를 급여한지 10일 후, 92.0 HU로 대조구(84.8 HU)에 비해 유의적으로 높았다. 클로렐라를 급여한 계란 난황의 황색도도 대조구에 비해 유의적으로 진한 황색을 나타내었다. 클로렐라를 급여한 계란의 무게는 급여 15일 후, 대조구에 비해 7.5% 증가하였으며, 단백질함량은 급여 10일과 15일 후, 대조구에 비해 각각 11.9%, 10.7% 증가하였다. 클로렐라 급여에 따른 계란 노른자의 지방산 함량의 변화를 조사한 바, 난황의 주요 지방산 조성은 oleic acid, trans-linoleic acid, palmitic acid, ${\alpha}$-linolenic acid, stearic acid, DHA, EPA, palmitoleic acid, heptadecanoic acid 순으로 나타났다. Palmitoleic acid는 클로렐라를 급여한 처리구가 대조구에 비해 감소하였다. 포화지방산은 클로렐라를 급여한 계란보다 대조구에서 높게 나타났고, 불포화지방산은 대조구보다 클로렐라를 급여한 계란에서 유의적으로 높게 나타났다. 불포화지방산과 포화지방산의 비율은 대조구보다 클로렐라를 급여한 계란이 높았다. 이상의 결과를 종합하여 볼 때 클로렐라 생균분말을 산란계의 사료에 첨가해서 급여할 경우 계란의 품질 향상과 난황의 불포화지방산 함량을 높이는데 긍정적인 영향을 미치는 것으로 판단된다.