• 식물조직배양학회지
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• 제24권2호
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• pp.93-97
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• 1997

Linoleic acid를 linolenic acid로 전환시키는 지방산불포화효소의 유전자(fad3)를 유채의 fad3 DNA probe를 이용한 plaque hybridization방법으로 $\lambda$ZAPII Arabidopsis thaliana cDNA expression library로부터 분리하였으며 1.8 kb-EcoRI DNA조각을 지니는 lambda clone을 pGEM7으로 subcloning하여 염기서열을 분석하였다. 그 결과로부터의 아미노산서열분석에 의하면 fad3 유전자는 open reading frame이 386개의 아미노산으로 이루어졌으며 44,075 Da의 분자량이 예측되고 있다. 엽록체의 $\omega$-3 지방산불포화효소(fad7)와 endoplasmic reticulum의 지방산불포화효소(fad2)와의 비교시 각각 70%와 58%의 유사성이 나타났다. 특히 82-151의 아미노산서열과 276-333의 아미노산지역은 보존성이 높았으며 이는 불포화지방산효소의 기능에 필수적인 지역으로 보여진다. 한편 대장균을 이용한 IPTG에 의한 Fad3단백질의 유도생성은 대장균의 생육에 독성효과를 나타내는 것으로 나타났다.

• Journal of Plant Biotechnology
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• 제41권3호
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• pp.146-158
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• 2014

카멜리나(Camelina sativa)는 십자화과(Brassicaceae)에 속하는 유지작물이다. 카멜리나 종자에는 건물 중의 약 40%에 해당하는 저장 오일을 가지고 있고, 이러한 저장오일은 식품뿐만 아니라 산업재료로 이용이 가능하다. Microsomal delta-12 fatty acid desaturase2 (FAD2) 효소는 oleic acid를 linoleic acid로 전환시키는데, 종자 내 oleic acid의 함량 차이를 보이는 품종들에서 FAD2 유전자의 polymorphism이 보고되었다. 본 연구에서는 카멜리나(Camelina sativa L. 품종 CAME)에 존재하는 3개의 FAD2 유전자를 발달하는 종자로부터 분리하였다. 3개의 카멜리나 FAD2 유전자의 염기서열 및 아미노산 서열은 카멜리나 품종 Sunesone과 SRS933으로부터 확인된 FAD2 유전자들과 여러 단자엽 및 쌍자엽 식물의 FAD2 유전자들의 염기서열 및 아미노산 서열과 상동성을 비교하였다. FAD2 효소의 활성을 결정짓는다고 알려진 histidine motif (HECGHH, HRRHH 그리고 HVAHH)와 효소 활성에 영향을 주는 SNP (single nucleotide polymorphism) 마커라고 알려진 소수성 아미노산 계열인 valine 혹은 isoleucine이 3 개의 카멜리나 FAD2에서도 잘 보존되어 있음을 확인하였다. 세개의 카멜리나 FAD2 유전자들 중 CsFAD2-1의 경우 카멜리나의 발달하는 조직에서 전반적으로 높은 발현 양상을 보이는 반면 CsFAD2-2와 CsFAD2-3.1은 꽃과 발달하는 종자에서 특이적인 발현을 보였다. 애기장대 fad2-2 돌연변이체에 3개의 카멜리나 FAD2를 각각 도입한 형질전환 식물체의 종자에는 애기장대 fad2-2 돌연변이체 종자대비 oleic acid의 함량이 감소하고, linoleic acid 함량은 증가하는 표현형이 관찰되었다. 이러한 결과는 카멜리나로부터 분리된 3개의 FAD2가 효소로서 활성을 가지고 있다는 것을 의미한다. 더불어 분리된 카멜리나의 FAD2 유전자는 종자 오일 성분이 변화된 유지작물을 개발하는데 응용될 수 있을 것이다.

• Plant Biotechnology Reports
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• 제4권3호
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• pp.185-192
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• 2010

The Arabidopsis ${\omega}$-3 fatty acid desaturase (AtFAD7) catalyzes the synthesis of trienoic fatty acids (TA). A transgenic tobacco line, T15, was produced by a sense AtFAD7 construct and showed a cosuppression-like phenotype, namely extremely low TA levels. The sequence similarity between AtFAD7 and a tobacco ortholog gene, NtFAD7, was moderate (about 69%) in the coding sequences. AtFAD7 siRNAs accumulated at a high level, and both AtFAD7 and NtFAD7 mRNAs are degraded in T15 plants. The low-TA phenotype in T15 was dependent on a tobacco RNA-dependent RNA polymerase6 (NtRDR6). We also produced tobacco RNAi plants targeting AtFAD7 gene sequences. The AtFAD7 siRNA level was trace, which was associated with a slight reduction in leaf TA level. Unexpectedly, this RNAi plant showed an increased NtFAD7 transcript level. To investigate the effect of translational inhibition on stability of the NtFAD7 mRNAs, leaves of the wild-type tobacco plants were treated with a translational inhibitor, cycloheximide. The level of NtFAD7 mRNAs significantly increased after cycloheximde treatment. These results suggest that the translational inhibition by low levels of AtFAD7 siRNAs or by cycloheximide increased stability of NtFAD7 mRNA. The degree of silencing by an RNAi construct targeting the AtFAD7 gene was increased by co-existence of the AtFAD7 transgene, where NtRDR6-dependent amplification of siRNAs occurred. These results indicate that NtRDR6 can emphasize silencing effects in both cosuppression and RNAi.

• BMB Reports
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• 제29권4호
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• pp.308-313
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• 1996

An 1.4 kb of the fad3 cDNA encoding microsomal linoleic acid desaturase catalyzing the conversion of linoleic acid (18:2, ${\omega}-6$) to linolenic acid (18:2, ${\omega}-3$) was introduced into tobacco plants by the Agrobacterium-mediated plant transformation, Among the transgenic tobacco plants conferring kanamycin resistance, five transformants showing increment in unsaturated fatty acid contents were selected and further analyzed for the transgenecity, In genomic Southern blot analyses, copy numbers of the integrated fad3 DNA in chromosomal DNA of the five transgenic tobacco plants were varied among the transgenic lines. By Northern blot analyses, the abundancy of the fad3 mRNA transcript directed by Cauliflower Mosaic Virus 35S promoter was consistent with the relative copy number of the fad3 DNA integrated in the chromosome of transgenic tobacco plants. When compared with the wild type, accumulation of linolenic acid in transgenic tobacco roots was elevated 3.7- to 4.7-fold showing a corresponding decrease in the linoleic acid contents; however, slight increments for linolenic acid were noticed in transgenic leaf tissues. These results indicated that the elevated level of fad3 expression is achieved in transgenic tobacco plants.

• 약학회지
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• 제23권3_4호
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• pp.147-152
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• 1979

Experiments were carried out to investigate for the interaction between FAD solution and synthetic resin containers made of polyvinylchloride(PVC), polyethylene(PE), and polycarbonate(PC), and for the effect of glycyrrhizine or malic acid on stabilization of FAD in aqueous solution by accelerated stability analysis. Analysis of FAD was determined by means of spectrometer and by separating by paper chromatography and metal ions were detected by atomic absorption spectrophotometer, which were extracted from containers by means of Food and Additive Regulation Standard. The thermal decomposition of FAD in aqueous solution was pseudo first order reaction and it was inhibited by adding glycyrrhizine or malic into the solution. PVC, PE and PC containers accelerated the decomposition of FAD in solution. It is assumed that bivalent heavy metals in resin containers may catalize the hydrolysis of FAD. The metals detected from the containers were Ca, Zn, Cu, Fe, Pb and Cd. And the total amounts of detected metals from PVC were 6.2mcg/cm$^{2}$, PE, 5.5mcg/cm$^{2}$, and PC, 2.7mcg/cm$^{2}$ which were proportional to the rate constant of FAD decomposition in aqueous solution.

• KSBB Journal
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• 제19권4호
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• pp.331-334
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• 2004

재조합 E. coli를 이용한 MCL-PHA의 생산에서 fatty acid pathway로부터 PHA 생합성 전구체 물질들이 만들어진다는 사실과 함께 이에 관여하는 enzymes이 밝혀지고 있다. 본 논문에서는 protein homology search로부터 탐색된 paaG와 ydbU genes의 PHA 생합성에서의 역할을 확인하기 위하여 paaG와 ydbU gene이 각각 knock-out된 mutant E. coli strains 를 제작하였다. 제작된 mutant E. coli들은 모균주들보다 낮은 PHA 농도와 함량을 가졌으며, 이러한 결과들로부터 paaG와 ydbU는 fatty acid pathway에서 PHA synthesis의 전구체 물질들을 공급한다는 사실을 확인하였다. 또한, 새로운 FadB homologous enzyme YgfG를 탐색하였으며, ygfG gene이 overexpression된 균주와 ygfG mutant를 제작하여 PHA 합성을 실험한 결과 ygfG도 paaG와 ydbU와 유사한 역할을 한다는 사실을 밝혔다. 이러한 연구결과들은 E. coli에서의 MCL-PHA 단량체들의 합성 경로를 확인하여 효과적인 PHA 생산 균주를 제작할 수 있게 할 것이다.

• 센서학회지
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• 제27권3호
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• pp.156-159
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• 2018

We developed a glucose sensor using glucose dehydrogenase flavin adenine dinucleotide (GDH-FAD). The structure of the three-layer glucose sensor was simplified, in which a single-layer design was used to lower the unit cost, and GDH-FAD was used to increase the measurement reliability. GDH-FAD has less impact on the 20 interfering substances that affect blood glucose measurement, such as galactose and maltose compared to glucose oxidase (GOD), and is not affected by the oxygen saturation; therefore, it is possible to measure both arterial or venous blood and thus less susceptibility to hematocrit. In this study, we developed a single-layer glucose sensor strip with low hematocrit effect using the GDH-FAD enzyme, and measured and evaluated the performance.

• BMB Reports
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• 제29권3호
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• pp.253-260
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• 1996

An enzyme that hydrolyzes flavin-adenine dinucleotide (FAD) was found to be present in rat liver lysosomal membrane prepared from Triton WR-1339 filled lysosomes (tritosomes) purified by flotation on sucrose. This FAD phosphohydrolase (FADase) exhibited optimal activity at pH 8.5 and had an apparent Km of approximately 3.3 mM. The activity was decreased 50~70% by dialysis against EDTA and this was restored by $Zn^{2+}$, $Mg^{+2}$, $Hg^{+2}$, and $Ca^{+2}$ ions inhibited the enzyme, but $F^-$ and molybdate had no effect. The enzyme was also inhibited by p-chloromercuribenzoate (pCMB), reduced glutathione and other thiols, cyanide, and ascorbate. The presence of ATP, ADP, AMP. ${\alpha}-{\beta}-methylene$ ATP, AMP-p-nitrophenyl phosphate (PNP), GMP, and coenzyme A (CoA) decreased the activity on FAD, but pyrimidine nucleotides, adenosine, adenine, or $NAD^+$ were without effect. Phosphate stimulated the activity slightly. FAD phosphohydrolase activity was separated from ATPase and inorganic pyrophosphatase activities by solubilization with detergents and polyacrylamide gel electrophoresis and by linear sucrose density gradient centrifugation suggesting that the enzyme is different from ATPase, inorganic pyrophosphatase, and soluble lysosomal FAD pyrophosphatase. Paper chromatography showed that FAD was hydrolyzed to flavin mononucleotide (FMN) and AMP which were further hydrolyzed to riboflavin and AMP by phosphatases known to be present in lysosomal membranes. Incubation of the intact Iysosomes with pronase showed that the active site of FAD phosphohydrolase must be oriented to the cytosol. The FAD hydrolyzing activity was detected in Golgi, microsome, and plasma membrane, but not in mitochondria or soluble lysosomal preparations.

• 한국약용작물학회지
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• 제12권5호
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• pp.424-427
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• 2004

Microsomal ${\omega}-3$ fatty acid desaturase (FAD3) is an essential enzyme in the production of the n-3 polyunsaturated fatty acid ${\alpha}-linolenic$ acid during the seed developing stage. To understand the regulatory mechanism of the gene encoding the ${\omega}-3$ fatty acid desaturase, a genomic fragment corresponding to the previously isolated perilla seed PfFAD3 cDNA was amplified from perilla (Perilla frutescens Britt) by GenomeWalker PCR. Sequence analysis of the fragment provided with identification of a 1485-bp 5'-upstream region and a 241-bp intron in the open reading frame. To determine the tissue-specificity of the PfFAD3 gene expression, the 5'-upstream region was fused to the ${\beta}-glucuronidase$ (GUS) gene and incorporated into Arabidopsis thaliana. Histochemical assay of the transgenic plants showed that GUS expression was restricted to seed and pollen, showing that PfFAD3 gene was exclusively expressed in those tissues.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2022년도 추계학술대회
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• pp.24-24
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• 2022

This present study was to identify a novel candidate gene that contribute to the elevated α-linolenic acid (ALA, ω-3) concentration in PE2166 from mutagenesis of Pungsannamul. Major loci qALA5_1 and qALA5_2 were detected on chromosome 5 of soybean through quantitative trait loci mapping analyses of recombinant inbred lines. With next generation sequencing of parental lines and Pungsannamul, and recombinant analyses, a potential gene, Glyma. 05g221500 (HD) controlling elevated ALA concentration was identified. HD is a homeodomain-like transcriptional regulator that may regulate the expression level of microsomal ω-3 fatty acid desaturase (FAD3) genes responsible for the conversion of linoleic acid into ALA in the fatty acid biosynthetic pathway. In addition, we hypothesized that combination of mutant alleles, HD and either of microsomal delta-12 fatty acid desaturase 2-1 (FAD2-1\ could reduce the ω-6/ω-3 ratio. In populations where HD, and FAD2-1A and FAD2-1B genes were segregated, combination of a hd allele from PE2166 and either of the variant FAD2-1 alleles were sufficient to reduce the ω-6/ω-3 ratio in seeds.