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Hur, Yoonkang,Vasconcelos, Aurea C. 충남대학교 생물공학연구소 1999 생물공학연구지 Vol.7 No.-
Spinach (Spinacia oleracea) cytosolic fructose-1,6-bisphosphatase (FBPase) was purified and the final preparation of protein has a specific activity of about 45 units/mg protein and a single band of molecular mass of 39 kDa. Polyclonal antibody against the protein did not cross-react with chloroplast FBPase, but showed strong cross-reactivity with all plant cytosolic FBPases tested. Studies of the FBPase expression characteristics at early stages of development demonstrated that it was controlled at both the transcriptional and translational levels, and its mRNA was detected even in etiolated cotyledons. This suggests that the expression is not light-inducible. A single transcript was detected in all spinach tissues tested. Western blot analysis revealed two protein bands in the etiolated cotyledons: one was the same size as that present in the mature leaf, and the other slightly smaller. A high enzyme activity was detected in etiolated cotyledons, especially compared to protein levels in Western blots. Expression of the cytosolic FBPase gene during leaf development showed no change in the steady-state level of mRNA, but the protein level and enzyme activity were higher in mature leaves than in young ones, suggesting that the increase in FBPase activity during development is due to an increase in protein synthesis. Young roots showed low enzyme activity, but an unexpectedly high activity was detected in old fiber roots.
Gene-specific marker development of cabbage for an efficient molecular breeding
Yoonkang Hur,Yong-Pyo Lim,Ill-Sup Nou 한국육종학회 2015 한국육종학회 심포지엄 Vol.2015 No.07
Molecular markers, such as PCR-based and SNP-based markers, are extremely useful for plant genetics and crop breeding. Marker-assisted selection (MAS) has been widely applied in plant breeding to improve crop yield, quality, and tolerance to biotic and abiotic stresses. To develop gene-based (or -specific) molecular markers, three different approaches have been used in Brassica species: Known-gene-based, RNA seq/Exon-based and RNA seq/Intron-based molecular marker development for several years. Using these techniques, molecular markers have been developed to identify flowering time, anthocyanin accumuation and abiotic stresses in B. rapa and B. oleracea. Markers were distributed in exons as well as introns, and coding sequences and untranslated regions (UTRs). All markers developed have been transformed into SNP marker after HRM confirmation. I will discuss efficiency, accuracy, and potential problems and contribution of these markers for Brassica breeding.
Production of Pokeweed Antiviral Proteins Nontoxic to Cells by Mutagenesis of PAP-cDNA
Hur, Yoonkang,Han, Ching-Tack,Park, Sangkyu 충남대학교 생물공학연구소 1999 생물공학연구지 Vol.7 No.-
Pokeweed antiviral protein (PAP), one of ribosome inactivating proteins (RIPs), has very strong toxicity both to prokaryotic and eukaryotic cells. To produce mutant PAPs nontoxic to cells, the PAP-cDNA was inserted into a yeast-E. coli shuttle vector under the control of galactose promoter, mutagenized using hydroxylamine, and transformed into yeast cells. Transformed yeast cells were selected on the uracil-deficient plate containing glucose or raffinose, and the yeast cells producing mutant PAPs nontoxic to cells were then selected on the galactose plate. Eighteen mutants were obtained by immunoblot analyses of 1,000 transformants: among them, three, ten and five mutants among them did not inhibit the yeast cell growth, and showed no or less inhibition of protein synthesis in vitro. Six among fourteen mutants were able to protect TMV infection in coinoculation experiment. The mutant PAPs showing an antiviral activity either without or reduced RIP activity contain neither the active site mutation nor C-terminal deletion mutation. These results suggest that both the RIP activity and the antiviral activity will require other amino acid residue(s) besides the active site and that the antiviral acitivity of PAP can be dissociated from its toxicity.
Expression Characteristics of Pokeweed Antiviral Proteins(PAPs) : Two Distinct Types of Proteins
Hur, Yoonkang,Han, Ching-Tack,Maeng, Jueson 충남대학교 생물공학연구소 1998 생물공학연구지 Vol.6 No.-
Pokeweed antiviral proteins (PAPs) become novel therapeutic agents in relation to application in human viral diseases and cancer, as well as potent tools in plant system for defending viral infection. We have studied the expression characteristics of PAAs in pokeweed plants by western blot analysis. PAP-Ⅰwas constitutively expressed in leaves, stems and roots of the pokeweed plant, while PAP-Ⅱ was not expressed in roots. The expression of PAP-Ⅱ began in May and then gradually increased with development of the plants. The PAP-Ⅱ expression was induced and/or stimulated not only by biotic stresses, such as insect pests and viral infection, but also by abiotic stresses, like drought. Interestingly, low-light intensity was found to be more effective than high-light in the expression of both PAP-Ⅰand PAP-Ⅱ. Our results suggest the PAP-Ⅱ appears to have an additive effect in terms of proteciton of the plant against pathogens during summer-time when the plant actively grows and is attacked by various pathogens.
Spinach Cytosolic Fructose-1,6-bisphosphatase : Ⅱ. Light Effect in Its Expression
Hur, YoonKang,Vasconcelos, Aurea C. 충남대학교 생물공학연구소 1999 생물공학연구지 Vol.7 No.-
The effect of light on the expression of spinach (Spinacia oleracea) cytosolic fructose-1,6-bisphosphatase (FBPase) was determined by the level of mRNA, protein content, and enzyme activity. It was found that its expression and activity were constant and stable during normal daily conditions as well as under continuous light or dark conditions. However, two different mRNAs were detected; one transcript was expressed all the time, while the other was detected only during prolonged dark periods. Analysis of the expression of the mRNAs at the protein level using an activity gel showed that this "darkness-specific" mRNA encoded a separate, distinct polypeptide. Thus, our data suggest that cytosolic FBPase is encoded by a small multigene family.
Yoonkang Hur,Jeongyeo Lee,Hayoung Song,Yong Pyo Lim 한국식물생명공학회 2006 JOURNAL OF PLANT BIOTECHNOLOGY Vol.33 No.2
hinese cabbage (Brassica rapa ssp. pekinensis) is one of the most important vegetable crops in Korea and other East Asian countries. Cytosolic fructose-1,6-bisphosphatase (cytFBPase) is a key enzyme in sucrose biosynthesis, which controls the sucrose levels as well as the productivity of plants. The Chinese cabbage cytFBPase gene, BrFBPase, encodes the 340 amino acid polypeptide, giving a theoretical molecular weight of 37.2 kD and a isolectric point of 5.4. BrFBPase showed high sequence identity with Brassica homologs and its functional domains, such as F2,6P2 binding site or active site and F6P binding site, were highly conserved in diverse sources of organisms. Although the genome of Chinese cabbage seemed to be triplicated, BrFBPase appears to be a single copy gene. The expression of BrFBPase was examined at transcript and protein levels under various conditions. BrFBPase expression was observed only in photosynthetic source tissue, not in sink tissue. The expression was slightly higher during the day than at night, and it showed a diurnal cycle with circadian rhythmicity. Short-term exposure to low temperature inhibited the expression of the BrFBPase, while long-term exposure increased the expression, supporting that sugar levels are high in late autumn when temperatures are low.