@phdthesis{oai:kitami-it.repo.nii.ac.jp:00007688,
 author = {Surina, Borjihan},
 month = {Sep},
 note = {In this doctoral thesis, the DNA analysis, structure of the carbohydrate and biological activities of polysaccharide in natural plants were studied.
Lacquer (urushi), originally produced in Asia, is the only natural paint that is polymerized by an enzyme, laccase, to give a beautiful coating surface. Rhus verniciflua that produces lacquer sap is both traditional and important industrial trees for an environment friendly paint of the next generation. Morphological, anatomical, and chemical characters of lacquer tree have been employed to delimit the lacquer plants, because, the genetic information is very limited.
Recently, valuable studies on the phylogeny of lacquer plants have been reported based on the chloroplast trnL (310bp) and trnL-F (470bp) and PS-ID gene sequences (plastid sub-type identity). However there are no reports on the analysis of ITS (internal transcribed spacer) and ETS (external transcribes spacer) regions of the nuclear DNA and 5S, rbcL (Large subunit of ribulose-1,5-bisphosphate carboxylase) and matk regions of the chloroplast DNA. We have analyzed the DNA sequences of lacquer trees in Abashiri A, B, and C areas to reveal the relationship between DNA sequences and geographical distance. In this study, we describe the sequence analysis of lacquer tree (Rhus verniciflua) in Hokkaido and the sequences were compared to those in Iwate and Fukushima prefectures, which are famous lacquer-growing districts at present and in the past in Japan. From the phylogenetic results of an intra-species genetic diversity in the length and GC content in the ITS region, we found for the first time polymorphism in lacquer tree cultivars. The lacquer trees in Abashiri C area were adapted to the cold environment from the point of sap production.
Lacquer laccase is a copper containing glycoprotein consisted of 533 amino acids and carbohydrates have 45% of the weight. For the analysis of carbohydrate structure and glycosylation sites of laccase glycoprotein, glycoprotein 1mg was digested with enzymes by trypsin and PNGase A and the digested sample was analyzed by using LC/MS/MS and MALDI-TOF/MS. The lacquer laccase is relatively heavily glycosylated glycopreotein. From MS/MS spectra, we found that twelve of the potential fifteen Asn-X-Thr/Ser sites were determined to be sites of N-linked glycosylation. Asn 5, 14, 28, 124, 180, 194, 233, 274, 284, 347, 381, and 398 were found to be bound carbohydrates in this study. Each N-glycan composition was elucidated and the possible structures were deduced.
In the last part, we studied on the natural polysaccharide, Konjac glucomannan which is a naturally occurring polysaccharide and difficult to dissolve in water and DMSO because of having high molecular weights. Before sulfation, hydrolysis by diluted sulfuric acid was carried out to decrease molecular weights to n=19.2x104-0.2x104. Sulfation with piperidine-N-sulfonic acid or SO3-pyridine complex gave sulfated konjac glucomannan with the molecular weights of n=1.0x 104-0.4x104 and degree of sulfation of DS = 1.3-1.4. It was found that the sulfated konjac glucomannans had potent anti-HIV activity of the 50% effective concentration of EC50=1.2-1.3 g/ml, which activity was almost the same as that of an AIDS drug, ddC, EC50=3.2 g/ml, and medium blood anticoagulant activity, AA =0.8-22.7 units/mg, which activities were almost the same activities as those of standard sulfated polysaccharides, curdlan (10 unit/mg) and dextran (22.7 unit/mg) sulfates. Structural analysis of sulfated konjac glucomannans was performed by a high resolution NMR measurement and the interaction between polylysine as a model compound of proteins and peptides was calculated preliminary from a surface plasmon resonance (SPR) measurement, suggesting that sulfated konjac glucomannans had a high binding stability on poly-L-lysine as a model compound of proteins and peptides.},
 school = {北見工業大学},
 title = {Study on DNA polymorphism and biological polysaccharides in plant resources},
 year = {2013}
}