Isolation, Purification And Action Mechanism On TMV Of Polysaccharide From Coprinus Comatus

Coprinus comatus was a well known edible and medicinal fungus which has been used traditionally in china to help digestion, increase appetite and treat hemorrhoids Both the fruiting bodies and mycelia of the fungus have been reported to contain some bioactive polysaccharides. It has been shown that the polysaccharides from Coprinus comatus exhibit such various pharmacological activities as hypoglycemic, enhancement of immune system, antitumor, etc. Most of these studies were performed on basis of the crude polysaccharides. And chemical characterization and primary structure of fraction polysaccharide from Coprinus comatus are rarely imported.This paper mained focused on suitable fermentative conditions of the mycelia growth , isolation ,purification and characterization , the anti-TMV activities both in vitro and in vivo ,and the action mechanisms on TMV were also systematically studied. The main results were shown as follows :The research established the suitable fermentative conditions of the mycelia growth of Coprinus comatus. The optical conditions for submerged fermentation culture was : sucrose 2%, maize meal 3%,wheat bran 4%, yeast 0.5%,KH₂PO₄ 0.25%,MgSO₄·7H₂O 0.10%.Crude polysaccharide(ccp ) was obtained by boiling water extraction and ethanol precipitation from fruiting bodies of Coprinus comatus. Two homogeneous fractions (ccp60a ,ccp60b ) were obtained by Sevag deprotein , DEAE Sephadex A-25 and Sepharose6B chromatography from ccp1. The Neutral sugar content of CCP1,CCP60,CCP60a and CCP60b were 95.22%,95.37%,97.22%,89.65%, respectively. The molar weight of CCP60a and CCP60b were 234 kDa and 5.0 kDa. Gas chromatography showed that CCP1 was composed of glucose, mannose, and galactose, the corresponding molar ration were 10.5:1.7:1, respectively. HPAEC showed that CCP60a was composed of glucose and galactose. Infrared spectra, GC-MS and nuclear magnetic resonance analysis (¹H NMR¹³ and C NMR) showed CCP60a mainly containedα-glucosidic bonds , and main chain was mainly [α-Glc(1-4)]_n-[α-Gal(2-6)]_m, probably involved some other residues and maybe some branch chain. CCP60a can form complexes with Congo Red ,but it cannot be confirmed that it was helix form. CCP60a can also form complexes with Celestine blue A.Activities of CCP60a had been determined, and the results were the following: the inhibition rate against Tobacco Mosaic Virus infection in Nicotiana glutinosa was over 90%. CCP60a could inactivate TMV in vitro and inhibit its multiplication in N. tabacum var. K₃₂₆.A primary study was done to analyse the mechanism of CCP60a inhibition to TMV. CCP60a didn't have direct viricidal effect on TMV particles, while they showed strong activity reducing TMV-RNA infectivity and could affect the polymerization of TMV coat protein in vitro. The tobaccoes were treated by the polysaccharide and by water, respectively, and enzymes included peroxidase (POD), polyphenol oxidase (PPO), phenylalanine ammonialyase (PAL), chitinase,β-1,3-glucanase in tobacco. Comparison with two methods in tobacco inoculated TMV, the enzyme activities of former one were obviously higher than the latter. The evidence indicated the increased enzyme activities should be associated with the development of induced resistance in tobacco. The results showed that applying with CCP60a made the endogenous SA contents increased in leaves of tobacco and SA took an important part in mechanisms of resistance to TMV in tobacco. It was showed that CCP60a could alleviate the symptom of plant infected by TMV in a certain extent by means of detecting their chlorophyll. Expression quantify of two genes of TMV-cp and TMV-replicase reduced 34% and 32% in tobacco leaves with polysaccharide by Real Time PCR. It showed that CCP60a could inhibit the expression of TMV-RNA.