| Plant virus disease is one of the most destructive diseases in agriculture worldwide,for example cucumber mosaic virus(CMV),potato virus Y(PVY),and tobacco mosaic virus(TMV).Currently,synthetic pesticide treatment has been the main method for controlling diseases.These pesticides are very harmful to the human and the environment.Sugar is a substance which exerts extensive bioactivity.Thus,they have wide application in the fields of medicine and agriculture.Carbohydrate pesticides have no residue,pollution,and toxicity to the environment and nontarget organisms when they are sprayed on crops.Dendrobium nobile Lindl(D.nobile Lindl),an orchid species,is one of the precious Chinese herbal medicine.Polysaccharides from D.nobile Lindl have been proven to possess multiple biological properties,including immunomodulation,antitumor,and antioxidant activities.However,few works reported that polysaccharide from D.nobile Lindl exerted antiviral activity.Therefroe,we do some work to study its antiviral activity.The conclusions are shown as following:1.Crude extract(W)was isolated by hot-water extraction.Then the crude extract W was dissolved in the deionized water,and extracted successively with ethyl acetate and N-butyl alcohol.The aqueous fraction was precipitated by ethanol,separated by the anion-exchange chromatography DEAE-Cellulose-52 to give E1,E2,E3,E4,E5,and E6.E4 and E6 were successively purified by the gel filtration chromatography Sephacryl S-200 and Sephadex G-100 to give eleven homogenous polysaccharides.We designated them as DNPE4(2),DNPE4(4),DNPE4(7),DNPE6(1),DNPE6(4),DNPE6(5),DNPE6(6),DNPE6(7),DNPE6(8),DNPE6(9),and DNPE6(11),respectively.Furthermore,the structures of these homogenous active polysaccharides were characterized by chemical and spectrographic techniques,including the analysis of monosaccharide composition,methylation analysis,periodate oxidation——Smith degradation analysis,partial hydrolysis with acid,FT-IR,and VU.2.The antiviral activities of the crude extract were evaluated.The result found that W exhibited excellent protective(57.6%)and curative(57.0%)activities against CMV at 500μg/m L,respectively,which was superior to those of Ningnanmycin(protective activity was 54.3%,and curative activity was 55.1%).W exhibited excellent protective(54.1%)against PVY at 500μg/m L,which was higher than that of Ningnanmycin(51.4%).Base on the results above,W was purified further,and antiviral activities of every fraction were tested.Detailed results were shown as follows:(1)Crude polysaccharide E2 exhibited notable protective activity of 64.5%against TMV at 500μg/m L,which was quite similar to Ningnanmycin(62.1%).Crude polysaccharide E1 exhibited excellent inactivation activity against TMV of 93.0%at500μg/m L,which was quite similar to Ningnanmycin(90.0%).Homogenous polysaccharides DNPE4(7),DNPE6(4),DNPE6(5),DNPE6(6),and DNPE6(9)possessed protective activities of 59.3%,69.9%,65.2%,57.0%,and 66.0%against TMV at 125μg/ml,respectively.Their protective activities were superior to those of Ningnanmycin(54.9%),COS(39.1%),and lentinan(52.3%).Moreover,homogenous polysaccharides DNPE4(2),DNPE4(4),DNPE4(7),and DNPE6(8)exhibited curative activities of 50.7%,51.4%,52.3%,and 52.3%against TMV at 125μg/m L,respectively.Their curative activities were higher than those of ningnanmycin(33.6%),COS(17.2%),and lentinan(48.8%).(2)Crude polysaccharide E6 exhibited notable protective activity of 43.9%,which was quite equal to those of COS(45.1%)and lentinan(43.0%).Crude polysaccharide E3 and E4 possessed curative activities of 40.2%and 50.2%against CMV at 500μg/m L,respectively,which were higher than those of COS(37.3%)and lentinan(37.8%).The inactivitation activities of E2,E3,and E6 against CMV at 500μg/m L were 49.7%,49.9%,and 54.5%,respectively,which were higher than those of COS(34.15)and lentinan(46.7%).DNPE4(3),DNPE6(1),and DNPE6(11)exerted excellent curative activities of 42.2%,39.3%,and 55.1%against CMV at 500μg/m L,respectively.Their curative activities were higher than those of COS(37.4%)and lentinan(37.8%).DNPE4(7),DNPE6(6),DNPE4(3),DNPE4(1),and DNPE6(11)possessed inactivation activities of 43.1%,45.7%,46.8%,60.6%,and 58.2%against CMV at 500μg/m L,respectively,which were superior than those of COS(34.1%)and lentinan(46.7%).(3)DNPE6(11)exhibited excellent protective activity of 56.6%against PVY at500μg/m L,which was higher than that of Ningnanmycin(43.6%).The curative activity of DNPE6(11)was 51.5%,which was similar to that of Ningnanmycin(50.8%).DNPE6(2)and DNPE6(3)possessed notable inactivation activities of 62.4%and 63.6%against PVY at 500μg/m L,respectively,which were higher than that of Ningnanmycin(60.7%).3.Given the excellent inactivating activity of DNPE6(11)against CMV,florescence titration and microscale thermophoresis techniques were used to measure the binding constant between DNPE6(11)and CMV CP.The result showed that K_abetween DNPE6(11)and CMV CP was 1.45×10~5 L/mol.K_d of DNPE6(11)with CMV CP was 18.1±6.4μM.These experimental results demonstrated that DNPE6(11)had a strong affinity capacity with CMV CP,which inhibited CMV infection the plant.Based on the notable protective activity of DNPE6(4)against TMV,superoxide dismutase(SOD),Peroxidase(POD),and L-phenylalanine ammonia-lyase(PAL)were tested after DNPE6(4)treatment in vivo.The result found that DNPE6(4)can improve defense enzyme activities,including SOD,POD,and PAL to increase tobacco resistance.The defense gene expressions of SOD and CAT1 were evaluated after DNPE6(4)treatment.Moreover,proteomics analysis was employed to investigate the differential proteins between the CK+TMV and TMV+DNPE6(4)groups,and GO term and KEGG were analyzed.The proteomes analysis showed that NADPH oxidase and NAD(P)H increased after DNPE6(4)induced.In addition,we found some Ca M(Calmodulin Nt Ca M11,Calmodulin Nt Ca M1,Centrin(probable calcium-binding protein CML20)and calcium-binding allergen Ole e 8-like),and some pathogenesis-related proteins and defense enzymes,such as(Thaumatin-like protein(pathogenesis-related protein R major form),Acidic endochitinase Q(EC 3.2.1.14)(Pathogenesis-related protein Q)(PR-Q),pathogenesis-related protein STH-2-like,and Peroxidase(EC 1.11.1.7))increase.Furthermore,we found DNPE6(4)can increase the expression levels of Isochorismate synthase protein(ICS1),EDS1,and Pathogenesis related protein 1(PR1),which involved in the systemic acquired resistance.All together,the proteomics analysis revealed that DNPE6(4),acting as an elicitor,trigged calcium signaling pathway and enhanced the plant defense against TMV. |
PDF Full Text Request