| Elicitor derived from pathogens(fungi,bacteria,oomycetes and viruses egg),non-pathogenic bacteria,host plants and non-host plants is a signaling molecule,which can induce the plant to generate the defensive reactions;Elicitor Includes proteins,glycoproteins,oligosaccharides,peptides and lipids,among which the most important is protein elicitor,specially fungal elicitor protein.Fungal protein elicitor which is widely present in the plant pathogenic fungi is a signaling molecules that can prossess broad spectrum of plant resistance and promote the growth of plants.Fungal elicitor protein is derived from the fungus and is a nonspecific of elicitor which can stimulate gene expression HR of plant defense response.Numerous studies show that fungal elicitor protein can induce expression of the plant's own resistance genes,activate the defense response and the immune system,improve the plant's own immune system,promote plant growth and seed germination by regulating plant growth and metabolism system and reduced HR of the plant.Therefore fungal elicitor protein can be used as a new type of biological pesticides used in agriculture.Root rot is not only a serious disease in croops but also a infractory disease in the cultivation of chinese hebal medical;there has yet not to find an effective cure measures.Chinese hebal medical root rot disease caused by infection of Fusarium including F.-oxysporumis,F.solani,F.avenaceum,F.equiseti F.semiterectum,Rhizoctnia solani et al.Underground part including roots,tubers,dormant buds et al of different medicinal plants which infected by pathogen were all decary necrosis,presented dry rot shape with yellow and brown.These syptoms caused great loss of medicinal plants themselves and their medicinal value.Soybean root rot disease caused by infection of soybean root rot-Fusarium oxysporumis widespread occurrence of soybean producing areas,Generally it happens in the soil moist or wet years,the incidence of the more serious plots,the higher disease index.There will have oval brown elongated and even irregularly depression spots in the root and stem base of infected plant until it expands to a large shaped patches surround the taproot.The disease does not cause plants dead,but it will cause a large number of reduction of yield.F.oxysporum derived from fabaceae,cereals rot and medicinal plant can cause the root rot of fabaceae and cereals.F.oxysporum derived from cereals has been almost studied thoroughly,butthere has not much research about protein elicitor from F.oxysporum derived from fabaceae especially soybeans.So far there is no study about the protein elicitor which has bioassay purified from F.oxysporum derived from soybeans.A novel protein elicitor witha molecular weight of 40kD was isolated and purified from F.oxysporum for first time using ultrasonication,centrifugation,the treatment of different temperature,anion exchange chromatography and size exclusion chromatography.This study with wheat and soybeans for biomaterials,investigates the influence of protein elicitor about the wheat seed germination and seedlings growth,the wheat root growth and root vigor,drought resistance in wheat and defense-related enzymatic activity of soybean.The total results were showed as follow:1 The culture of the F.oxysporum and the preliminary purification of the protein elicitorThe F.oxysporum was cultured in PD medium and then Tris-HCl buffer extracted the total proteins.The total proteins were concenirated by treatment with room temperature and boiling water about 30min,then Centrifuged,The supernatant purified certain elicitor by anion exchange chromatography-Hitrap Q HP column.The influence of wheat seed germination and seedling growth by elution peak as a physiological index,the crude protein extracts and elution peak have a certain activity of protein elicitor.2 Purification and activity of the crude extract of 40kD protein elicitor of F.oxysporumThe crude protein extracts treated by different temperatures(room temperature,40?,60?,80?,100?),and then desalted,purified by anion exchange chromatography-Source 30Q Column.Activity assay showed that when the wheat seedling growth through 6d,the elution peak treated with the room temperature,40?,60? compared with the control,the purified peak significantly promotes the growth of seedlings,especially 60? treatment.So A novel protein elicitor with a molecular weight of 40kD which has bioassay was purified with size exclusion chromatography-Superdex 200 3/200 GL column of elution peak which treated 60? of Source 30Q Column.These results showed that the protein which was first time isolated and purified from soybeans root rot-F.oxysporum is a novel protein elicitor.3 The study of biological activity of the 40kD protein elicitor of F.oxysporumThe study of biological activity of the 40kD protein elicitor of F.oxysporum includes the influence of protein elicitor about the wheat root growth and root vigor,drought resistance in wheat and defense-related enzymatic activity of soybean.The wheat seeds were soaked with the purified protein elicitor at different concentration for 24h and the wheat seedlings height were measured after 5d growth;the height of seedlings were promoted 5.64%,11.25%and 14.69%at concentration of 1ug/ml,5ug/ml and 10ug/ml;and the root vigor were promoted 1.78%,3.52%,7.79%and 14.37%at concentration of 1ug/ml,5ug/ml,lOug/ml and 20ug/ml.The coefficient of wheat drought tolerance increased 59%comparing with CK when the wheat seeds were soaked with protein elicitor at concentration of 5ug/ml.The result showed that the purified protein could enhance the drought resistance of wheat seedlings which may have a certain relationship with increase of SDH.Phenylalanine ammonialyas(PAL),Catalase(CAT)and Polypheol oxidase(PPO)are all related to the resistance of plants.The activity change of PAL,CAT and PPO from the mesophyll of soybean seedlings which treated with purified protein were measured.The activity of PAL,CAT and PPO were increased after treated.The activity of PAL was increased 62.5%comparing with CK at 24h;the activity of CAT and PPO were separately increased 98.7%and 150%comparing with CK at 48h.The experimental results showed that the 40kD protein elicitor could promote the growth of wheat seedling roots and the root vigor of wheat,enhance drought resistance in wheat and improve the defense-related enzymatic activity of soybean.The purified protein is biologically active protein elicitor.4 MS sequence analysis of the 40kD protein elicitor of F.oxysporumThe band of The active protein by SDS-PAGE electrophoresis which was cutted was identified by mass spectrometry,then gotten the peptide fingerprinting of the protein.The peptide fingerprint searched in database(NCBI),there has no matching result showed that this protein is a new protein.According to mass spectrometry to do MS/MS,there has gotten different peptides,then searched with NCBI database through cross-species got two match points higher protein gi | 691788124 and gi | 302696543,these two protein all derived from macro fungi are hypothetical proteins which can hydrolyze protein or peptide and specifically hydrolyze aspartic acid.A novel thermal stability protein elicitor with a molecular weight of 40kD detected by SDS-PAGE was firstly isolated and purified from F.oxysporum.The results of Mass spectrometry showed that the protein is a new protein elicitor.The protein elicitor could promote the growth of wheat seedling and wheat seedling roots,the root vigor of wheat,enhance drought resistance in wheat and improve the defense-related enzymatic activity of soybean.These bioassay results provide new idea for new fungal protein elicitors with bioassay.For preventing the diseases of medicinal plants and croops by Fusarium has potential developing value. |
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