Study On Purification And Charaeterization Of An Antifungal Protein From Pumpkin(cucurbita Moschata D.) Seeds

Plants need to absorb the full spectrum of nutrients from the environment riched in microorganisms during their growth,however,microorganism infections of plants are fairly rare.Plants form respective antifungal path-way during the long-term development,including secondary metabolites and protein which have antifungal activity,and antifungal protein of them play the key role of protection against infection.The targets of the experiment are purifying an antifungal protein from seeds of Cucurbita moschata D.and analysing its physical and chemical properties and biological activity aimed at establishing basis for cloning its cloning its gene,studying the antimicrobial mechanism and applying in in agricultural and medical realm.The experiment was carried out on comparing the total protein content extracted by low constant temperature and stirring method,homogenate method and ultrasonic cell disrupter method.By comparing,the optimal method was tissue homogenation.Then,the protein of BaoKu I(bare seed),YinHui ?,XueYu ?,JinHui ? was extracted by homogenate method.Comparisons between the four varieties of protein were performed on protein content,distribution of molecular weight,and antifungal activity.The obtained results showed that the best one was BaoKu ? which was selected as experimental material in following studies.The total protein of BaoKu ? was extracted by using extraction buffer(10mmol/L pH7.0 PBS,0.15mol NaCl,10mmol mercapto-ethanol).After that,the grinding pumpkin seeds were marinated followed by centrifuged,and then precipitated by using saturated(NH4)2S04.Then,four parts of precipitation.Test their antifungal activity and go on automatic column chromatography purification with the parts which had remarkable activity.The 40%-55%and 55%?95%parts of precipitation was purificated by cation exchange column SP-Sepharose(10/30),and the flow rate and eluent ionic strength of column were studied.The best condition was 10mmol,pH=4.5 acetic acid buffer with NaCl 50mmol,and flow rate was 0.8mL/min.We obtained three fractions and four fractions from the 40?55%and 55?95%parts of component,respectively,by testing their antifungal activtity.The components with strongest activities are as follows:SP2,SP3,SP5 and SP6.Then the four compounds were analysed by SDS-PAGE,and the SP3 was showing a single band.Then,SP3 was repurificated by cation exchange column SP-Sepharose(10/30),and the fraction was collected,dialyzed,andfreeze-dried.The pure antifungal protein showed a clear band on SDS-PAGE.Then,we picked protein spot from the SDS-PAGE gel,the molecular of the protein mass was 14.88kDa.By drawing standard curve of molecular weight and rate of flow of marker,determination of molecular weight of SP3 was 14.84kDa.Its purity was about 87.3%.After digestion with trypsin,the proteins changed to peptides.MALDI-TOF-MS showed that their peptide Mass spectrum.On internet,compared them to the NCBInr protein database and we discovered that they were new protein.Amino acid composition analysis of SP3 suggested this protein especially enriched in Glu and Arg and content was 16.4%and 17.4%,respectively.The methods analyzing biological activity of SP3 in the present study was mentioned asfollows:1)Antifungal activity and antifungal hemi-inhibitory concentration(IC50):protein SP3 should be a strong inhibitory activity to Fusarium oxysporum.sp.cucumainum Owen,Cercospora sojina Hara,Alternaria alternate,besides,the IC50 values of SP3 to these three fungi were 16.64?mol/L,20.91?mol/L,21.22?mol/L,respectively.2)Anti-bacterial activity:SP3 has a strong inhibitory effect to Staphyloccocus albus,Staphyloccocus aureus,Bacillus subtilis,but its inhibition to Escherichia coli is very weak.3)Determination of temperature-sensitive:the antimicrobial activity of SP3 is relatively stable under 50?.4)Determination of the pH sensitivity:SP3 keep its antimicrobial activity better in the buffer with pH between 5.0-9.0.5)Determination of UV sensitivity:SP3 keep its antifungal activity better under the UV irradiation within 12 h.6)Determination of carbohydrate content:SP3 is non-glycoprotein,for low carbohydrate content,and carbohydrate content may be original carbohydrate of pumpkin seeds.7)Antioxidant activity:the ability of removing DPPH radicals is Vc>SP>Ve,and the IC50 value is 31.8 ?g/mL.8)SOD-like activity:the unit of SOD-like activity of SP3 is 24.31U/mg.