南極海泥5種放線菌的分離鑒定及其生物活性

南極海泥5種放線菌的分離鑒定及其生物活性

1通過論使用細則國際通用的抗辯國體染色,科學(xué)發(fā)現(xiàn)和保護nractityraperitys.u2005.3.3.3.3.3.3.3.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.3.3.3.3.3.3.3.3.3.3.3.3.4.3.3.4.3.4.3.4.3.4.3.4.3.3.3.4.3.4.3.4.3.4.3.3.4.3.3.4.3.3.3.4.3.3.3.3.3.4.3.3.3.3.3.4.3.4.3.3.3.3.3.3.3.3.3.3.3.3.3.3.4.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3活動人士是一個受歡迎的例子。它主要展示鹽的參與、修訂和微修訂。它與母字母皮膚不同。它是一個短而帶敏的句子。它是一個短而帶敏的句子。這些是授權(quán)的和二級蒙太奇。他們是短而非正統(tǒng)狀態(tài)潘基文的成員。Duetotheenergeticdemandsforcommercialenzymesandrapidemergenceoffatalantibiotic-resistantpathogens,itisnecessarytocontinuethesearchfornovelbioactiveagents.However,intherecentyears,thishasresultedinthereducedchancesoffindingnewbiologicallyactivemoleculesfromactinobacteria.Therefore,itisimportanttoexplorenewhabitatswithunusualenvironmentandpoorlystudiedareasoftheworldtodiscoveryofnovelactivecompounds,especiallyenzymes.Consequently,theAntarctica,oneoftheharshest,coldestandpoorlyexploredareasonEarthwasconsideredasahighlypotentialareaforthedevelopmentofnovelbioactivecompoundsfromactinobacteria.SomenovelspecieswerediscoveredfrommarinesedimentsinAntarctica,suchasPseudonocardiaantarcticasp.Nov.,Friedmanniellaantarctica,Micromonosporaendolithicaandsoon.Manynewsecondarymetabolites,producedbymarineactinobacteria,possessnovelbiologicalactivitiesandhavethepotentialtobedevelopedastherapeuticagents.Additionally,sequencingmarineactinobacteriagenomesmayprovideusefulinsightsinthediscoveryofnovelagents.TheaimofthisarticlewastoisolateandtoscreenforcultivableactinobacteriafromsedimentofAntarctica,andwastopreliminarilystudytheenzymeproduction.2藝術(shù)與文化2.1因違反dried-pcr-n,which一個單一性/wras-dried-ure-關(guān)于日TwosampleswerecollectedfromsiteintheAntarcticGreatWallStation(62.22°S,58.96°W),inwhichonesamplewasfrommarinesedimentsamplesandtheotherwasfromshore.Thesedimentsampleswereair-driedasepticallyinlaminarflowhood,andweredividedintoeach5gonepackageaswellaswerestoredat-80°C.2.2lyctorol,n3,kno3,khpo4,5.3,5.v.3,5.v.3,5.v.7.3,5.v.7.3,5.v.7.3,5.v.7.3,7.2o,7h2o,7h2o,7h2o,7h2o,7h3o,7h2oFivedifferentmediawereusedfortheisolationofactinobacteriafromsedimentsamples:M1:starch(1%,W/V),casein(0.03%,W/V),K2HPO4(0.2%,W/V),KNO3(0.2%,W/V),NaCl(0.2%,W/V),MgSO4爛7H2O(0.005%,W/V),CaCO3(0.002%,W/V),FeSO4爛7H2O(0.001%,W/V);M2:glucose(0.4%,W/V),yeastextract(0.4%,W/V),maltextract(1%,W/V);M3:Oatmeal(2%,W/V),FeSO4爛7H2O(0.01%,W/V),MnCl2爛4H2O(0.01%,W/V),ZnSO4爛7H2O(0.01%,W/V);M4:glycerol(0.05%,W/V),starch(0.05%,W/V),casein(0.03%,W/V),asparagicacid(0.01%,W/V),sodiumpropionate(0.05%,W/V),KNO3(0.01%,W/V),K2HPO4(0.05%,W/V),FeSO4爛7H2O(0.0001%,W/V);andM5:glucose(1%,W/V),N-ZamineA(0.2%,W/V),beefextract(0.1%,W/V),yeastextract(0.1%,W/V).AllmediawereadjustedtopH7.2~7.5andcontainedbaysalt(2%,W/V),20.0gagar,and1000mLdistilledwater.Inaddition,allmediaweresupplementedwithactidione(20μg/mL),nystatindihydrate(50μg/mL),nalidixicacid(10μg/mL)andpotassiumbichromate(0.002%,W/V),respectively,inordertosuppressfungiandfast-growinggram-negativebacteria.Sedimentsamples,storedat-80°C,wereprocessedintwodifferentways.Oneisthatfivegramsofdrysedimentsampleswereaddedto50mLsterileartificialseawater(sterilewaterwith2%baysalt)with10acid-washedglassbeadsof0.1mmdiameterandwerehomogenizedfor3hat180r/minat25°C.Afterserialdilutionsfortwotimes,200μLaliquotswereplatedontotheisolationmediaintriplicate.Theothermethodisfollowingadispersionanddifferentialcentrifugationtechnique(DDC)withcarboxymethylcellulosesodium(CMC-Na).Alloftheplateswereincubatedat25°Cforupto4weeks,andtheisolateswerepurifiedintheiroriginalisolationmediabytherepeatedstreaking.2.3pcr-pcr相關(guān)程序TotalgenomicDNAfromthemarine-derivedactinobacteriawasextractedbymodifiedmethodWeisberg:4mLsuspensionculturewascertificatedat12000r/min,5mintorinseintriplicatewith400μLTEbuffer(10mmol/LTrishydrochlorides,pH8.0,1mmol/LEDTA).Theproduction,actedbylysozyme(2.5mg/mL)andRNase(0.05mg/mL)upto16hat37°C,wasmixedwith50μLof20%sodiumdodecylsulfate(SDS)for30min,andthenwithapproximately400μLofphenol,saturatedwiththeabovebuffer.Aftershaking,thesamplewasseparatedbycentrifugation10minat12000r/minandtheaqueousphasewasprecipitatedwithethanol.Thepelletwassuspendedin20μLto50μLofTEbuffertobeusedforPCRamplification.ThecontentofgenomicDNAwasmonitoredbymeasuringtheratevalueoftheextinction260nmand280nm.ThePCRamplificationswerecarriedoutin50μLreactionvolumescontaining20~50ngofgenomicDNAtemplate,10×TaqDNApolymerasebuffer(5μL),0.2mmol/LdNTP,10mmol/Lprimerand5UofTaqDNApolymerase.Theprimersusedforthe16srDNAamplificationwere27f:5-AGAGTTTGATC-CTGGCTCA-3and1492r:5-GGTTACCTTGTTACGACTT-3.ThePCRconditionsfor16srDNAreactionswere:onecycleofdenaturationfor5minat95°C;30amplificationcyclesconsistingofdenaturation(94°Cfor45s),primerannealing(57°Cfor45s),andprimerextension(72°Cfor2min);andafinalextensionof10minat72°C.The16srDNAamplificationswereassessedfollowingseparationina1%(W/V)agarosegelandwererecycledbyusingtheTIANgelmaxpurificationkit.2.4o對于apos的oun-manufactorr&apos的介紹Theclonelibrariesof16srDNAwerecreatedforthelaboratorysamples.PCRproductswereligatedintopUCm-Teasyvectorsfollowingthemanufacturer'sprotocolwithusing5ngofeachPCRproduction.Theligationswereincubatedat4°CandshippedovernightfortransformationbyusingDH5α.Thenthemethodconformingtoclonelibrarieswasblue-whiteselectionandPCRwiththeplasmidsfromtransformationDH5αbyusingthe27fand1492rprimer.ThesuccessfultransformationwasusedtosinglepassSanger.2.5國際習(xí)慣法規(guī)范wraceTheobtained16srDNAsequenceswerecomparedwithsequenceswithNCBIBLASTalgorithmandthenucleotidesequencesofreferencespeciesweredownloadedfromNCBIGenbank.AllofthemwerealignedandtrimmedusingMolecularEvolutionaryGeneticAnalysis(MEGA5.1).Theevolutionaryhistorywasinferredusingtheneighbor-joiningmethod.Scorebarrepresents1nucleotidesubstitutionper1000nucleotides.Bootstrapvaluesof50andabovewereshown.2.6enzmecdivategecivatechivi分離sivi體制Theisolateswereinoculatedonsuitablemediumbyspotinoculationmethodtoscreentheirenzymeactivities,suchasprotease,lipase,amylase,tyrosinase,urease,cellulaseandchitinaseactivity.Theplateswereincubatedat25°Cfor7days.ThemediaascriteriaforenzymeactivatesweregiveninTab1.2.7英國宣告法u2005,k病.ThetestorganismsincludeEnterococcusavium,Sarcinalutea,Staphyloccocusaureus,Pesudomonaspyocyaneum,EscherichiacoliandKlebsiellapneumoniaewereusedtotesttheantimicrobialactivityofpotentstrains.Theisolateswereincubatedinfermentationmediumwithsoyabeanfor7days.Afterincubation,platediffusionmethodwasusedfortheantimicrobialtestingandtheplateswereincubatedat37°Cfor18handanalyzedforzoneformation.3whichsot兩設(shè)置Thecharacteristicofthesedimentsamplesaccordingtothelocationswasasfollows:No.1samplewasredandrelativelydrysoilandhadmuchcoarsegranule;No.2samplewasblackandwedsoilandhadsingle-sizegranuleandsomeplantroot.Atotalof9actinobacteriastrainswereisolatedfrom2sedimentsamplesbythe5differentisolationmediaand2methods.The4isolatesof9showedsimilargrowthpatternson25°C,whileotheroneisolatewasdependentonlowtemperature(4°Cperformswell).ThecharacteristicsoftheseisolatesshowedinTab1,suchasisolationmedium,sizeofcolony,vegetativemycelia,aerialmycelia,sporemorphologyandpigment,whichwereobservedinM2mediumafter10dcultivation.Differentmethods,isolationmediaanddifferentantibioticshadaneffectontheperformanceoftheisolates.Withthedistributionanalysisofthetotalisolates,themoremicroorganismswereisolatedbyDDCmethod(60.2%)thangradientdilutionmethod(39.8%),whichexplainedthatDDCcouldwellseparateparticles.Thedistributionofthetotalisolatesaccordingtotheisolationmediumrevealedthattheperformanceoftheisolationmediumwasasfollows:136plates(hadisolates)fromM1(71.2%),96platesfromM2(50.3%),102platesfromM3(53.1%),130platesfromM4(68.1%),and115platesfromM5(60.4%).Additionally,consideringtheeffectofantibiotics,therateofisolatesperformanceis83.3%inthepresenceofactidione,51.2%,43.8%and45.8%withnalidixic,fungicdinacidandpotassiumbichromate,respectively.The5isolatesweresequencedfortheir16srDNAgenesfollowingPCRamplification,purification,andcloning.Theconcentrationofproductionwasmonitoredbyagarosegelelectrophoresis(1%)andtheclearbandwasseeninFig1.Afterpurification,cloningandsinglepassSanger,thesequenceswereloadingtoGenbankandtheGenbankaccessionnumberswereasfollows:KF938603,KJ488981,KJ488980,KJ488979,andKJ488978.16srDNAsequencedatawereusedforBLASTanalysisandconstructionofaphylogenetictree(Fig2).Accordingtotheseveralscreeningmedia,theabilitytoproduceenzymeofisolateswerecheckedandtheresultwasshowninTab2.Thehighactivitywassupportedbymorethan1cmtransparentcircleandwasrepresentedasthreeplussigns.Meanwhile,themoderateactivitywasexpressedby0.5cmtransparentcircle.Outof5isolates,1isolate,theXE-1,exhibitedantibacterialactivityagainstEnterococcusaviumwith0.5cminhibitionzoneby50μLfermentationbroth,whileotherstrainshadnoantibacterialactivityagainstanytestedbacteria.4通過數(shù)字method國際習(xí)慣法檢測貿(mào)易摩擦產(chǎn)生的基因表達Inrecentyears,thepolarmarinesedimentshaveproventobevaluablefromscientificpointofviewforwidediversityofbioactivenaturalproducts,suchasantibiotics,biologicalenzymesandantitumoragents.Mostofthemicroorganismsinsoilwereuncultivablewithlimitofpretreatmentandbyisolationmethods.Differentisolationmethodssuchasultrasonictreatment,gradientdilutionmethod,dispersionanddifferentialcentrifugationtechnique(DDC),dryheat(120°C,60min)andacidandheat-shocktreatmentspriortoinoculationcanbeusedtoincreasetherelativenumbersofactinobacteria.Gradientdilutionmethodwaswidelyusedtoisolatemicroorganisms.Themainreasonobviouslywasthatitwasasimplemethodandhadnoeffectonsoilcomposition.Whenthesituationofsoilsample,suchasbiomassandcategory,wasunknown,thegradientdilutionmethodwasprimarilyused.However,whendifficultiesinseparatingparticlesresultedinlowerrecoveriesfromsedimentsamples,themethodofDDCmaybeassociatedwithrelativelydensesoilmaterialandbecameasuperiormethod.Theunknownenvironmentandpossiblylowrateofmicroorganismswereconsidered.Sothesampleswereair-driedinlaminarflowhood,andwerehandledbyDDCmethodandgradientdilutionmethodinthisresearch.Inthisisolation,thesampleNo.2hadrelativelyhigherrecoveriesthanNo.1,whichwassuggestedthatacertainrateofwaterandplantrootmaybeasignalofrelativelyhighrecoveries.Italsomeansthatthecharacteristicsofsoilwasneededtoanalyzebeforeisolationtopredictthebiomass.Manydifferentmodifiedmediacanbeusedtoisolateterrestrialactinobacteriafromsedimentsandothermarinesources,whichwereresultedbymicroorganismfavorincarbonandnitrogensources.Inourstudy,5mediawereusedforisolation.Thecompositionofmediahadaneffectonthenumberofmicroorganisms.Accordingtoourresearch,crudenutrientssuchascaseinandoatmealcanincreasethenumberofit,whichwassimilartothatofthesomeotherreports.Noticeable,theculturetemperatureforpolarmicroorganismmaybelower.Inthisresearch,someinterestmicroorganismshadnogrowthatabove25°C,butithadstronggrowthofgram-negativebacteria.Consideringofit,the25°Cwasusedascultivationtemperature.Thestrains,isolatedfromthisstudy,weremostlygeneStreptomycesandwerediversityofcolors,suchasbrilliantyellow,red,andwhite.AndthestrainXPwassimilartoMicrococcusyunnanensisiYIM65004T(96.29%),whichwasworthoffurtherstudyowingtopotentialpossibilityofanewstrain.Theenzymeactivityofthoseisolates,suchasprotease,cellulase,chitinases,amylases,urease,andlipasewaschecked.ThestrainXE-1canhighlyproduceproteaseat