Expression of cry1Ab gene from a novel Bacillus thuringiensis strain SY49-1 active on pest insects

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Environmental

Microbiology

Expression

of

cry1Ab

gene

from

a

novel

Bacillus

thuringiensis

strain

SY49-1

active

on

pest

insects

Ugur

Azizoglu

a,∗

_,

_Abdurrahman

_Ayvaz

b

_,

_Semih

_Yılmaz

c

_,

_Salih

_Karabörklü

d

_,

Rıdvan

Temizgul

b

a_{ErciyesUniversity,DepartmentofCropandAnimalProduction,Kayseri,Turkey} b_{ErciyesUniversity,DepartmentofBiology,Kayseri,Turkey}

c_{ErciyesUniversity,DepartmentofAgriculturalBiotechnology,Kayseri,Turkey} d_{DüzceUniversity,DepartmentofFieldCrops,Düzce,Turkey}

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Articlehistory:

Received5June2015 Accepted24November2015 Availableonline20April2016 AssociateEditor:LucySeldin

Keywords: BtSY49-1 Genecloning RecombinantCry1Ab Insecticidalactivity

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Inthisstudy,thecry1AbgeneofpreviouslycharacterizedandLepidoptera-,Diptera-,and Coleoptera-activeBacillusthuringiensisSY49-1strainwascloned,expressedandindividually testedonEphestiakuehniella(Lepidoptera:Pyralidae)andPlodiainterpunctella(Lepidoptera: Pyralidae)larvae.pET-cry1Abplasmidswereconstructedbyligatingthecry1AbintopET28a (+)expressionvector.ConstructedplasmidsweretransferredtoanEscherichiacoliBL21(DE3) strainrenderedcompetentwithCaCl2.Isopropyl␤-d-1-thiogalactopyranosidewasusedto

inducetheexpressionofcry1AbinE.coliBL21(DE3),andconsequently,∼130kDaofCry1Ab wasobtained.BioassayresultsindicatedthatrecombinantCry1Abatadoseof1000␮gg−1 caused40%and64%mortalityonP.interpunctellaandE.kuehniellalarvae,respectively. How-ever,themortalityratesofBtSY49-1strains’spore–crystalmixtureatthesamedosewere observedtobe70%onP.interpunctellaand90%onE.kuehniellalarvae.Theresultsindicated thatcry1Abmaybeconsideredasagoodcandidateintransgeniccropproductionandasan alternativebiocontrolagentincontrollingstoredproductmoths.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Introduction

Bacillus thuringiensis (Bt) is a Gram-positive aerobic or fac-ultativeaerobicspore-formingentomopathogenicbacterium thatcaneasilybeisolatedfromavarietyofenvironmental sources.1_{Ithasspecifictoxicityagainsttargetinsectsandis} safetonon-targetorganisms.Cry1toxinsarethemost com-moncrystalproteinscharacterizedsofarinBtstrainsandhave

∗ _{Correspondingauthor.}

E-mails:azizogluugur@hotmail.com,azizoglu@erciyes.edu.tr(U.Azizoglu).

specific insecticidal activity against lepidopteran insects.2 They form typical bipyramidal parasporal inclusions with 130kDamolecularweight.3_{Biotechnologicaldevelopmentsin} agriculturehavecaused scientiststoseek newsolutionsto insectpestproblems.Transgenictechnology,involvingawide range ofpesticidal genes from Bt,dominates the scenario of agriculturalbiotechnology. The improvementof broader spectrumbiopesticidesusingnovelBtstrainsagainsttarget insectsisanimportantaspectforimprovingtheirpersistence

http://dx.doi.org/10.1016/j.bjm.2016.04.011

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

onplants.ThepyralidmothsEphestiakuehniella(Lepidoptera: Pyralidae)andPlodiainterpunctella(Lepidoptera:Pyralidae)are global pests, particularly of stored grains, legumes, dried fruits,nuts,dates,and cocoabeans,andtheyare themost commonlyreportedpestsofstoredgrains.Larvaecancause extensivedamagetocropsandavarietyofprocessedfoods. Thepresenceofliveinsectsandinsectpartscanresultinthe depreciationofthegrainwhensold.Chemicalinsecticidesare commonlyusedincontrollingtheseimportantpestinsects worldwide.However,duetothe risksofchemicalpest con-troltotheenvironmentandhumanhealth,thecloningand expressionofcrygeneswithactivitytolepidopteranpestsis animportantissueconcerningspecies-specificcontrol.Inthe currentstudy,weamplifiedandclonedthecry1Abgeneofa novelBtSY49-1strain.4_{Thestrainhadinsecticidalactivityto} avarietyoflepidopteran,dipteranandcoleopteranpests,and wedesiredtoimprovetheefficiencythroughtransformingthe

cry1AbgeneintoE.coliBL21(DE3).Expressionofthecry1Ab

geneand itsinsecticidal activity againstthe serious stored productpestsE. kuehniellaandP.interpunctellawere investi-gated.Theresultswillreveal useful informationforstored product pestcontrol by providing Cry proteinsfrom novel strains.

Materials

and

methods

Strainsandplasmids

The Bt SY49-1 was isolated from a soil sample collected fromAdana, Turkeyin2008usingsodiumacetateenriched medium.5 _{Thestrains B.thuringiensisSY49-1,Escherichia coli} DH5␣ (kindlysupplied byMiddleEast Technical University, METU,MolecularBiologyLaboratory),cloningvector pGEMT-Easy,expressionvectorpET28a(+)andE.coliBL21(DE3)were usedinexperimentalprocedures.BtandE.coliwerecultured inLuriaBertani(LB,10g/LTriptone,5g/Lyeastextract,5g/L NaCl)mediumat30◦Cand37◦C,respectively.

Electronmicroscopy

Forelectronmicroscopy,aBtSY49-1spore–crystalmixturewas suspendedindH2Oonamicroscopeslideandfixedafterair dryingatroomtemperature.Thesamplewassputter-coated with 10nm Au/Pd using a SC7620 Mini-sputter coaterand viewedusingascanningelectronmicroscope(LEO440)at20kV beamcurrent.

Insectcultures

The larvae of E. kuehniella were rearedon a diet contain-ingamixtureofwheatflour, wheatbranand glycerol,and theIndian mealmothP. interpunctellalarvaewere obtained from naturally infested dried apricot in Kayseri province. The larvae of P. interpunctella were maintained continu-ously on a dietcontaining 10% glycerol, 50% driedapricot and 40% wheatflour–wheat bran mixture. Throughoutthe experiments, insect cultures were maintained at constant temperature (27±1◦C), photoperiod (14L:10D) and relative humidity(60±5%).6,7

Amplificationofcry1AbgenefromBtSY49-1

TotalDNAofBtSY49-1wasextractedaccordingtothemethod ofBravoetal.8 _{andusedastemplateforpolymerasechain} reactions (PCR). The cry1Ab gene was amplified using the primerpairsF-5-GGATCCATGGATAACAATCCGAAC ATC-‘3;R-5-GTCGACTTATTCCTCCATAAGAAGTAA-3.9_BamHI andSalIrestrictionenzymerecognitionsequenceswereadded tothe5endoftheforwardandreverseprimers,respectively. PCRmixescontainedthereagentsatafinalconcentrationof 2.3mMMgCl2,1×Taqbuffer,0.2mMdNTPmix,0.3pmolof eachprimer,0.5UMaxTaqDNApolymerase(Vivantis,PL2201), and30–100ngtemplateDNA.ThePCRamplificationwas per-formedunderthefollowingconditions:Initialdenaturationat 95◦Cfor5min,followedby30cyclesat94◦Cfor1min,52◦Cfor 1min,72◦Cfor3.5min,andafinalextensionstepat72◦Cfor 10min.9_{Adeninewasaddedtothe3}_{endofthePCRproducts} afteramplificationfortheTAcloningprocess.

Transferofcry1AbgeneintopGEM-TEasycloningvector

Toconstructthe pGEM-cry1Ab,PCRproducts(∼3.5kb) were purified and ligated into pGEM-TEasy vector. Theligation procedure was conducted according to the manufacturer’s protocol(Promega,vectorsystemI),andthenligatewas trans-formedintoCaCl2-renderedcompetentE.coliDH5␣.

Expressionofcry1AbgeneinE.coli

BamHIandSalI-treatedcry1AbPCRampliconandpET28a(+) vectorwereligatedaccordingtothemanufacturer’sprotocol (pETmanualsystem).ThepET-cry1AbwastransferredintoE. coliBL21(DE3)cellsrenderedcompetentwithCaCl2.The posi-tivecloneswereselectedusingthePCRmethodandincubated at37◦CuntilreachingOD600=0.5–1in100mLofLBmedium containing 30␮g/mL kanamycin. The50mL ofculture was induced by1mMIPTG for4hand followedbycentrifuging at4◦Cand5000rpmfor5min.Thepelletwassolubilizedin 12mLof20mMTris–HCl(pH7.5)andcentrifugedasdescribed above.Theremainingpelletwassolubilizedin5mLof20mM Tris–Cl (pH 7.5) containing 50␮Llysozyme (10mg/mL) and incubatedfor15minat30◦C.Thecellswerethensonicated for1mintoreleaseproteinsfromlysate.Subsequently,itwas centrifugedat4◦Cand14,000rpmfor10min,andthepellet wasresuspendedin2mLof20mMTris–HCl(pH7.5)for SDS-PAGEanalysis.Thesameprocedurewasappliedforisolating theproteinsfromE.coliBL21(DE3)andBtSY49-1.Total pro-teinquantitationwasdeterminedaccordingtotheBradford10 method.

Cry1Abproteinquantification

TheSDS-PAGE imageofthe approximately 130kDaCry1Ab band was used for determining the quantities. Cry pro-tein concentrations were calculated by the following for-mula: Cry protein concentration (␮g/mL)=(␮g/mL total protein)×(proportionofCryproteintototalprotein).11 _The proportion of Cry protein to total proteinwas determined

usingtheBioradChemiDoc MPImagingSystemImageLab version5.1(Biorad).

InsecticidalactivityofCry1Ab

LyophilizedsamplesofBtSY49-1,E.colicarryingpET-cry1Ab, andplasmid-freeE.coliBL21(DE3)wereappliedto10 third-instarlarvaeofP.interpunctellaand E.kuehniellaatdoses of 10,25,50,100,250,500and1000␮gg−1suppliedinthediet. BioassayexperimentsonP.interpunctellaandE.kuehniellawere performedaccordingtothemethodofObeidatetal.12 Experi-mentswerecarriedoutasthreereplicates.Thedatafromthe experimentsweresubjectedtoanalysisofvariance(ANOVA), andmeanswereseparatedatthe5%significancelevelbyusing theTukeyHSDposthoctest.LC50 wereestimatedbyprobit analysis.13

Results

Amplificationofcry1Abgene

ThetotalDNAoftheBtSY49-1strainwasscreenedbythePCR methodusingthecry1Ab-specificprimerpairsforamplifying thefull-lengthgeneregion(Fig.1).

Spore–crystalmixtureandelectronmicroscopy

BtSY49-1wasincubated inT3sporulationmediumat30◦C for4daystoobtainaspore–crystalmixture.Investigationsof themixtureindicatedthatbipyramidalcrystalswere compat-iblewiththepresenceofcry1geneproducts.Spherical,cubic andirregularlyshapedcrystalswerealsoobservedviaelectron micrograph(Fig.2).

Cry1AbgeneTAcloning

PCR product corresponding to the open reading frame of

cry1Ab gene (∼3.5kb) was amplified and inserted into the pGEM-TEasyvectorsystemtopreservethegeneforfurther use. Theresulting combination was transferred into E. coli

DH5␣.Subsequently,pGEM-cry1Ab from a positive cloneof

Marker

3.5kb

10kb 6 4 3 2 1

Fig.1–cry1Abgeneamplicon,Marker(Fermentas,SM 0331).

I C

Sp

B

MAG = 50.00 KX Detecter = SE1 Date: 17 Dec 2009 1μm

EHT = 20.00 kV

S

Fig.2–Spore–crystalmorphologyofBtSY49-1strain.B, bipyramidal;C,cubic;S,spherical;I,irregularlyshaped spherical;Sp,spore.

E.coliDH5␣wasdigestedwithEcoRItovalidatetheligation (Fig.3).

Expressionofcry1AbinE.coliBL21(DE3)

Thecry1Ab geneexcisedwithBamHI andSalIwas inserted into expressionvector pET28a(+)forobtainingpET-cry1Ab. The resulting pET-cry1Ab was transformed into the E. coli

BL21(DE3)strain,andpositivecloneswerevalidatedbycolony PCR (Fig. 4).A positiveclone, E.coli BL21 (DE3)pET-cry1Ab, was cultured in kanamycin containing LB medium until OD600=0.5–1 was achieved and subsequently induced with IPTG.Theexpressionofrecombinantproductswasanalyzed bySDS-PAGE,verifyingthepresenceofa130kDaproteinband (Fig.5).

Cry1Abquantification

Theproteinconcentrationwascalculatedaccordingtothe fol-lowingformula:Cry1Abconcentration(␮g/mL)=(␮g/mLtotal protein)×(%proportionofCry1Abtototalprotein).Here,the totalproteinwas7.63␮g/mL,theproportionofCry1Abtototal

Marker 10kb 6.515 kb pGEM-Tcry1Ab 3.5 kb cry1Ab 3.015 kb pGEM-T 6 3.5 3 2

Fig.3–ValidationofrecombinantpGEM-cry1Abby restrictiondigestionwithEcoRI,Marker(Fermentas,SM 0331).

10kb

Marker

3.5 3

1

Fig.4–Validationofcry1Abgeneinpositiveclonesusing colonyPCR,Marker(Fermentas,SM0331).

proteinwas2.58␮g/mL,andtheproportionofCry1Abtototal proteinwas33.82%.

ToxicityofpET-cry1Ab,BtSY49-1andplasmid-freeE.coli BL21(DE3)

TheLC50 valuesofrecombinantCry1Abon third-instar lar-vaeofE.kuehniellaandP.interpunctellawerefoundtobe685.67 and 1320.84␮gg−1, respectively. Thetoxicity ofthe source strainSY49-1washighercomparedwithrecombinantprotein (LC50=365.17␮gg−1 forE.kuehniellaand582.179␮gg−1 forP.

interpunctella).Plasmid-freeE.coliBL21(DE3)didnotexert sig-nificantactivityonbothpestlarvae.Themortalityratesare suppliedinFigs.6and7.(Fig.6;(A)F=7.710;df=7;P≤0.0001;

(B) F=22.136; df=7; P≤0.0001; (C) F=0.635; df=7; P=0.721; Fig.7;(A)F=4.082;df=4;P≤0.037;(B)F=8.799;df=4;P≤0.003; (C)F=0.474;df=4;P=0.754).

Discussion

Bt SY49-1 is a novel strain, and the toxicity of its wet-tablespore–crystalpowderwaspreviouslydeterminedagainst insectpestsfromdifferentorders.4_{E.kuehniellaandP.}

inter-punctellaaretwotroublesomepestsposingseriousproblems instoredproductsworldwide.Inthepresentstudy,thecry1Ab

gene(∼3.5kb)ofapreviouslycharacterizedBtSY49-1strain wasusedthoroughcloningandexpressioninE.coliBL21(DE3). Itiswell knownthat cry1Ageneshavespecificinsecticidal activityagainstlepidopteranpests.14,15 _{BtSY49-1producesa} 130–140kDaCry1bandcorrespondingtothebipyramidal crys-talstructure(Fig.2).Cry1Ab(∼130kDa)wasoverexpressedin IPTG-inducedE.coliBL21(DE3),anditsbioactivitywastested on E. kuehniella and P. interpunctella. The results indicated thatrecombinantCry1Abexhibitedconsiderablemortalityon pestlarvae.However,increasingdosesofCry1Abfrom50to 500␮gg−1 had littleadditional toxicityon the larvaewhen comparedwiththespore–crystalmixtureofsourceorganism. SimilartrendswerereportedwiththerecombinantCry3Aaon

Hypothenemushampei.16_Zhangetal.17_{alsoreportedthatthe} activityofrecombinantcry8Ab1haslowertoxicitycompared withthesourceorganism,suggestingthatthisphenomenon mayoriginatefromlessCry8Ab1expressioninthehost organ-ismandmorethanonetypeofdifferentlysizedcrystalsbeing expressedinwild-typeBt.Comparativelylowertoxicitywas

M M 6 5 4 3 2 1 200 kDa 150 kDa 150 kDa ∼130 kDa ∼130 kDa 120 kDa 120 kDa 100 kDa 100 kDa 85 kDa 85 kDa 70 kDa 70 kDa 60 kDa 60 kDa 50 kDa 50 kDa 7

Fig.5–SDS-PAGEanalysisofcry1AbgeneexpressedinE.coliBL21(DE3).M;Marker(FermentasSM0661),Lane1: IPTG-inducedE.coliBL21(DE3)pET-cry1Ab;lane2:E.coliBL21(DE3)pET-cry1Ab;lane3:IPTG-inducedE.coliBL21(DE3) pET-cry1Ab;lane4:E.coliBL21(DE3)pET-cry1Ab;lane5:E.coliBL21(DE3);lane6:IPTG-inducedE.coliBL21(DE3)IPTG;lane7: B.thuringiensisSY49-1.

100

A

B

C

90 80 70 60 50 P ercent mor tality 40 30 20 ab Control 10μg/g 25μg/g 50μg/g Concentration 100μg/g 250μg/g 500μg/g 1000μg/g Control 10μg/g 25μg/g 50μg/g 100μg/g250μg/g 500μg/g1000μg/g ab a abc abc pET-cry1Ab bc bc a ab bc c c c Bt SY49-1 dc e c 10 0 100 90 80 70 60 50 P e rcent mor tality 40 30 20 10 0 a _a a a a E. coli BL21(DE3) a a a 100 90 80 70 60 50 P ercent mor tality 40 30 20 Control 10μg/g 25μg/g 50μg/g Concentration Concentration 100μg/g 250μg/g 500μg/g 1000μg/g 10 0

Fig.6–ToxicityonE.kuehniellalarvae.(A)TpET-cry1Ab;(B)BtSY49-1spore–crystalmixture;(C)E.coliBL21(DE3)total protein. 100

A

C

B

pET-cry1Ab Bt SY49-1 90 80 70 60 50 40 P ercent mor tality 30 20 10 0 100 E. coli BL21(DE3) 90 80 70 60 50 40 P ercent mor tality 30 20 10 0 100 90 80 70 60 50 40 P ercent mor tality 30 20 10 0 Control a a a a ab a a a a ab ab ab ab ab ab a a a a b bc bc c b 10μg/g 25μg/g 50μg/g Concentration Concentration 100μg/g250μg/g500μg/g1000μg/g Control 10μg/g 25μg/g 50μg/g Concentration 100μg/g250μg/g500μg/g1000μg/g Control 10μg/g 25μg/g 50μg/g 100μg/g250μg/g500μg/g1000μg/g

Fig.7–ToxicityonP.interpunctellalarvae.(A)pET-cry1Ab;(B)BtSY49-1spore–crystalmixture;(C)E.coliBL21(DE3)total protein.

alsoreportedbysomeotherresearchersonE.kuehniellausing Cry1Aa,Cry1Ac,andCry2Aa18_{andonChironomustepperiusing} Cry11AandCry4B.19_{Ontheotherhand,Parketal.}20_reported similartoxicitytotheparentstrainagainstCulexmosquitoes withCry11BfromB.thuringiensissubsp.,jegathesan.Similarly,

BtSY49-1harborscry1,cry2,cry4,cry5andcry9genes,andits higheractivitymaypossiblyresultfromthecombinedactivity ofthesegene-correspondingproducts.Therefore,thisstudy wasintendedtodeterminetheeffectivenessofCry1Ab, inde-pendentofothergenesencodinginsecticidalproteins.

In the present study, the individual toxicity of Cry1Ab (expressedinE.coli)wasevaluatedonE.kuehniellaandP. inter-punctellatoavoidpotentialsynergisticinteractions between sporesandcrystals.21_{Asfarasweknow,thisisthefirstreport} evaluatingthe toxicityofE. coliBL21(DE3)pET-cry1Ab onE. kuehniellaandP.interpunctella.Significantdifferenceswerenot observedwithrespecttotoxicitybetweenE.coliBL21(DE3)and waterycontrol.TheindividualtoxicityofrecombinantCry1Ab onthethird-instarlarvaeofthesetwostoredproductpests waspreciselyestimated.

Inconclusion,thecry1AbgeneoftheBtSY49-1strainwas successfullycloned,expressedandtestedonE.kuehniellaand

P.interpuctellalarvae.Theresultsindicatedthatcry1Abmaybe consideredasagoodsourceintransgeniccropproductionand asanalternativebiocontrolagentincontrollingstoredproduct moths.

Ethical

statement

Theauthorsdeclarethattheexperimentscompliedwiththe currentlawsofthecountryinwhichtheywereperformed.

Conflict

of

interest

Theauthorsdeclarethattheyhavenoconflictofinterest.

Acknowledgements

TheauthorsthankDr.MikailAkbulutandZehraBüs¸raAtciyurt fortheirvaluableassistance.SpecialthankstoMETU Molecu-larBiologyLaboratoryforsupplyingE.coliDH5␣.Thisproject wasfundedbythe ErciyesUniversityScientificProjectUnit under the codes of FBD11-3634 and ÖNAP-3638 and also fundedbyBilim,SanayiveTeknolojiBakanlı ˘gıTürkiye (TGSD-0802).

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