Molecular Cloning, Recombinant Expression and Antifungal Activity of BnCPI, a Cystatin in Ramie

15 Pages·2017·3.39 MB·English

G C A T genes
T A C G
G C A T
Article
Molecular Cloning, Recombinant Expression and
Antifungal Activity of BnCPI, a Cystatin in Ramie
(Boehmeria nivea L.)
YongtingYu1,*,† ID,GangZhang2,†,ZhiminLi1,YiCheng1,ChunshengGao1,LiangbinZeng1,
JiaChen1,LiYan1,XiangpingSun1,LitaoGuo1andZhunYan1,*
1 InstituteofBastFiberCropsandCenterforSouthernEconomicCrops,ChineseAcademyof
AgriculturalScience,Changsha410205,China;[email protected](Z.L.);[email protected](Y.C.);
[email protected](C.G.);[email protected](L.Z.);[email protected](J.C.);[email protected](L.Y.);
[email protected](X.S.);[email protected](L.G.)
2 CollegeofPharmacyandShaanxiProvincialKeyLaboratoryforChineseMedicineBasis&
NewDrugsResearch,ShaanxiUniversityofChineseMedicine,Xi’an712406,China;
[email protected]
* Correspondence:[email protected](Y.Y.);[email protected](Z.Y.);Tel.:[phone](Y.Y.)
† Theseauthorscontributedequallytothiswork.
Received:30July2017;Accepted:3October2017;Published:11October2017
Abstract: Phytocystatinsplaymultiplerolesinplantgrowth,developmentandresistancetopests
andotherenvironmentalstresses. Aramie(BoehmerianiveaL.)phytocystatingene,designatedas
BnCPI,wasisolatedfromaramiecDNAlibraryanditsfull-lengthcDNAwasobtainedbyrapid
amplificationofcDNAends(RACE).Thefull-lengthcDNAsequence(691bp)consistedofa303bp
openreadingframe(ORF)encodingaproteinof100aminoacidswithdeducedmolecularmassof
11.06kDaandatheoreticalisoelectricpoint(pI)of6.0. ThealignmentofgenomeDNA(accession
No. MF153097) and cDNA sequences of BnCPI showed that an intron (~104 bp) exists in the
codingregion. TheBnCPIproteincontainsmostofthehighlyconservedblocksincludingGly5-Gly6
at the N-terminal, the reactive site motif QxVxG (Q49V50V51S52G53), the L79-W80 block and the
[LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-x-[EDQV]-[HYFQ]-N(L22G23R24F25A26V27D28D29H30N31)block
thatiscommonamongplantcystatins. BLASTanalysisindicatedthatBnCPIissimilartocystatins
from Glycine max (77%), Glycine soja (76%), Hevea brasiliensis (75%) and Ricinus communis (75%).
TheBnCPIwassubclonedintoexpressionvectorpSmart-IandthenoverexpressedinEscherichiacoli
BL21 (DE3) as a His-tagged recombinant protein. The purified reBnCPI has a molecular mass of
11.4kDadeterminedbysodiumdodecylsulfatepolyacrylamidegelelectrophoresis(SDS–PAGE).
Purified reBnCPI can efficiently inhibit the protease activity of papain and ficin toward BANA
(Nα-benzoyl-L-arginine-2-naphthyamide),aswellasthemyceliumgrowthofsomeimportantplant
pathogenic fungi. The data further contribute to our understanding of the molecular functions
ofBnCPI.
Keywords: BoehmerianiveaL.;BnCPI;cysteineproteaseinhibitors;intron;functionalexpression
1. Introduction
Cysteineproteaseinhibitorsorcystatinsofplantoriginarecalledphytocystatins;theydisplay
multiple functions in seed formation and germination, plant growth and development, as well as
plantresistance/tolerancetobiotic/abioticstresses[1–3]. Foralmost30years,phytocystatinsandtheir
rolesinplant–pestinteractionsattractedextensiveinternationalconcernandstudy. Adeeperand
morenuancedunderstandingofphytocystatinswascreatedfromtheincreasingamountofdataand
relatedresearch. Somephytocystatinscandirectlyimpairherbivoregrowth,asevidencedbyimpaired
Genes2017,8,265;doi:10.3390/genes8100265 www.mdpi.com/journal/genes
Genes2017,8,265 2of15
growthofthesepestswhenrearedonanartificialdietcontainingcystatinsortransgenicplantswith
exogenous cystatin genes [4–6]. Cystatins of rice, maize and taro can increase plant resistance to
variousphytopathogenicnematodes[7–9]. Manyphytocystatinscanalsodirectlyinhibitthegrowthof
phytopathogenicfungi[2]. Somephytocystatins(suchasCeCPI,HvCPI-1)withantifungalactivity
alsoinhibitthegrowthofnematodesandherbivores[7,10–12]. Transgenictobaccoover-expressing
potatosporaminandtarocystatindisplayedincreasedresistancetobothinsectsandphytopathogens,
including Helicoverpa armigera, Erwinia carotovora and Pythium aphanidermatum [13]. These reports
supplydirect,substantialevidenceforthepotentialofphytocystatinsinimprovingplantresistance.
The inhibition mechanism of phytocystatins on different pests has also been explored.
Cysteine protease (CP) is a major digestive enzyme in many herbivores and nematodes; it plays
an important role in reproduction, development, tissue invasion, pathogenesis and immune
invasion [14–17]. The harmful effect of phytocystatins on nematodes and herbivores was thought
to inhibit the activity of CP of these animals. However, the mechanism of growth inhibition of
phytocystatinsonfungiisstillunclear. Someresearchershaveproposedthatitblocksindigenous
proteinaseactivityoffungi[11,18],whereasothersfoundthattheinhibitionisnotassociatedwithits
cysteineproteinaseinhibitoryproperties[10].Thesediscoveriesindicatethattheinhibitionmechanisms
ofdifferentphytocystatinstowardfungiaredifferent.
Ramie (Boehmeria nivea L.) is a perennial herbaceous plant of the family Urticaceae. It is also
called “China grass” since it has been cultivated in China for over 6000 years [19]. Ramie is an
importantnaturalfibercropplantedmainlyinChina,IndiaandotherSoutheastAsianandPacific
Rim countries [20]. Traditionally, ramie was planted solely for harvesting bast fibers. Recently,
ramie leaves and shoots have also been used as fodder for beef cattle and geese because of the
plant’s high crude protein content [21]. Root lesion disease (RLD), a destructive root disease that
iscausedbythenematodePratylenchuscoffeae,severelyimpairsthegrowthandyieldoframie[22].
The lack of knowledge on ramie RLD resistance mechanisms or genes severely hinders efforts in
effectively breeding and utilizing nematode-resistant ramie. More recently, a ramie cystatin gene
BnCPI (Cysteineproteaseinhibitor, Unigene11292)wasfoundtoberegulatedinP.coffeae-infected
resistant ramie but not in a susceptible cultivar, which suggested that BnCPI may be involved in
pestresistance[23]. InordertodecipherthebiologicalfunctionofBnCPI,itisveryessentialtoclone
andcharacterizethegeneandactiveprotein. Thisstudyreportedthemolecularcloning,sequence
analysis,recombinantexpressionandbiochemicalandantifungalactivitystudyofthisgeneandits
correspondingprotein.
2. MaterialsandMethods
2.1. PlantGrowthandSampling
Ramie varieties Qingdaye (QDY), Zhongzhu No.1 (ZZ1) and Heipidou (HPD), which were
resistant,moderatelysusceptible,andsusceptibletoP.coffeae,respectively,wereusedinthisstudy.
Seedlingswerepreparedwiththecuttingpropagationmethod[23]. Twoweekslater,whentheroots
were about 20 cm length, roots of a single plant were sampled and immediately frozen in liquid
nitrogenandstoredat−80◦C.
2.2. DNAandRNAExtractionandcDNASynthesis
Roottissuesoframieina2mLEppendorftube(pre-chilledinliquidnitrogen)weregroundto
finepowderusingaTissuelyser-24Multi-SampleTissueGrinder(ShanghaiJingxing,Shanghai,China).
TotalDNAandRNAwereextractedusingaPlantDNAMiniKitandanEASYspinPlusTotalRNA
Kit (both from Aidlab, Beijing, China), following the manufacturer’s protocol. A Nanodrop 2000
spectrophotometer(ThermoFisherScientific,Waltham,MA,USA)wasusedtomeasureDNAand
RNAconcentration,andfirst-strandcDNAsamplesweresynthesizedfromabout1µgofthetotal
RNAusingaRevertAidFirstStrandcDNASynthesisKit(ThermoFisherScientific).
Genes2017,8,265 3of15
2.3. MolecularCloningoftheBnCPIGene
A787-bpcDNAfragmentoftheBnCPIgenewasobtainedfromtranscriptomesequencingdata
oframie[23]. Thesequencescontainedanopenreadingframe(ORF)-encodedramiehomologofthe
cysteine protease inhibitor. Based on this sequence, 3(cid:48)-RACE (rapid amplification of cDNA ends)
was performed to amplify the 3(cid:48)-end of this gene using a SMART™ RACE cDNA Amplification
Kit(Clontech,MountainView,CA,USA)accordingtothemanufacturer’sguidelines. Twointernal
gene-specificforwardprimers,cpi3-1(5(cid:48)-GATGGCGGTGTCAAGAAGGTTTACGA-3(cid:48))andcpi3-2
(5(cid:48)-AAGGTCTGGGAAAAGTTGTGGTTGAA-3(cid:48)),wereassignedfromtheknowncDNAfragment
ofBnCPI.Briefly,first-strandcDNAwassynthesizedwith3(cid:48)-CDSprimerA,andthenamplifiedby
PCR using cpi3-1 as a forward primer and UPM (Universal Primer A mix) as the reverse primer.
Subsequently,theproductofthefirstroundofPCRwasdiluted50times,andusedasatemplatein
thesecond-roundPCRamplificationusingcpi3-2andUPM.ThePCRproductswereseparatedon1%
agarosegels,purified,clonedintopMD-18vectorandsequencedbyShanghaiSangonBiotechnology
Co. Ltd. (Shanghai, China). Theresultingfull-lengthcDNAofBnCPI wasdepositedintheNCBI
(NationalCenterforBiotechnologyInformation)GenBankwithaccessionnumberKT438742.1.
2.4. SequencesAnalysisandPhylogeneticAnalysis
TheonlineORFfinderprogramofNCBI(https://www.ncbi.nlm.nih.gov/orffinder/)wasused
tosearchtheentireORFsequencesofBnCPI,whiletheonlineBLASTprogramofNCBIwasusedto
searchproteinhomology. Theoreticalmolecularweight(MW)andisoelectricpoint(pI)forBnCPIwere
predictedusingtheExPASytool(http://web.expasy.org/compute_pi/);signalpeptidepredictionwas
performedusingSignalP4.1(http://www.cbs.dtu.dk/services/SignalP/);andsecondarystructure
waspredictedusingSOPMA[24]. TheconserveddomainwassearchedagainsttheNCBIConserved
DomainDatabase[25].
Asetofprimers(cpif: 5(cid:48)-CGCAGAAAAGTAAAAGCA-3(cid:48) andcpir: 5(cid:48)-TCCACCAAAGAC
GAA TGA-3(cid:48)) was assigned for PCR amplification of ORF fragments in different ramie cultivars.
Primerscpifandcpirwerelocatedupstreamofthestartcodonanddownstreamofthestopcodonof
theORF,respectively. PCRwasperformedinanETC-811PCRinstrument(Eastwin,Beijing,China).
Thereactionmixturecontained25µLof2×TaqPCasterMix(Aidlab,Beijing,China),4µLofeach
primer(10µmol/µL),2µLofgenomicDNAorcDNA(10µg/µL),andthetotalvolumewasadjusted
to 50 µL with ddH O. The PCR amplification procedure consisted of 94 ◦C for 4 min, 35 cycles of
2
denaturationat94◦Cfor45s,annealingat50◦Cfor1min,andextensionat72◦Cfor1min,with
final extension at 72 ◦C for 10 min. The PCR products were separated by electrophoresis on 1.5%
agarosegelsandvisualizedbystainingwithGoldView(Applygen,Beijing,China). Ampliconswere
sequenced by Tsingke Company (Tsingke, Beijing, China). BnCPI and phytocystatins from other
plantsdownloadedfromtheNCBIwebsitewerealignedusingMEGA6.0software[26]. Theresulting
alignmentwastrimmedandaphylogenetictreewasconstructedusingtheneighbor-joiningmethod
with100bootstrapping. ThededucedaminoacidsequenceofBnCPIwasalsoalignedwiththoseof
otherphytocystatinsavailableintheEMBL-EBIdatabaseClustalW2(http://www.ebi.ac.uk/Tools/
msa/muscle/),setatdefaultparameters.
2.5. ExpressionofaRecombinantBnCPIinEscherichiacoli
TheORFofBnCPIcontainsmanyrarecodons(FigureS1a)thatwillimpairexpressionefficiency
inarecombinantexpression. Genecodonoptimizationwasperformedbysynthesizingsequences
encoding amino acids identical to those encoded by BnCPI but without rare codons (Figure S1b).
ORFgenesynthesiswasperformedbytheTsingkeCo. (Tsingke,Beijing,China). Theoptimizedgene
fragmentwasclonedintotheexpressionvectorpSmart-I(GeneralBiosystems,USA),whichcarried
6×His-tagandSUMOtofacilitatesolutionandpurificationoftherecombinantexpressedprotein.
TherecombinantvectorwassubsequentlytransformedintoEscherichiacoliBL21(DE3). Positiveclones
Genes2017,8,265 4of15
containingtherecombinantvectorwerescreenedonampicillin. Cellsofpositiveclonesweregrown
at 37 ◦C in LB liquid medium with 220 rpm until OD600 reached 0.6–0.8. Subsequently, IPTG
(isopropyl β-D-1-thiogalactopyranoside) was added to a final concentration of 0.5 mM to induce
expressionofBnCPIat37◦Cfor4horat15◦Cfor16h.
After culture, cells were collected by centrifugation, suspended in 25 mM Tris buffer
(containing300mMNaCl,pH8.0),andsonicatedbyanultrasonicprocessorfor2min.Thefragmentized
cell suspension was centrifuged at 12,000 rpm at 4 ◦C for 15min. The resultant suspension
and precipitate were separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS–PAGE)tochecktherecombinantexpressedBnCPI(reBnCPI).Then,recombinantproteinwas
treatedwithSUMOproteasetoremovetheSUMOproteinandpurifiedbyaNi–NTAaffinitycolumn
(GEHealthcare,Russellville,AR,USA)usinganÄKTA-primesystem(GEHealthcare).
2.6. ProteaseInhibitoryActivityAssay
CysteineproteaseinhibitoryactivityofreBnCPItowardpapain(EC3.4.22.2),ficin(EC3.4.22.3)
andbromelain(EC.3.4.22.32)(allfromSigma-Aldrich,St. Louis,MO,USA)weretestedusingamethod
describedpreviously[15]. TheinhibitoryactivityofreBnCPIwasrecordedasresidualenzymeactivity
in the presence of inhibitor. In the control treatment, equal volumes of the corresponding buffer
wereusedinsteadofreBnCPI.Therewerethreereplicatesineachtreatmentandtheexperimentwas
repeatedtwice.
2.7. AntifungalActivityAssayofreBnCPI
Two pathogens of the ramie plant, Pythium vexans causing brown root rot [27] and Alternaria
alternatacausingleafspot[28],aswellastwootherimportantplantpathogens(Fusariumoxysporum
andBotrytiscinerea,isolatesofwhicharekeptinourlaboratory),wereusedforthegrowthinhibition
assay. The invitro growth inhibition assays were performed as described by Pernas et al. [29].
Fungalstrainsweregrownonpotatodextroseagar(PDA)mediumfor3–7days. ForF.oxysporum,
A. alternata and B. cinerea, spores were collected with 1/3 PDB (PDA medium without agar) and
dilutedtoaconcentrationof104 spores/mL.ForP.vexans,hyphaewerecollectedwith1/3PDBin
a2-mLEppendorftube,homogenizedanddilutedtoaconcentrationof104CFU/mL.ThereBnCPI
(after filtration sterilization) was added to the suspension to produce a final concentration of
20–80 µg/mL. Fungicides carbendazim, jinggangmycin, fluazinam and mefenoxam were added
tothesuspensionsofF.oxysporum,A.alternata,B.cinereaandP.vexans(finalconcentration50µg/mL),
respectively,toserveaspositivecontrols. FungalsuspensionswithoutfungicidesandreBnCPIwere
usedasnegativecontrols,andbuffer(25mMTris,300mMNaCl,pH8.0)usedtodissolvereBnCPIwas
addedinsteadofreBnCPIorfungicides. Then,sporesorhyphaesuspensionof200µLwerecultivated
on a sterile 96-well microtiter plate at 25 ± 1 ◦C for 48 h. Fungal growth was then monitored by
measuringabsorbanceat492nm(Infinit200Pro,Tecan,Männedorf,Switzerland)andcheckedunder
aNikonAZ100microscope(NikonCo.,Tokyo,Japan). Resultswereexpressedasthepercentageof
relative growth in the absence of the reBnCPI. Each treatment was replicated three times, and the
experimentwasrepeatedtwice.
3. Results
3.1. SequenceAnalysis
ThecDNAsequenceoftheBnCPIgenewas691bpinlength,including123bpof5(cid:48) untranslated
region(UTR),303bpofORFand265bpofthe3(cid:48) UTR.TheORFlikelyencodedadeduced100-residue
aminoacid(AA)sequence(Figure1). Thematureproteinhadamolecularweightof11.06kDaand
pIof6.0,aspredictedbytheExPASytool. NosignalpeptidewasfoundwhentheAAsequencewas
analyzedwithSignalPsoftware.Aconserved-domaindatabase(CDD)searchshowedthatthededuced
BnCPIproteinsequencewasidenticaltotheconservedcystatin-likedomain(CYdomain: cd00042).
Genes2017,8,265 5of15
BnCPIcontainedmostofthehighlyconservedblocksthatareessentialforcysteineproteinaseactivity,
Ginencelsu 2d0i1n7,g 8,t h26e5 G G(Gly5-Gly6),thereactivesitemotifQxVxG(Q49V50V51S52G53),theWresidue5(W of 8104)
andthehighlyconserved[LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-x-[EDQV]-[HYFQ]-Nblock(L22G23R24
(FF2i5gAu2r6eV 12)7. DTh28eD s2e9cHon30dNar3y1 )s(tFruigcuturere1 )o.fT thhee sceocdoendd aprryotsetirnu cmtuarienloyf cthoensciosdteedd opfr oαt-ehienlimceasi nalnydc orannsdisotemd
coofilαs- h(Feilgicuerse aSn2da)r.a Tnhdeo mterctoiairlsy (sFtirguucrteurSe2 ap)r.edTihcetiotenr toiaf rtyhest rcuocdteudr epproretediinc tiionndiocafttehde tchoadt etdhep rsoptaecine
sintrduicctautreed mthaaitntlhy ecospnataciensetdru αc-thuerleicmesa,i nβl-ytucronnst aaninde dβ-αsh-heeeltisc e(Fs,igβu-rtue rSn2sba).n dβ-sheets(FigureS2b).

FFiigguurree 11.. NNuucclleeoottiiddee aanndd ddeedduucceedd aammiinnoo aacciidd sseeqquueenncceess ooff tthhee idideennttiiffiieedd ffuullll--lleennggtthh ccDDNNAA ooff BBnnCCPPII. .
NNuucclleeoottiiddee iinn bboolldd tteexxtt iinnddiiccaatteess tthhee ssttaarrtt aanndd ssttoopp ccooddoonnss.. SSeevveerraall mmoottiiffss ooff pphhyyttooccyyssttaattiinnss wweerree
ddeetteecctteedd iinn tthhiiss sseeqquueennccee,, ssuucchh aass GGGG,, LLAARRFFAAVV,,Q QVVVVSSGGa annddL LWW..T Thheer erespspecetcitviveem mootitfisfsa raereb booxexded..
Both the genome DNA and the cDNA sequences of BnCPI were amplified from different ramie
BoththegenomeDNAandthecDNAsequencesofBnCPIwereamplifiedfromdifferentramie
cultivars using the primers cpif and cpir (Figure 2a). Alignment of cDNA and the genome DNA
cultivars using the primers cpif and cpir (Figure 2a). Alignment of cDNA and the genome DNA
sequences indicated the presence of a 104 bp intron between the sequence encoding the LARFAV
sequencesindicatedthepresenceofa104bpintronbetweenthesequenceencodingtheLARFAVmotif
motif and the reactive site QxVxG (Figure 2b). The ORF sequences of BnCPI from the cultivars QDY
andthereactivesiteQxVxG(Figure2b). TheORFsequencesofBnCPIfromthecultivarsQDYand
and HPD were found to be identical, whereas two nucleic acids (positions 225 and 236) were
HPDwerefoundtobeidentical,whereastwonucleicacids(positions225and236)wereidentifiedin
identified in cultivar ZZ1 that differed from those in QDY and HPD (Figure 3a), while the encoded
cultivarZZ1thatdifferedfromthoseinQDYandHPD(Figure3a),whiletheencodedaminoacids
amino acids were identical (Figure 3b).
wereidentical(Figure3b).

(a) M 1 2 3 4 5 6
))
Genes2017,8,265 6of15
Genes 2017, 8, 265 6 of 14

(a) M 1 2 3 4 5 6
1337 bp
525 bp

(b)

FFigiguurere2 2.. ((aa)) EElelecctrtroopphhooreressisis ofo fPCPRC Ramapmlipflicifiactiaotnio fnrafgrmagemntesn otsf BonfCBPnIC fProImfr ocDmNcAD NanAd gaenndomgeen DomNeA
DoNn A1.5o%n a1g.5a%rosaeg agreol.s Meg, eDl.NMA ,mDaNrkAerm IIaI;r k1‒e3r, IfIrIa;g1m-3e,nfrtas gammepnlitfsieadm fprolimfie DdNfrAo mof DZZN1A, HoPfDZZ a1n,dH QPDDY
arnedspQecDtiYverleys;p e4c‒t6i,v eflrya;g4m-6e,nftrsa gammepnlitfsieadm fprloifime dcDfrNomA coDfN ZAZ1o,f ZHZP1D, HaPnDd aQnDdYQ, DrYes,preecstpivecetliyv; el(yb;)
(bA)liAgnlimgnemnte onft goefngomeneo DmNeAD N(aAcce(sascicoens snioo.n MNFo1.53M09F71) 5a3n0d9 7cD) aNnAd (caDccNesAsio(anc nceos. sKioTn43N87o4.2)K sTe4q3u8e7n4c2e)s
soeqf uBennCcPeIs forfoBmn rCaPmIifer o(cmulrtaivmaire. Q(cuDlYti)v. aTr.hQe sDhYa)d.eTdh neushclaedice dacnidusc lienidciaccaitdes ainn dinictraoten aonf 1in0t4r obnp oinf 1le0n4gbtph.
inThleen sgetqhu.eTnhceesse eqnuceondciensge tnhceo dLiAngRFthAeVL mARoFtiAf Vanmd otthief arneadctthiveer esiatcet iQvexVsixteGQ axreV xuGndaerreliunnedde arnlidne ddoaunbdle
duonudbelerluinnedde, rrleinspedec,trievseplyec. tively.
Genes2017,8,265 7of15
Genes 2017, 8, 265 7 of 14

(a)

(b)
Figure 3. (a) Alignment of cDNA and (b) deduced amino acid of BnCPI from three ramie cultivars,
Figure3. (a)AlignmentofcDNAand(b)deducedaminoacidofBnCPIfromthreeramiecultivars,
QDY, HPD and ZZ1. Two nucleic acids (positions 225 and 236) in ZZ1 differ from QDY and HPD
QDY,HPDandZZ1. Twonucleicacids(positions225and236)inZZ1differfromQDYandHPD
(nucleic acid shaded). The deduced amino acids of three cultivars are identical.
(nucleicacidshaded).Thededucedaminoacidsofthreecultivarsareidentical.
3.2. Homology Analysis of BnCPI
3.2. HomologyAnalysisofBnCPI
Analysis of the BLAST results (11 May 2017) revealed that the deduced amino acid sequence of
AnalysisoftheBLASTresults(11May2017)revealedthatthededucedaminoacidsequenceof
BnCPI shared the highest identity (76%) with sequences of cystatin from Glycine max (accession nos.
BnCPIsharedthehighestidentity(76%)withsequencesofcystatinfromGlycinemax(accessionnos.
XP_003543365 and AAA97905) and Ricinus communis (accession no. XP_002523225), followed by
XP_003543365 and AAA97905) and Ricinus communis (accession No. XP_002523225), followed by
sequences of cystatin from Glycine soja (accession no KHN35444), G. max (accession no.
sequencesofcystatinfromGlycinesoja(accessionnoKHN35444),G.max(accessionNo. NP_001239817)
NP_001239817) and Hevea brasiliensis (accession no. ACZ02398), all of which shared 75% identity with
and Hevea brasiliensis (accession No. ACZ02398), all of which shared 75% identity with BnCPI.
BnCPI. These were followed by sequences of cystatin from Vigna unguiculata (accession no.
These were followed by sequences of cystatin from Vigna unguiculata (accession No. CAA79954)
CAA79954) and Cajanus cajan (accession no. XP_020236689), both of which shared 74% identity with
andCajanuscajan(accessionNo. XP_020236689),bothofwhichshared74%identitywithBnCPI.
BnCPI.
AsshowninFigure4,alignmentofBnCPIwithsequencesofphytocystatinsfromotherplants
As shown in Figure 4, alignment of BnCPI with sequences of phytocystatins from other plants
confirmedthatBnCPIandR.communiscystatinweremostsimilar. BnCPIcontainedmostofthetypical
confirmed that BnCPI and R. communis cystatin were most similar. BnCPI contained most of the
featuresofotherphytocystatins,suchastheGGdoubletandLARFAVmotifintheN-terminalregion,
typical features of other phytocystatins, such as the GG doublet and LARFAV motif in the N-terminal
andthecentralsignaturemotifQxVxG(alsoastheactivesite),butitlackedthePWmotifcontainedin
region, and the central signature motif QxVxG (also as the active site), but it lacked the PW motif
someoftheothercystatinsfromplantsandanimals. Here,itwasrevealedbymultiplealignmentsthat
contained in some of the other cystatins from plants and animals. Here, it was revealed by multiple
thePWmotifwasreplacedbytheLWmotifintheBnCPIaminoacidsequences,whichisidenticalto
alignments that the PW motif was replaced by the LW motif in the BnCPI amino acid sequences,
thephytocystatinofpapaya(accessionNo. CAA50437)andragweed(accessionNo. AAA32672).
which is identical to the phytocystatin of papaya (accession no. CAA50437) and ragweed (accession
no. AAA32672).
Genes2017,8,265 8of15
Genes 2017, 8, 265 8 of 14

FFiigguurree 44.. AAlliiggnnmmeenntt oof fththee ddeedduucecedd amaminino oacaicdi dsesqeuqeunecnec oefo BfnBCnPCIP (IA(LAGL3G83384374) 7w)iwthi tthhet hseeqsueqenuceensc oefs
pohfyptohcyytsotcaytisntast i(nwsith(w mitohlemcuolalerc mulaasrs mofa assboouft 1a2b okuDta1) 2frokmD ao)thferor mplaontthse; rthpelya nartse; Htehveeya barraesilHieenvseisa
HbrbaCsiPlieI n(sAisCHZ0b2C3P9I8)(,A MCaZn0ih2o3t9 e8s)c,uMlenantaih MoteeCscPuI l(eAntAaFM72e2C0P2I),( PAeAlaFrg7o2n2i0u2m), hPoerltaorrguomni uPmhChPoIr t(oAruBmG8P1h0C97P)I,
T(AriBfoGliu81m0 9s7u)b,teTrrriafnoeliuumm TssuCbtPeIr r(aGnAeuUm21T0s3C7P),I R(uGmAeUx 2o1b0tu3s7i)f,oliRuus mReoxCoPbIt u(CsiAfolDiu2s14R4o1C), PMIe(dCicAagDo2 1sa4t4iv1a),
MMseCdiYcaSg1o (AsaAtiZva98M79s1C),Y CSa1st(aAneAa Zsa9t8i7v9a 1C),YSC a(sCtaAnAea11s8a9t9iv)a, PCopYuSlu(uCpAhrAat1ic1a8 9P9e)C, PPIo (pAulGuXup2h8r1a3t9ic),a GPlyecCinPeI
m(AaGx XG2m81C39P)I, G(KlyHciNne3m54a4x4G),m GClyPcIin(Ke HsoNja3 5G4a4C4)P,IG l(yKcHinNes3o5ja44G4a)C, PVIig(KnaH uNn3g5u4i4c4u)l,aVtai gVnauCunPgIu (iCcuAlaAta7V9u95C4P)I,
R(CicAinAu7s 9c9o5m4m),uRnicisin RuscCcoPmI m(XuPn_is00R2c5C2P32I2(X5)P, _D00ia2n5t2h3u2s2 c5a),ryDopiahnytlhluuss cDaCry-oCpPhIynll u(sADACK-3C0P00In4)(,A AAraKb3id0o0p0s4i)s,
tAhraalibaindao psAistCthYaSli1a n(aCAAtAC0Y3S9129()C, ACAar0i3ca9 2p9a),paCyaar icCaspt a(pCayAaAC5s0t4(3C7)A Aan5d0 43A7m)barnodsiaA marbtermosiisaiiaforltieam AisiaifColPiaI
(AAaACAP3I2(6A7A2)A. 3T2h67e 2)i.deTnhteiciadl enatnidca lpaanrdtiapl-acrotinasle-crovnatsieornv arteiosnidrueessi daurees asrheadsheadd eind ibnlabclakc kanadn dggrraayy,,
rreessppeeccttiivveellyy.. RReedd bbooxxeess sshhooww ccoonnsseerrvveedd mmoottiiffss ooff pphhyyttooccyyssttaattiinnss.. TThhee aalliiggnnmmeenntt wwaass ppeerrffoorrmmeedd bbyy
CClluussttaallWW pprrooggrraamm aanndd wwaass sshhaaddeedd uussiinngg tthhee GGeennee DDoocc ssooffttwwaarree..
3.3. Recombinant Expression of BnCPI
The ORF sequence was optimized and subsequently introduced into the pSmart-I vector, then
expressed in E. coli BL21 (DE3). Overexpression of BnCPI was induced by adding IPTG (final
Genes2017,8,265 9of15
3.3. RecombinantExpressionofBnCPI
Genes 2017, 8, 265 9 of 14
The ORF sequence was optimized and subsequently introduced into the pSmart-I vector,
tchoennceenxtpraretisosne d0.5in mEM. )c otoli tBhLe 2m1e(dDiuEm3).. ExOpvreersesexdp rreescsoiomnbionfanBtn BCnPCIPwI awsaisn ndoutc seedcrbeyteda dtod itnhge lIiPqTuGid
(mfineadliucmon. cBenactrteartiioaln c0e.l5lsm wMer)et ocothlleecmteedd biuym c.enEtxripfuregsasteiodnr,e hcoommboigneannizteBdn CbyP Iuwltarassnoontics eccrraesthe dantod
tphuerliifqieudid bmy eadffiiunmity. Bcahcrtoemriaaltocgelrlaspwheyr. eAcfotlelre crteemdobvyincgen StrUifMugOa taionnd, fhuormthoegr epnuizriefdicbatyiounlt, rtahseo nreicsucrltaasnht
apnrdotpeuinr iwfieads abnyaalyffizneidty ucshinrogm SDatSo–gPraApGhEy. eAlefctetrrorpemhoorveisnisg. SAU sMinOglea npdroftueritnh beranpdu rwifiictha taio mn,otlheecurelasru mltaansts
porfo atebionuwt 1a1s.a4n kaDlyaz wedaus soibntgaiSnDeSd– (PFAigGuEree l5e)c, twrohpihcho rwesaiss. sAligshintlgyl ehpigrhoteeri nthbaann tdhew ditehdaumceodl epcruoltaerinm saiszse
o(1f1a.b0o6u ktD1a1). 4fokrD BanCwPaIs (odbetsacirnibeded( Faibgouvree).5 T),hwe hreicshulwtsa isndsliicgahtetldy thhiagth seurctcheasnsfuthl epdroekdaurcyeodticp reoxtperiensssiizoen
(o1f1 .B0n6CkPDI aw)faosr aBchniCePvIed(d. escribedabove). Theresultsindicatedthatsuccessfulprokaryoticexpression
ofBnCPIwasachieved.

FFiigguurree 55.. AAnnaallyyssiiss ooff rreeccoommbbiinnaanntt eexxpprreesssseedd BBnnCCPPII ((rreeBBnnCCPPII)) oonn 1122%% ssooddiiuumm ddooddeeccyyll ssuullffaattee
ppoollyyaaccrryyllaammiiddee ggeell eelleeccttrroopphhoorreessiiss ((SSDDSS––PPAAGGEE)). .MM: :pprorotetienin mmarakrekre; r1;: 1r:eBrenBCnPCI PwIitwh iSthUMSUOM aOnda Hndis
Htaigs t(abgef(obreef oernezeynmzyem deigdeisgteiosnti)o; n2) ;a2nadn d3: 3r:erBenBCnPCIP Iwwitihtohuotu tSSUUMMOO aanndd HHiiss ttaagg,, rreessppeeccttiivveellyy ((aafftteerr
eennzzyymmee ddiiggeessttiioonn));; 44:: SSUUMMOO aanndd HHiiss ttaagg ((aafftteerr eennzzyymmee ddiiggeessttiioonn))..
AA pphhyyllooggeenneettiicc ttrreeee ((FFiigguurree SS33)) ccoonnssttrruucctteedd wwiitthh tthhee aammiinnoo aacciidd ((AAAA)) sseeqquueenncceess ooff BBnnCCPPII aanndd
5511 ootthheerr ccyyssttaattiinnss ffrroomm ddiiffffeerreenntt ppllaanntt ssppeecciieess rreevveeaalleedd tthhaatt tthhee BBnnCCPPII sseeqquueenncceess aarree mmoosstt cclloosseellyy
rreellaatteedd ttoo ccyyssttaattiinn ffrroomm VV.. uunngguuiiccuullaattaa,, ffoolllloowweedd bbyy ccyyssttaattiinn ffrroomm VViiggnnaa rraaddiiaattaa,, GG.. mmaaxx aanndd GG.. ssoojjaa..
TThheessee rreessuullttss aarreei inna aggrreeeemmeennttw witithht thhoosseeg geenneerraatteeddb byyt htheeB BLLAASSTTr eresseeaarcrchha annaalylysissi.s.
33..44.. PPrrootteeaassee IInnhhiibbiittoorryy AAccttiivviittyy ooff rreeBBnnCCPPII
TTood deetteerrmmiinneew whheetthheerr tthhee rreeccoommbbiinnaanntt BBnnCCPPII iinnhhiibbiittss ccyysstteeiinnee pprrootteeiinnaassee,, tthhee ppuurriififieedd rreeBBnnCCPPII
((≈≈1100 μµgg)) wwaass pprree--iinnccuubbaatteedd wwiitthh ppaappaaiinn,, ffiicciinn aanndd bbrroommeellaaiinn,, aanndd tthheenn tthhee rreessiidduuaall eennzzyymmee aaccttiivviittiieess
oonn NNαα--bbeennzzooyyll--DDLL--aarrggiinniinnee ββ--nnaapphhtthhyyllaammiiddee ((BBAANNAA)) wweerree tteesstteedd aass aabboovvee.. FFoorr ppaappaaiinn aanndd fificciinn,,
oonnllyy aabboouutt 1188..22%% aanndd 2299..00%% eennzzyymmee aaccttiivviittiieess rreemmaaiinneedd aafftteerr ttrreeaattmmeenntt,, rreessppeeccttiivveellyy,, wwhheerreeaass ffoorr
bbrroommeellaaiinn,,r erBeBnCnCPIPsI hsohwoewdendo noob voiobuvsioiunhs ibinithioibnitaicotniv iatcyti(vFiitgyu r(eFi6g).uTreh e6s)e. dTahtaesien ddicaatate itnhdaitcpauter ifitheadt
rpeuBrniCfiPedI hreaBsnstCroPnI ghainsh sitbriotnogry inefhfiebcittsooryn eefnfzeyctms eosnw enitzhycmysetse winiethp rcoytsetieniansee parcotitveiitnya,saen adctthivaittyfu, nanctdio tnhaalt
efxupnrcetsiosinoanl ewxapsreascshiioenv ewda.s achieved.
Genes2017,8,265 10of15
Genes 2017, 8, 265 10 of 14
Genes 2017, 8, 265 10 of 14

Figure 6.FiAgusrsea 6y. Aosfsaiyn hofi binihtiiobintioan catcitvivitityy ooff ppuruifrieifid eredBnrCePBIn tCowPaIrdt opwapaarind, fpicainp aanidn ,brfiomcienlaiann. Tdheb romelain.
Theinhibiitnihoinbitwiona swaesx epxrperesssseedd aass rerseidsuidalu eanlzyemnez aycmtiveitya icnt itvheit pyreisnentche eofp BrneCsPeIn act e10o0 fμBg/nmCL,P aInda tN1α0-0µg/mL,
benzoyl-DL-arginine β-naphthylamide (BANA) was used as substrate. Vertical bars represent
andNα-besntaznodyarld-D dLe-vairagtiionnisn. eDβiff-enreanpt hstmhaylll almettiedrse o(Bn AbNarsA r)ewpreassenuts esidgnaifsicsauntb sdtirffaetree.ncVee artt ic5a%l bleavresl represent
standard d(Deuvnicaatni'os nmsu.ltipDlei frfaenrgeen tetsts)m. all letters on bars represent significa nt difference at 5% level
(Duncan(cid:48)sFmiguurleti p6.l eAsrsaany goef itnehsitb)i.tion activity of purified reBnCPI toward papain, ficin and bromelain. The
3.5. Antifungal Activity of reBnCPI
inhibition was expressed as residual enzyme activity in the presence of BnCPI at 100 μg/mL, and Nα-
3.5. AntifungalAbAenntczitfouiyvnl-igDtayLl- oaarfcgtriivneBiintyne aCβsP-snaIaypsh wtheyrlea mpiedref o(rBmAeNdA w) itwha ds ifufeserden at sc osnucbesntrtartaet.i oVnesr toicfa pl ubrairfise dre rperBesneCntP I on
four pstlaanndta prda thdoevgieantiiocn fsu. nDgifif e(Fre. notx ysmspaolrl ulmet,t eArs. aotner nbaatras, rBe.p crienseerneta saignndi fPic.a nvet xdainffse)r ceunclteu aret s5. %T hlee vreels ults
Antifiunndigca(aDtleuadnc cttahinav'sti mtryeuBlatnispCslPea Iry ainsnghwei bteeistrete)d. ptheer fgorromwtehd owf ailtlh foduirf fpehryetnoptactohnogceennst, rbautti othnes ionhfipbiutorriyfi eefdfercte BnCPIon
fourplantvparaietdh odgepeennidcinfug nogn ith(Fe .fouxnygaslp ospreucmie,s A(F.igautreer n7a).t aA,mBo.ncgi ntheerseea faonurd fuPn.gvie, xBa. ncsin)erceua lwtuarse ms.osTt heresults
3.5. Antifungal Activity of reBnCPI
sensitive to reBnCPI. Concentrations of reBnCPI of 20 μg/mL (1.81 μM) slowed the growth rate of B.
indicatedthatreBnCPIinhibitedthegrowthofallfourphytopathogens,buttheinhibitoryeffectvaried
cinereAa ntoti 5fu4n%g oalf athcatitv eitxyh aibsistaeyds b wye Br.e c pineerrfeoar mcueldti vwaittehd dinif fmereednitu cmon wceitnhtorautti orenBsn oCf PpIu; raitf iceodn rceeBnntrCaPtiIo onns
dependingofof ou8rn0 pμtlgha/nemt Lfpu a(7tnh.2go3ga μelnMsicp) ,fe tuhcneige Bis .( cF(iF.n oiexgreyuas prgoerrou7wm)t.,h A Ar.a matete owrnnaasgt ao,t nBhly.e c2sin1e%erfe ooaf ua trnhdaft u Pcn.u vlgteiixv,aanBtse.)d cc iiunnl tmeurreeedasi.u wTmha ews rimethsuoolusttst sensitive
toreBnCPriIne.BdCnicCoaPtneIcd. eG tnhrotawrt atrhteiB ornantCesPs Ioo fif nFrh.e iobBxiytnespCdo PrtuhIme o gafrno2dw0 Athµ. a goltf/e ramnlal tLafo w(u1re .r8pe1 hryeµdtoMupcae)tdhs oblgoye wanbseo, dubtu t5th0 t%eh ega tri nao hcwiobntithcoernryta retaeftfieoocntf B.cinerea
to 54% ofovtfha rraieetBdne CdxePhpIi ebonfid t8ein0d gμ bgo/nym tBLh;e. tchfuiannt egirsa,el atshpcee u5cl0iet%isv (gaFrtiogewudrthei n7in).hm Aibemitdiooinnug m( EthCwe5s0)ei tffhoorou turh tefusrene gtBwi, noBC .p PcaitnIh;eorageate nwcso awns acmse o8ns0tt rations of
μsegn/msitLiv. eM toor reeoBvneCr,P tIh.e C ionnhcibeinttiorant ieofnfisc ioefn rceyB onfC rPeIB onfC 2P0I μ(agt/ m80L μ (g1/.m81L μ) Mw)a ss lsolwigehdtl yth hei gghroewr tthha rna tteh aotf oBf.
80µg/mL(7.23µM),theB.cinereagrowthratewasonly21%ofthatcultivatedinmediumwithout
jciinngegreaan tgom 5y4%cin o (f5 t0h aμtg e/xmhLib)i. tIend t bhye Bca. scein oerf etah ceu rlotiovta rtoedt p inat mhoegdeinu mP. wveixthaonus,t grreoBwnCthP rIa; taets c foenllc ebnyt r5a0t%io nats
reBnCPI.GroerfB o8n0wC μPtghI/ cmroaLnt c(ee7sn.2to3ra fμtiFMo.n)o,s x tohyfe s2 Bp0.o μcrigun/emmreLaa agnnrdodw A3t5h.% ar aaltttee c rwonnaacste aonntwrlayet i2ro1en%sr oeoffd 8tuh0a cμte gcd/umlbtLiyv, aiantebddio ciuant timn5ge0d t%hiuamta P tw. avitehcxooanunstc entration
of reBnCPirsIe BonnofCt 8Pv0I.e rGµyr gos/wenmthsi Lrtiav;teet sh tooaf t FB.in soCx,yPtsIhp. oeFruo5mr0 a%anlld g fAoru.o arwl tfeutrhnnagtiia,n whheoirbwei ertveioderun, cet(hdEe bC yg5 ra0ob)wofutoht r5r0at%hte e atsd eae ccltoiwnnecoden ptarsaa tttihhoeno gens was
80 µg/mLco.of MnrceeoBnnrteCraoPtivIo oenfrs , 8o0tfh rμeegB/inmnCLhP;i Ibt ihniatctir oeisan,s teehdfe.fi 5 c0i%e ngcroywothf irnehBibnitCioPnI (E(aCt50)8 0forµ tgh/esme tLw)o wpaatshosgleignsh wtlays h80ig her than
μg/mL. Moreover, the inhibition efficiency of reBnCPI (at 80 μg/mL) was slightly higher than that of
thatofjinggangmycin(50µg/mL).InthecaseoftherootrotpathogenP.vexans,growthratesfellby
jinggangmycin (50 μg/mL). In the case of the root rot pathogen P. vexans, growth rates fell by 50% at
50%atreBnCPIconcentrationsof20µg/mLand35%atconcentrationsof80µg/mL,indicatingthat
reBnCPI concentrations of 20 μg/mL and 35% at concentrations of 80 μg/mL, indicating that P. vexans
P.vexansisisn noott vveerryy sseennssitiitviev etot oBnBCnPCI. PFIo.rF aolrl afolulrf ofuunrgfiu, nhogwi,ehveorw, tehve egrr,otwhteh grraotew dtehclrinaetde dase cthlien edasthe
concentratcioonncsenotrfartieoBnns CofP reIBinnCcPreI ainscereda.sed.

Figure 7. Growth inhibition assays of four fungal pathogens by reBnCPI. Fo, Fusarium oxysporum; Aa,
Alternaria alternata; Bc, Botytis cinerea; and Pv, Pythium vexans. reBnCPI was added to the fungal
suspension to a final concentration of 0, 20, 40, 60 or 80 μg/mL. Fungicides carbendazim,
jinggangmycin, fluazinam and mefenoxam were added to the suspension of Fo, Aa, Bc and Pv (final
concentration 50 μg/mL), respectively, to serve as positive control. Vertical bars repre sent standard
deviations. Different small letters on bars represent significant difference at 5% level (Duncan's
Figure7. mGFiugrloutirwpel e7th .r Gainrnoghwe ittbehs iittn)i.ho inbitaiosns aasyssayos foff foouurr ffuunngagl aplatphaogthenosg beyn rseBbnyCPrIe. BFon, CFuPsaIr.iuFmo ,oxFyuspsoarruimu;m Aao, xysporum;
Alternaria alternata; Bc, Botytis cinerea; and Pv, Pythium vexans. reBnCPI was added to the fungal
Aa,Alternariaalternata;Bc,Botytiscinerea;andPv,Pythiumvexans.reBnCPIwasaddedtothefungal
suspension to a final concentration of 0, 20, 40, 60 or 80 μg/mL. Fungicides carbendazim,
suspensionjintoggaanfignmaylccion,n fclueanztirnaatmio anndo fm0e,f2en0o,x4a0m, 6w0eorer a8d0dµedg /tom thLe .sFuuspnegnsicioind eosf Fcoa,r Abae,n Bdca aznidm P,vj i(nfignagla ngmycin,
fluazinamcaonncdenmtraetfioenn 5o0x μagm/mwL)e, rreespaedcdtiveedlyt, otot sheervseu ass ppeonsistiivoen coonftrFolo. ,VAerati,caBl cbaarns drepPrvese(nfitn satalncdoarndc entration
50 µg/mLd)e,vrieatsiponesc. tDivifefleyre,ntto smsearllv leettaesrs poons ibtairvse recpornesternotl .sigVneifrictaicnat ldbifaferrsenrcee parte 5s%en ltevsetla (nDduanrcdan'ds eviations.
multiple range test).
Differentsmalllettersonbarsrepresentsignificantdifferenceat5%level(Duncan'smultiplerangetest).

The inhibitory effects that reBnCPI exhibited on the growth of these four phytopathogenic
fungi were also checked under microscopy (Figure S4). In the negative control (without reBnCPI)
treatments,allfourfungalspeciesgrewfast,withmoremyceliathanthatpresentwithBnCPIand