Whiteclover(Trifoliumrepens)isanallotetraploid(2n=4x=32)outcrossingplantspecies
(AtwoodandHill1940)andasreportedbyEllisonetal.,(2006),thetwodiploidancestorsare
proposedtobeT.occidentale(Schreb.)andT.pallescens(Schreb.)whichwererecognized
usingphylogenetictoolsbasedonnuclearribosomalDNAITS(internaltranscribedspacer)and
chloroplasttrnLintronsequences.Therefore,asinglegenotype(aplantcomingfromasingle
germinatedseed)wasselectedrandomlyandusedfortheidentificationofKunitzproteinase inhibitor(KPI)genes,andthissamegenotypewasusedinmostexperimentsbymeansof
vegetativepropagation.
TosearchforKPIgenesinwhiteclover,twoapproacheswereundertaken.Thefirstinvolved
screeningtheAgResearchGrasslands,PalmerstonNorth,NewZealandwhitecloverEST
database,whilethesecondinvolveddesigningdegenerateprimers,basedonKPIsequences
expressedinotherlegumespecies,andthenobtainingputativeKPIsequencesusingPCR.
3.1.1ApproachI:SearchingtheAgResearchESTdatabase
ToidentifyKPIgenes,theAgResearchwhitecloverESTdatabasewasscreenedusinga
MedicagotruncatulaKPIsequence(AF526372.1)bytblastn.Amonglegumes,M.truncatula,
whichhasasmallgenomeofapproximately500millionbasepairs,hasbeenwidelyadoptedas
amodelforgenomicresearch.ItwasfoundpreviouslybyDonaldHunter(1999)thatACC
oxidasegenesofwhiteclovershare90to95%identitywiththe3’UTRregionofM.truncatula
ACCoxidasegenes.Therefore,initiallyaM.truncatulaKPIsequence(AF526372.1)wasusedto
virtuallyprobeforKPIgenesinthewhitecloverESTdatabase.
TheAgResearchdatabasegavehitstoabout27sequences(Appendix2).Thefirstgroup
comprisedsevenfulllengthsequencesandshowed80%identitywiththeM.truncatula
sequence,withminorvariationsatthetranslationallevel.AClustalWalignmentofthewhite
cloversequencesshowed100%identity(Appendix2;GroupA)indicatingthattheyarethe
sameprotein.Thesecondgroup(Appendix2;GroupB)comprisedfivepartialsequencesand
showed50%identitywiththeM.truncatulaprotein.Alignmentofthesesequencesalso
showedthattheyarethesamegene.Theotherhitswereeithersmallpartialsequencesorthe
reversecomplementstrandofthefirstgroupwithminoridentitieswiththeMtruncatulaKPI
wasselectedtobethetypicalmatchfortheM.truncatulasequenceasitwasfulllengthand
thiswassubsequentlydesignatedasTrͲKPI1(Figure3.1).
3.1.2ApproachII:Usingdifferentsetsofdegenerateprimers
TogetadditionalKPImembers,degenerateprimersweredesignedbasedonconserved
regionsofM.truncatula(AF526372.1),Glycinemax(EU444603.1)andCicerarietinum
(AJ276263.3)KunitzgenesfoundintheNCBIdatabase(Appendix3;Group1)andone
degenerateprimerwasdesigned.TheM.truncatuladatabasewasalsosearched
[http://www.jcvi.org/cgiͲbin/gb2/gbrowse/mtruncatula/M.truncatulagenomerelease
version3.0(Mt3.0)]with‘Kunitz’asthekeyword.TheMedicagoGBrowsegave45hitsandit
wasfoundthatKunitzgenesareclusteredonchromosome1,3,5and6ofthisplantspecies.In
thedatabaseonlythegenessittinginchromosome6wereannotatedas‘KunitzProteinase Inhibitor’.Therefore,thegenesonchromosome6werefurtherinvestigated,alignedand
threefurtherdifferentsetsofdegenerateprimersweredesignedintheconservedregionfor
fishingoutthegenesfromwhiteclover(Appendix3,Group2,3,4).
Thehighlightedregions(Appendix3)wereselectedasthebestregionforallfourdegenerate
primerstoavoidprimerselfͲcomplementarities(i.e.,abilitytoformsecondarystructuressuch
ashairpinsandthetendencytoformhomoͲdimerswithitselforheteroͲdimersinthe
reaction).Thoughtheselectedregionsarenotcompletelyconservedinthesensethatthere
aresomesinglenucleotidedifferences,onlythisregionshowedasimilarmeltingtemperature
fortheforwardandreverseprimers(64.10Cand64.30C)andareasonableproductsize(more
than300kbproduct).
AsinglePCRproductranginginsizefrom336to450bpwasobtainedfromcDNAcombined
fromdifferenttissuesofwhitecloversuchasdryseed,imbibedseed(6h,24hand48h),leaf,
petiole,stolonandrootwhenusingeachofthefourprimers.ThepurifiedPCRproductswere
clonedintothepGEM®TEasyvectorandtransformedintoE.colistrainDH5Dandtheinserts
weresequenced.Atleast25insertsweresequencedforeachgroupofsequencesamplified
witheachdegenerateprimer.TheORFsofthesecDNAsequencesyieldedaminoacidproducts
of112to140aawhichwereselectedfromsixframetranslationsandthesewereproposedto
beputativeKunitzProteinaseInhibitor(KPI)genesandwerenamedasTrͲKPI2,TrͲKPI3,TrͲ KPI4,TrͲKPI5TrͲKPI6,TrͲKPI7andTrͲKPI8(Figure3.1A).
(A)
Tr-KPI1 DINGNAIFPGGEYYILPALRGPG-GGGVRIGKTGD--LKCPVTVLQDRREVKNGLPVKFT 57 Tr-KPI8 DINGNAIFPGGEYYILPALRGPG-GGGVRIGKTGD--LKCPVTVLQDRREVKNGLPVKFT 57 Tr-KPI6 DIHGTPIFPGGEYYILPALRGPG-GGGVRIGKTGD--LKCPVTILQDRREVKNGLPVKFT 57 Tr-KPI3 DLHGTPIFPGGKYYIFPVSHDDTYGGGLRLAKTGD--SKCEVTALQDDNIVIDNIPVKFS 58 Tr-KPI7 DKHGIPIFPGRKYYIFPVSHDDTYGGGLRLAKTGD--SKCEVTALQDDNIVIDNIPVKFS 58 Tr-KPI2 GYKWQPPYSGGKYYIFPVSHDETYGGGLRLAKTGD--SKCDVTALQDDNIVIDNIPVKFS 58 Tr-KPI4 DLNGNPIFYSTRFYIMPSIFGAA-GGGLKLGETGK--LTCPLTVLQDYSEVINGLQLKFT 57 Tr-KPI5 DKNGNPVVSGKQYFIFPATDNPK-KGGLTLNNVGDDDSKCPVTVLQNN--AITGLPVKFT 57
. : . . .::*:* . **: : :.*. .* :* **: . .: :**: Tr-KPI1 IPD-ISTGIIFTGTPVE-IEFFKKPNCAKSSKWLVFVDNVIKKACVG 102
Tr-KPI8 IPD-ISTGIIFTGTPVE-IEFFKKPDCAKSSKWLVFVDNVIKKACVG 102 Tr-KPI6 IPD-ISTGIIFTGTPIE-IEFFKKPNCAKSSKWLVFVDNVIKKACVG 102 Tr-KPI3 IPG-ISPGIIFTGTPIE-IEFTKKPSCVESSKWLIFVDDVIQKACVG 103 Tr-KPI7 IPG-ISPGIIFTGTPIE-IEFTKKPSCVESSKWLIFVDDVIQKACVG 103 Tr-KPI2 IPG-ISPGIIFTGTPIE-IEFTKKPSCVESSKWLIFVDDVIQKACVG 103 Tr-KPI4 PPGEIFVDLISTDQPLKGIEFVEKPECAESSKWVVVEDDDFPRPYVG 104 Tr-KPI5 IPQ-TTTDNIVTGTDLD-IEFTEKPDCAESSKWLLVTDDNTQQSYVG 102 * . * *. :. *** :**.*.:****::. *: :. **