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1、Functional characterization of SPO11 homologue(s) from bread wheat Sachin Rustgi and Diter von Wetsin Department of Crop and Soil Scincs Washington Stae University Pulln WA, 99164, USArustgiwsu.edu, ditrwsu.edu Introduction Meiot reombination n plants involves induction of DSBs in the chromosomal DN
2、A by SPOrulation 11 (SPO11), proten thabelongs o thefamlyofype II topoiseras. SP11 ws first isolated fromyes ia screen for utants howing reduced porultion. Lter theprotn ws dicovered n ntial compone the doubl strand break repai(DSBR) pathwy. Mutation iSPO11 notonly displayed decread bility o generat
3、 Bs, but also howed corresponding defet in SC formtion. These defcs were caused by decine in the number of ZIP3 cplexes, whih re thoughtto presnt its ofSC nitaon hrough their rol promoting the cruimnt ofhe transvers flmnt protein ZIP1. Unique from other txa, plntsposse multiple copies ofSPO11: Arabi
4、dopsis and maize hathreecopiswhile the ricgenomontainsfour copies. ut ofthethree Arabidopsis homologu, only SPO11-1 and SPO11-2 have amiotcfuncion. TphenotypeofArabidopsis po11-1 and spo11-2 utntspper to overlap onsderably. None the above to mutnt could form crosovers or re ble to synapse chromosome
5、s, lding to only univales at metaphase I (Fig. 1)The lack of functional redundancy betwen these wo genes ugges ha DSB formation i catlyzed by aPO11 heterodimr in plnts nd not homodias thoughtto occur in other eukaryotes. In S. cervisae, SPO11 requi nine otr protins to catlyzDSB formation. nly four o
6、f hese are conserved in plants (Rad50, M11, Nbs1, Ski8), butnone have consd function in thistep ofreombination. However, forward meiotc utant screning has ld to the dentiftion of AtPRD1, which isnesry for meiot DSB formation in Arabidopssand interacs wth tSPO11-1 in yest two hybrid asy, providing th
7、e first evidence for the xienc ofa B forming complex in higher eukaryotes. The otr actors in this complex remain to be identifed. Aording to he phenotype oftDSB-defevemutants decribed bove, the SDS gene, which encodes a mioss-pecif cyclinke protein my lo long to this clas. Fig. 1DAPI staing ofspo11-
8、 PMCs during meioss. At th end of the propase, clsontig 10 fully condensed chromosomes mostof the te arobserved (A). When anaphase migraton occurs,chromosome bridges can sometibe seen (B, arrow). Segregation of unpaired chromosomes anaphase I leads to random partitioning ofchroosoes (CE). The second
9、 meiotic division produces cells with more thanfurlsof DNA,crespndig ta vrible umberofchroosoes (F). Scale bar = 10m.Considering the iportance ofSPO11 in homologous recombinaton it sworthwhile to intfy andfunctonaly charaterizwheat ologues ofSPO11.In order to dewt homologues used relvant riequences
10、to blast gains heat ESTdabase. Theanalyss alowidentifction of wheat homologueofhree(OsSP11A, OSP11B nd OSP11C) of the our riSP11genes. Two the ric 11 homologues, SP11A nd sSPO11Btha showed simlrity ith and 12 wheatESTs correponds tothemeiotcaly ctive Arabidops AtO-1 and AtSPO11- 2 gene, repetively.T
11、antisns oligos weredesigned fromhe conserved regionsofts howng silrity with SP11A and OsSP11B. nc fragment correponding with both of SPO11 genes slectd it was usd for blast rchesagainst hea EST database to chek for it uniquenes,and lso erchedfor t bsenof PacInd NotIreriion site (whinheoligo equenc).
12、 T fraent husslctd ilbe synthesizd ontraform Sigma nd wil be usd or construct preparaon and VIGS. Example Following the method described as above, we identifed DMC1 (disrupted miotc cDNA1) homologues in breawhet, nd designed asns oligosto uppres DMC1expresion through VIGS. T resulof the VIGS xperimn
13、tare shown iFigure 2. Suppreof DMC1 expresion through resulted in the phenotype very slar to the phenotypes obsrved for dmc1 mutants in Arabidopss and mize (Fig. 2). Figure 2.Chromose spreads of meiotic metaphs I pollen moerclfrm: (a)negative contol (rubbed with FES), (b)positve control(rubbed wthp.
14、MC), (c and ) TaDMC1 splants (rubbed ith p.TaD1as). MCS = multie clonig site.Proposed bnefits Dtails understanding of meiotc proces in bread wheat il eventualy benefit plant breedersin mnipultithe reombinaton proces. For nstanc, bydesigningnrtifclfusonprncomprisdofSPO1ndDNAbindgomainoecndrmbiatiiste
15、-pcimar.TheNA- rcuitshfusioptebdgintheDNAaiucesrosverftiasites.Thisechniquehasbeenshowntoworkellinyeast,althoughheinductionofdoubl-stranreaksocursmolyinbidngitinpchromatinregionsandnot,ole,innaturalcoldpots,suchaseromeers.Timed,whchasbeenproposedforuseinplants,ouldprovideapowerfultoolforinductionofs
16、ite-spcifrsovers.SPO1coldbefdtvrityofdiferntDNAbingdoman,thultingisuitfusionpreinshcul,hory,ecruitSPO11tvaritheDNA.VIGS pocedu Antis PO11 oligos designd for VIGSCJ562962AACTCTTGGTTGTGAATACCAAGGTGATTGTTTGATCAGACAAGCAACTACAAGTGGTGTAACCAAAACA AAAGGAGACTCTTAGCCTAGCACAATTCTGCATAGATACAGGTCTATGCAATCCTATATGTACCTCCCTA GTCTGATCTGTTGGGGGATGTACTCTTGCGACAGAAACGAAATGGAACTTGCTGACAATGCTTCAA
