Cloning And Functional Identification Of EIF4E Genes From Sugarcane

Sugarcane(Saccharum spp.Hybrid)is the most important sugar crop around world,especially in China,Sugarcane mosaic disease(SMD),caused by Sugarcane mosaic virus(SCMV)or Sorghum mosaic virus(SrMV),is one of the most serious viral diseases in sugarcane and wide-spread in sugarcane planting area.SCMV and SrMV belong to genus potato virus Y(Potyvirus)family Potyviridae.The genome of potyviruses is positive sense single-strand RNA.There is no cap structure at the 5'end of potyvirus genome.A virus-encoded protein,virus genome-linked protein(VPg),binds to the 5' end of potyvirus genome.It is the VPg,acting as cap structure,interacts with eukaryotic translation initiation factors,especially 4E,to initiate the replication of potyvirus genome.When the plant host genes encoding eIF4E or eIF(iso)4E was silenced by RNA interference(RNAi),the replication of virus genome was interrupted.And the transgenic plants,which showed normal growth and development as wild types,gained broad-spectrum and durable resistance to viral infection.So far 12 known recessive resistance genes in plants are all mutants of eIF4E.However,for the most important sugar crop,sugarcane,the molecular mechanism for SCMV infection is yet absent.There is no report on the isolation of genes encoding eIF4E from sugarcane,no mention of its interaction with SCMV-VPg.Therefore,some innovated study was accomplished as following:1.Isolation of eIF4E from sugarcane and SCMV-VPgReverse transcripticon PCR(RT-PCR)and rapid amplication of cDNA ends(RACE)were applied to isolate the genes encoding eIF4E from sugarcane leaf.Three genes were obtained.Two of them,Sc-eIF4E3 and Sc-eIF4E,are full-length genes.The other one,Sc-eIF4E2,is not full length,but the complete open reading frame(ORF)was gained.The Sc-eIF4El is 886 bp in length with a 663 bp ORF encoding 220 amino acids.The partial Sc-eIF4E2 is 786 bp in length with a 633 bp ORF encoding 210 amino acids.The Sc-eIF4E3 is 978 bp in length with a 690 bp ORF encoding 229 amino acids.The SCMV-VPg gene was cloned from the SCMV isolate infecting sugarcane planted in the field.The SCMV-VPg is 567 bp in length and encodes 189 amino acids.2.Identification of interaction between Sc-eIF4Es with SCMV-VPgThe yeast two-hybrid system(Y2HS)was applied to testify the interaction between SCMV-VPg and Sc-eIF4E1,Sc-eIF4E2 or Sc-eIF4E3,respectively.Bait vector containing SCMV-VPg gene was constructed.Trap vectors containing Sc-eIF4E1,Sc-eIF4E2 and Sc-eIF4E3 gene respectively were constructed too.They were co-transformed NMY51 yeast strain.Then the transformed NMY51 was cultured on the auxotrophic culture and screened by chromogenic reaction.The results showed that SCMV-VPg interacted with all three Sc-eIF4Es.However,the interaction between SCMV-VPg and Sc-eIF4E1 runs strongest,Sc-eIF4E3 runs second,the Sc-eIF4E3 is the weakest.Further,bimolecular fluorescence complementation(BiFC)was used to testify the interaction between SCMV-VPg and Sc-eIF4E1,Sc-eIF4E2 or Sc-eIF4E3,respectively.SCMV-VPg was cloned into vector Yn or Yc to construct the vectors Yn-VPg orYc-VPgvector.Sc-eIF4E1,Sc-eIF4E2 and Sc-eIF4E3 were cloned into vector Yn or Yc to construct Yn-Sc-eIF4E1,Yc-Sc-eIF4E1,Yn-Sc-eIF4E2,Yc-Sc-eIF4E2,Yn-Sc-eIF4E3 or Yc-Sc-eIF4E3.They were co-transformed into EHA105 agrobacterium strain.Positive transformed strains were selected to inject the leaves of benthamiana.After 48h-60 h,confocal laser scanning microscopy was used to detect the interaction between them.The results showed that the SCMV-VPg interacted with all three Sc-eIF4Es.It is consistent with the results of the Y2HS.3.Quantification of Sc-eIF4E by Real-time PCRReal time PCR was applied to study the transcriptional difference of Sc-eIF4E between control and sugarcane challenged by SCMV.The results showed that both 3 Sc-eIF4E were up regulated in treated samples.In summary,three genes encoding Sc-eIF4E were firstly isolated from sugarcane and their interaction with SCMV-VPg was identified by Y2HS and BiFC.The research work finished in this study is helpful in revealing the molecular mechanism for infection of SCMV on sugarcane.Furthermore,this study provided a molecular target for RNA interference(RNAi)to create new sugarcane germplasm charactering as broad-spectrum and durable resistance to sugarcane mosaic disease.Also,this study will deploy a new strategy for the research of transgenic sugarcane resistant to pathogen and benefit the development of cross discipline between plant breeding and plant protection.Generally,this study is important for its scientific significance and application value.