Screening And Verification Of Transcription Factor LpNAL Interprotein In Perennial Ryegrass

Perenial grass(Lolium perenne L)is one important turf and pasture grass in the temperate and subtropical regions of China,which is widely used in landscaping and mixed-sowing grassland.However,perennial ryegrass is not resistence to many adverse environmental conditions,and its leaves are often induced into premature aging by various stresses affecting its biomass yield,and turf and forage quality.NAC is a family of plant-specific transcription factors that are involved in stress-induced leaf senescence.Our laboratory found that overexpression of a novel transcription factor LpNAL(NAP-Like)in both Arabidopsis thaliana and ryegrass resμlted in a stay-green phenotype.Therefore,we constructed a yeast two-hybrid cDNA library of perennial ryegrass,and used yeast two-hybrid technology to screen LpNAL interaction proteins.The unraveled protein interaction network provides an insight into the molecμlar mechanism of LpNAL and its interaction proteins cooperatly regμlating the progression of leaf senescence of perennial ryegrass,and will also be usefμl for perennial ryegrass breeding in the future.In this study,the LpNAL gene cloned by the laboratory was truncated to delete its C-terminal suppression domain.LpNAL1-525 construct plasmid pGBKT7-LpNAL1-525 as the bait protein plasmid.A perennial ryegrass cDNA library was also constructedfor the yeast two-hybrid(Y2H)screening.After the screening,the res μ ltant putativeclones were further confirmed by yeast backcrossing experiments.The the res μ ltant positive clones were sequenced,and the coding sequences from these positive clones were obtained by matching the transcriptome data of perennial ryegrass and the NCBI website database,and the fμll length of these genes was further isolated form the cDNA of reygrass.The verification of interaction between LpNAL and individual full length protein was further verified by Y2H assay,and by the bimolecpμar fluorescence complementation(BiFC).The resμlts are listed as follows.(1)Senescent leaves of perennial ryegrass were used to construct the high quality perennial ryegrass cDNA Y2H library.The Y2H library is of 100%positive with cDNA inserations,and the library capacity was 1×107 CFU with the average insertion size of 1.5kb.The LpNAL gene was truncated from the open reading frame(ORF)1-525 bp to delete its transcriptional suppression domain at the C-terminus,and the bait plasmid pGBKT7-LpNAL1-525 was constructed and transferred into yeast cells.The bait plasmid had no self-activation activity and no toxicity to yeast cells.Throμgh 3-AT background screening experiments,it was found that 5-mM 3-AT can completely inhibit the leakage expression of HIS3 in yeast.(2)The bait plasmid and the cDNA library were co-transformed into Y2H Gold Yeast by one step transformation.About one million monoclonal yeast had been transformed and screened,and a total of 42 candidate positive clones were obtained.The yeast plasmid was extracted and transferred to E.coli.Yeast backcross experiments were performed and the positive clones were sequenced.A total of 12 protein-encoding fragments that interacted with LpNAL1-525.The obtained coding sequence were used as quries to search against the NCBI database using the BLAST program and against the local transcriptome database of perennial ryegrass to retrieve their full coding sequences.Subsequently,the f μll length coding sequences of these genes were cloned and subloned into Y2H bait and prey vectors.Throμgh the confirmation with full length coding sequences Y2H,four interacting proteins were obtained,namely LpMIEL1,LpPR5,LpBG1(β-glucosidase)and LpNRPB3(DNA-directed RNA polymerases Ⅱ,Ⅳ and Ⅴ subunit 3).(3)After subcellμlar localization experiments of ryegrass protoplast,it was shown that LpNAL,LpMIEL1,LpPR5,LpBG1,and LpNRPB3 all located in the nucleus.The BiFC res μ It also confirmed that LpNAL and the four genes encoding the interacting proteins can be observed in the nucleus with cyan fluorescence signals under a laser confocal microscope,and their subcellμlar organelles are consistent with the localization information.In summary,throμgh Y2H screening and confirmation of protein interactions,we found four interacting proteins with LpNAL,namely LpNAE,LpPR5,LpBGl,and LpNRPB3,and thus established a preliminary LpNAL interaction network.This study provided an insight into the futher study of molec μ lar signaling network ofLpNAL involved in the reg μ lation of leaf senescence of perennial ryegrass,and it also provides new genetic information for ryegrass breeding.