Transposition Efficiency And Copy Number Analysis Of Maize Endogenous Ac-Ds Transposons In Transgenic Lines Overexpressing Ac Transposase And CDNA Isolation And Overexpression Analysis Of Maize GRAS Transcription Factors

Maize is the world’ s most important food and forage crops and it is one of the mostcultivated crops in the world. Maize plays a key role in biology and genetics and have a rapiddevelopment, especially in cytogenetics.Mutant is a prerequisites for genetics research in Maize.It is difficult to obtain mutantthrough traditional method due to its character.We discuss the transformation system formaize through Agrobacterium-mediated and bombardment transformation. We could assaythe copy number and transposition of endogenous Ds and accumulate mutant by expression ofAcTPase.(1) Agrobacterium-mediated transformation system and Bombardment transformationsystem for maize.We take the ecotype A188and Qi-319as the material,which is cultures onN6basic media and tissue culture are built with the immature embryo between9and13daysafter pollination.The instantaneous expression experiment indicated that it is better to chooseinfect medium with OD between0.8and1.0, water bath at46℃for6min,infection for5minand cocultivation for3days for better infection efficiency and less damage to immatureembryo. Bombardment transformation system is different method. We optimize thepreparation of microparticle and explore a system. Now the frequency of instantaneousexpression experiment is about80%.We find that promatine can retard the degradation ofDNA in the cell with protamine coated plasmid DNA-gold particles. In the particlebombardment using cassette DNA,we find that the degradation of cassette DNA is a littlefaster to plasmid DNA.The frequency of particle bombardment using cassette DNA is about30%.(2) We could accumulate the new excion and re-insertion events of Ac-Ds at a high level with expression AcTPase. We could accumulate the new excion and re-insertion eventsthrough htbrids with single plant which are bombarded using P-35S::AcTPase::Tnos cassetteand Ds-GFP(Ds5’-P-ZmUbi::GFP::Tnos-Ds3’) cassette.GRAS proteins belong to a plant-specific transcription factor family and it is found inmore than30species in20flora.GRAS proteins play an important role involved in root andmeristem development, phytochrome signaling, gibberellin signal transduction.Now theprotein related to NFL in GRAS proteins was found and it is a evidence for GRAS preteins astranscription factors. Bioinformatic analysis identified57and32GRAS genes in rice andArabidopsis, respectively. We assay the sequence of GRAS proteins,clone the GRAS geneand identify the function through transformate Brachypodium distachyon.(1) The GRAS proteins share5distinct seqence motifs.The5distinct seqence motifs isLHRⅠ、VHIID、LHRⅡ、PFYRE and SAW motif.We found the conserved motif VHIID andother conserved amino acid in GRAS proteins, it indicated that GRAS proteins in maize sharethe distinct seqence motifs.(2) The locations of GRAS gene in maize are on chromosomes1,2,3,4,7,9and10.Weidentify the location of GRAS gene through MaizeGDB BLAST and found that the GRASgene are on chromosomes1,2,3,4,7,9and10,nearly no location on chromosomes5,6and8.(3) The subfamily of GRAS proteins.We make a Neighbor-Joining tree of GRASproteins between Maize and Arabidopsis and make the family classification on the basis of thehomology of proteins.Now the GRAS proteins belong to6subfamilys: LISCL、PAT1、SHR、HAM、DELLA and SCR.(4) Agrobacterium-mediated transformation of Brachypodium distachyon.GRAS gene isexpressed in shoot,root,ear,leaf and meristem in maize.We clone the gene and produce vectorsand now we have cloned16members,9vectors are finished and5dre transformed. We findalbino seedling in ZmGRAS22plant and dwarfed seedlings in ZmGRAS1plant.