The Use Of Mannose Selection System To Transgenic Chrysanthemum Morifolium 'Huoyan'with AtWRKY57 Gene

During the growth of life plants will be affected by various external factors,including biotic stress and abiotic stress.Drought is one kind of plant grow-th encountered during abiotic stress when subjected to drought stress,will regulate certain transcription factors and stress or drought-related genes,through a series of transcription,translation produces plays an important protective role in the cell such as proteins.AtWRKY57 is one of the most important regulatory gene in response to drought stress plays an important role,can activate many stress-inducible gene expression,enhanced plants to drought stress endurance.Mannose phosphate isomerase gene(PMI)is the biological safety marker genes,reduced the harm of antibiotic or herbicide to the environment In this study,we use the Gateway technology and mannose phosphate isomerase gene as a selection marker gene PMI,build AtWRKY57 gene expression vector,has been established in the field chrysanthemum 'flame' in vitro regeneration system will the AtWRKY57 into chrysanthemums,for the realization of a security tag is improved drought resistance characteristics of chrysanthemum varieties basis.The results of the study are as follows:Gene cloning:The AtWRKY57 gene is cloned by using RT-PCR,and the full length of cDNA sequence is 1141 bp.The complete open reading frame is 915 bp,codes 304 Amino acid.The Protein molecular formula is C1471H2272N430O482S11.The Molecular weight is 34045.4.The isoelectric point is 6.22.The instability index is 52.84(>40).So the protein is belong to labile protein and hydrophilic protein.Construction of expression vector:We use Gateway technology to build pCAMBIA1301-PMI-AtWRKY57 plant expression vector.The result is verified by PCR and restriction enzyme digestion,indicate that building plant expression vector is successfully,then put it into Agrobacterium tumefaciens EHA105.The plasmid is extracted and verified by PCR showed pCAMBIA1301-PMI-AtWRKY57 plant expression vector into Agrobacterium EHA105 is successfully.chrysanthemum 'flame' genetic transformation:We choose robust chrysanthemum 'flame'feet.The feet is disinfectsd by hypochlorous acid and alcohol,then inoculate on the MS culture medium,to gain sterility chrysanthemum seedings.lor 2 months later,the leaf is cut into small pieces(8 mm×8 mm)as explant.Two days later we use the small pieces will to be material,and put the agrobacterium tumefaciens to make the OD600?is 0.6,then use it infect the small pieces 10min.Later put the small pieces on the co-culture medium,with cephalosporin drug concentration of 100 mg/L for bacteriostatic concentration,under 25 0C and dark condition cultivated two days,then put it into containing mannose concentration for 8 g/L of the early stage of the medium.When the adventive shoots is appeared,put it into the containing mannose concentration for 10 g/L of the late stage of the medium.At last put it into the containing mannose concentration for 12 g/L of the rooting medium,until the plant that grew well and take root appears.In this experiment the number of resistance seedlings is 219.After the PCR identification the number is 7,so the transformation efficiency is 3.2%.