Cloning Of AtCOR15?,AtCBF3 And CmICE1 And Genetic Transformation In Chrysanthemum

As one of the most important ornamental plants used widely in Northeast of China,Chrysanthemum plays a combined role in greening,coloring and beatifying.Yet the serve chilling in Northeast of China always limits the growth and production of chrysanthemum.So for a long period,to cultivate or improve the ability of plants which can show good out looking during the late autumn or winter is a main breeding target.After surveyed the characteristics of the forty-eight hybrids in our lab,Chrysanthemum morliflorum variety 'Huoyan' was chosen to be the material for the following experiments.RNA extracted from seedlings of Arabidopsis and Chrysanthemum under treatment of 4 C was as template to amplify the whole length sequences of AtCOR15a,AtCBF3 and CmICEl.Primer pairs were designed by the sequences of COR15a,CBF3 of Arabidopsis and ICE1 of Chrysanthemum dichrum.Furthermore,three recombination vectors were constructed,named pCAMBIA1301-PMI-A tCOR15a,pCAMBIA1301-PMI-AtCBF3 and pCAMBIA1301-PMI-CmICE1.After transformation toleaflets of 'Huoyan' by Agrobacterium tumefaciens,there were positive lines detected.The main experimental results were showing as the following.(1)According the survey on the characteristics of chrysanthemum about the width,height,color and blooming period,forty eight hybrids were divided into three groups,early blooming,middle blooming and the late one.Among them,'Huangjinzhan','Yanghuang'and 'Dushiliren'showed high quality when they conquer the chilliness and frost.On the contrary,some hybrids such as 'Fendie','Xuetao' and 'Zidanban' were damaged severely after facing the temperature lower than zero.Variety 'huoyan' which show the moderate growth was chose to be material of the subsequent experiments.(2)Primers were decided by the sequences of the COR15a,CBF3 of Arabidopsis and ICE1 of Chrysanthemum dichrum from database.The whole length sequences of AtCOR15a,AtCBF3 and CmICE1 were amplified by PCR.The analysis on the CmICE1 indicated the opening reading frame is 1410 bp,encoding 469 amino acids.CmICE1 protein is hydrophobic,without signal peptide,and located in cell nucleus.The homology of ICE1 from Chrysanthemum morliflorum and Chrysanthemum dichrum is up to 98%.Amino acid sequence contains threes conserved domains,DNA binding domain,E-box/N-box specific sites and dimerization domain.The protein is belonging to bHLH-type protein.The bioinformation suggests that the protein can interact with MYC element of the promoter in CBF,thereby to activate expression of CBF.(3)After construction of the recombined vectors of pCAMBlA1301-PMI-AtCOR15a,pCAMBIA1301-PMI-AtCBF3 and pC AMBIA1301-PMI-CmICEl,these vectors were transformed into Agrobacterium tumefaciens successfully.Subsequently transformation and screening help to obtain two positive transformed AtCOR15a lines,AtCOR15a-43 and AtCOR15a-44.which was named CFCOR-43.One positive transformed AtCBF3 lines,AtCBF3-6,named CFCBF-6 latter.Unfortunately,no positive line transformed CmICE was identified.(4)The positive transformed lines CFCOR-43,CFCBF-6 and untransformed line were cultivated under 4?.The activity of SOD,POD and content of soluble sugar were detected from treatment for 2 hours to 8 hours steps by two hours.The activities of SOD and POD presented to decrease after increasing at the early stage.And the activity of SOD and POD present higher in the transformed lines than the untransformed ones.Yet the amount of MDA presents a trend of decrease followed by increase.And the amount of MDA of the transformed lines was significantly lower than the untransformed ones.The content of the soluble sugar were increasing during the treatment period and the amount of the transformed lines was higher than the untransformed ones.These results indicated CFCOR-43 and CFCBF-6 were the transformed lines and they would present much more endurance with the untransformed line under the low temperate stress.