Genetic Transformation System Basis And Function Verification Of Populus Ussuriensis Kom.PuCBF

CBF is one of the plant resistance transcription factors.In order to reveal gene fuction of CBF,and provide genetic material and theoretical support,Populus ussuriensis Kom was used as experimental material.PuCBF gene from P.ussuriensis Kom was cloned and bioinformatics analysis was done.To reveal the function of PuCBF gene,overexpression vector was constructed and transferred into tobacco.Cold and drought resistance experiments were done with these transgenic tobaccos.In addition,orthogonal experiment design was used to optimize the tissure culture system.The main results of this study were as follows;1.The sequence of PuCBF was very similar to CBF in P.trichocarpa.The PuCBF gene encoded 231 amino acids.This amino acid peptide chains showed hydrophilic.The NJ phylogenetic tree was constructed.Fluorescence quantitative PCR showed that this gene had higher expression under drought stress.2.Concentration of NAA had the strongest inducing effect on adventitious buds.Concentration of NAA also had strong effect during proliferation of adventitious buds which was compared with types of culture medium.Hormone effect was stronger than culture medium type during rooting.In addition,types of culture medium and the concentration of hormone had significant effect on the regeneration system.The most suitable medium for differentiation of adventitious buds was WPM + 0.5mg/L 6-BA + 0.05mg/L NAA,and the inducing rate was 100%.The most suitable medium for buds proliferation was MS + 0.05mg/L 6-BA + 0.01mg/L NAA,and the plantlets were healthy.The most suitable medium for rooting was 1/2 MS + 0.1 mg/L IBA.3.The reults of drought resistance experiment were showed that,PuCBF overexpression could increase the stability of the plant cell membrane.The chlorophyll content of transgenic lines was higher than that of non-transgenic lines in 0-24 h.The content of MDA in transgenic lines was much lower than that of non-transgenic lines.In summary,Pu CBF overexpression could increase the ability of drought resistance.4.The results of cold resistance experiment were showed that,the content of MDA in transgenic lines was lower than that of non-transgenic lines.Transgenic lines and nontransgenic lines were almost identical at 25°C.But as the temperature decreasing,the content of POD and SOD in transgenic lines were higher than non-transgenic lines and the magnitude was relatively large.In summary,PuCBF overexpression could increase the ability of cold resistance.