The Histone Modification Of Cold Responsive Genes And Genetic Transformation Of Taraxacum Kok-saghyz Rodin

T Taraxacum kok-saghyz Rodin(Tks)is a perennial herb of the genus dandelion of Compositae.It is known as one of three largest gum-producing plants along with silver chrysanthemum and Hevea brasiliensis.Compared with Hevea brasiliensis,Tks has many advantages,such as short life cycle,large number of seeds,small seed size and relatively easy genetic transformation,so it has become an ideal model plant to study the mechanism of rubber production and drainage.In previous studies,we found that epigenetic modification of cold stress responsive genes is an important mechanism for rubber trees to adapt to chilling injury.In order to analyze the molecular mechanism of cold stress response in rubber-producing plants,Tks was used as experimental material to study the histone modification of cold responsive genes.The genetic transformation system of Tks is constructed to enable the transformation of cold related genes,the following results were obtained:1.Physiological and biochemical properties of Tks under cold stress:4 ? cold treatment induced accumulation of a large amount of anthocyanin in leaves,which changed leaves color to purple;The electrical conductivity of leaves increased at 3 days after cold stress treatment;the level of proline(Pro)and malonic dialdehyde(MDA)increased significantly under cold stress,reached 2.5folds of the control,and then decreased rapidly;cold stress also increased superoxide dismutation Superoxide dismutase(SOD)level.2.Differential expression of cold stress response genes in Tks :we found that the expression levels of cold responsive genes reached a maximum value 5d after treatment,then the expression of decreased,but the expression of Tks CBF1 increased to the highest level,then the expression recovered to the normal value.The expression level of Tks COR15 a,which is a sucrose synthesis related gene,remained at a high transcription level after the peak to maintain the sucrose level of Tks to cope with the continuous cold stress.3.Histone modification of cold stress responsive genes in Tks: two types of stress-related histone modification on the cold related genes,i.e.H3K27me3 and H3K27me3,were impacted in the process of cold stress,and the main time point of change is in the first 5 days of the cold stress at the time point of 3d and 5d,with the continuous progress of time,except for the H3K27me3 of Tks ICE1,which still exists after 5d.In the second half of the period,the concentration level of other histones recovered in varying degrees.4.TKs transcriptome sequencing(RNA-seq)and chromatin immunoprecipitation sequencing(CHIP-seq)associated analysis.In this study,Tks leaf at 10 days after10? cold treatment were used for RNA-seq and H3K27me3 enriched CHIP-seq sequencing analysis.In RNA-seq,8802 coldinduced differentially expressed genes were found,including 4075 up-regulated and 4727 downregulated.Through association analysis with CHIP-seq,a total of 44 genes were down-regulated in both CHIP-seq and RNA-seq,and 19 genes were up-regulated in both CHIP-seq and RNA-seq.Thirty-two genes were up-regulated in CHIP-seq and down-regulated in RNA-seq,and 21 genes were up-regulated in CHIP-seq and down-regulated in RNA-seq.These results indicate that cold-induced chromatin histone H3K27me3 changes have a correlation with gene expression regulation.5.Establishment of genetic transformation system of Tks :the condition of adventitious bud induction and differentiation of Tks: when the concentration of NAA is 0.1 mg/L and the concentration of 6-BA is 0.5 mg/L,it is conducive to the differentiation and regeneration of three explants,leaf,petiole and root.The conditions of rooting induction of Tks: when the concentration of NAA is 0.3 mg/L,it is beneficial for rooting of differentiation seedlings of Tks.Using this transformation system,the HBICE1 gene was successfully transformed into TKS and the transgenic plants were obtained.In conclusion,Tks showed obvious changes in the physiological and biochemical indexes,the expression of cold responsive genes and the modification of H3K4me3 and H3K27me3 after cold treatment.The modification changes of H3K27me3 which are induced by cold correlated improve the expression of genes,indicating that chromatin modification was an important molecular mechanism for Tks responding to cold signals.Additionally,optimizing the genetic transformation system of Tks and successful transformation of Hb ICE1 laid a foundation for further study of the molecular mechanism of cold stress in rubber producing plants.