Rose RcTCP9 Gene Transformation System Optimization And Functional Research

Rose（Rosa chinensis）is one of the most important woody flowers in the world,with important ornamental and economic value.TCPs are a family of transcription factors that are unique to plants.Existing studies have shown that their members are involved in various stages of plant growth and development.They are a class of genes with great research value.In this study,a green fluorescent protein（e GFP）-containing editing vector based on CRISPR-Cas9 gene editing technology was constructed to explore the effect of RcTCP9gene of rose on plant growth and development and environmental stress;Agrobacterium-mediated genetic transformation was carried out with the wild-type Arabidopsis（Arabidopsis thaliana,ecotype Columbia）and somatic rose embryos as materials,and the regeneration system of somatic rose embryos was optimized at the same time;Using the transformed Arabidopsis as material,drought and low temperature stress were carried out,and the main research conclusions obtained are as follows:i)Using bioinformatics to analyze the rose RcTCP9 gene,the results show that the chemical formula of the protein encoded by the gene is:C₁₇₄₉H₂₇₉₁N₅₂₁O₅₈₁S₁₅ which is an unstable protein and has hydrophilicity;α-helix,β-sheet,β-turn and random coil are the main ones Structure;subcellular localization results show that it is most likely to be located in the nucleus;it contains two conserved domains,and there is no transmembrane domain;after homology comparison,it is found that the protein homology with Fragaria vesca has the highest homology similarity.ii)Successful construction of gene editing vector CRISPR-Cas-e GFP-RcTCP9iii)The gene-edited Arabidopsis plant was successfully obtained,and the growth phenotype was observed with the wild type as the control.It was found that the main changes were:the transformed type emits dazzling green fluorescence under the excitation light source;the leaf edge of the transformed leaf is wavy;the transformed leaf epidermis Trichoids on cells increased;roots in transformed Arabidopsis were shortened and easily broken.iv)Using transformed and wild-type Arabidopsis as materials,abiotic stress experiments of drought and low temperature were carried out at the same time,and the phenotypic changes were observed.The results showed that the wild-type Arabidopsis thaliana could recover rapidly after 8 days of drought stress,but the transformed Arabidopsis lost the ability to recover from drought;the transformed Arabidopsis also lost its cold resistance under cold stress.v)The effects of three hormones,6-BA,KT,and TDZ,on the regeneration of somatic embryos of rose B103 were explored.The results showed that the somatic embryo germination rate of rose B103 was the highest when the concentration of 6-BA and TDZ was0.5 mg/L,both of which were 20%.12%higher than the previous one.vi)The somatic embryos of rose B103 were genetically transformed by Agrobacterium tumefaciens,and the transformation rate was 15.7%at 7 days.The somatic embryos were detected by excitation light source,and green fluorescence in clusters was found.