Cloning And Preliminary Functional Analysis Of PLTs Gene In Populus Euphratica

PLT(PLETHORA)transcription factors belong to the ERF / AP2(ETHYLENE RESPONSE FACTOR / APETALA 2)family and play important roles in plant embryogenesis,organ development,and meristem maintenance.Related studies have elucidated the mechanisms by which PLT3,PLT5 and PLT7 genes control adventitious shoot regeneration in Arabidopsis,but their functions in woody plants are unclear.Populus euphratica is an important ecological tree species distributed in the desert area of northwest China.It is difficult to propagate by cuttings,and the characteristics of the regenerated seedlings are severely separated.Therefore,the study of adventitious shoot regeneration mechanism is key to solve Populus euphratica asexual propagation problem.In this study,the homologous genes PePLT3,PePLT5 a,and PePLT5 b in Populus euphratica were obtained by BLAST searches,molecular cloning and functional analysis were performed to investigate the functions of PLT genes in Populus euphratica.The purpose of this study was to investigate the functions of PLT genes in adventitious shoot regeneration in woody plant Populus euphratica,while providing new ideas and methods for Populus euphratica in vitro.The main studies are as follows:1.Cloning and bioinformatics analysis of PePLT3,PePLT5 a,PePLT5 b genes: Three genes were cloned using molecular cloning-related technologies.The conserved domains,protein structure,physicochemical properties,protein phosphorylation sites,protein subcellular localization,and phylogenetic tree of PLT proteins in different species were analysis by online tools and software.It was found that the three genes in Populus euphratica and Populus tomentosa were most closely related.2.Subcellular localization of PePLT3,PePLT5 a,PePLT5b: By constructing 35S::GFP expression vector and transiently infecting tobacco,it was found that all three proteins were localized in the nucleus.This result was the same as the predicted subcellular localization which laying the foundation for the subsequent study of the three genes.3.Antibiotic resistance test of Populus tomentosa genetic transformation system: The leaves of Populus tomentosa were cultivated by using media with different concentrations of cefalexin,timentin,and kanamycin,and the leaf survival rate,regeneration rate,and antibiotic inhibition at different concentrations were counted.30-50 mg/L kanamycin was selected as the selected concentration,and 200mg/L timentin was selected as the bacteriostatic concentration.4.Obtaining transgenic plants of Populus tomentosa: Populus tomentosa that transformed with35S::PePLT3,35S::PePLT5 a,35S::PePLT5 b were obtained by direct type and indirect type respectively.5.Phenotypic analysis of transgenic Populus tomentosa: The number of adventitious shoots and the regeneration rate of adventitious shoots were measured.According to the analysis of experimental results,it was found that the PePLT genes of Populus euphratica were related to the regeneration of adventitious shoots.Among them,the adventitious shoots regeneration rate of PePLT3 overexpression plants increased and the average number of shoots increased,indicating that PePLT3 promoted the regeneration of adventitious shoots in poplar trees.The adventitious shoots regeneration rate of PePLT5 a and PePLT5 b overexpressed plants reduced and the average number of shoots decreased,indicating that PePLT5 a and PePLT5 b inhibited adventitious shoot regeneration.6.Genetic transformation of Populus euphratica: Genetic transformation of Populus euphratica has been carried out,in which some resistant seedlings have taken root and are under continuous screening.In summary,this study cloned three PLT genes of Populus euphratica and performed functional analysis.The experimental results showed that PePLT genes have functions related to the regeneration of adventitious shoots in poplar.Overexpression of PePLT3 promotes regeneration of adventitious shoots,and overexpression of PePLT5 a and PePLT5 b inhibits regeneration of adventitious shoots,which is different from the function in the model plant Arabidopsis,indicating that the homologous genes may play different functions in different species.This study is dedicated to revealing the function of PLT genes in Populus euphratica,and provides experimental ideas for the subsequent research on the molecular mechanism of adventitious shoot regeneration of Populus euphratica and in vitro culture of Populus euphratica and the construction of transgenic plants.