Cloning And Genetic Transformation Of PlPP2C Gene Related To Dormancy And Germination Of Paeonia Lactiflora Seeds

Peony（Paeonia lactiflora Pall.）is an herbaceous flower of Paeonia in the family Paeoniaceae,which is a traditional Chinese flower and an unique gardening plant.However,the seeds of peony have typical characteristics of double dormancy of the up and down-hypocotyls,and the growth cycle is too long.In the process of seed reproduction,the germination rate is often low due to the inability to break the dormancy,which brings great obstacles to practical production,especially to the breeding of new varieties and hybrid breeding work.In this study,the hybrid seeds of the peony cultivars?Fen yu nu?and?Fen yu lou?were used as materials.The transcriptomes were sequenced in the four key stages of seed germination,and the key gene PlPP2C was selected for seed germination.The full-length cDNA of this gene was cloned and analyzed by bioinformatics and its expression pattern during seed germination.A plant expression vector was constructed and the Arabidopsis thaliana was genetically transformed by inflorescence method to reveal the gene pair,which laid a foundation for revealing the effect of PlPP2C on seed germination of peony.The main results are as follows:1.The transcription data were analyzed,dormancy and germination related genes were mined,and PlPP2C,a regulatory gene in abscisic acid signal transduction pathway was identified.The PlPP2C gene of peony seed was cloned by PCR based on CDS sequence obtained by transcriptome sequencing.The ORF sequence length of PlPP2C gene is 1233bp and encodes 410 amino acids.The amino acid sequence contains PP2Cs gene characteristic sequence FFGVYDGHG and conserved domains,belonging to the PP2Cs gene family.Comparison of amino acid sequence homology revealed that the PlPP2C gene has high homology with plants such as Morus notabilis,Vitis vinifera,Eucalypyptus grandis,and Durio zibethinus.The phylogenetic tree showed that the genetic relationship between PlPP2C gene and Arabidopsis thaliana AHG1 was the closest.The PlPP2C gene encodes a protein with a molecular weight of 45kDa and a theoretical isoelectric point of 5.04.It is an unstable hydrophobic protein that does not has transmembrane structure and located on the nucleus.2.The real-time quantitative RT-PCR method was used to analyze the expression of PlPP2C gene in the four key stages of seed development of peony.The results showed that the expression level of PlPP2C gene was highest in the germination stage of down-hypocotyls,and the expression level was lowest before the seeds imbibition.It is speculated that the gene may promote seed germination by regulating ABA content.3.The plant expression vector of PlPP2C gene was constructed and named as pBI121-PP2C.The plant expression vector successfully constructed was transformed into Agrobacterium tumefaciens GV3101 by freeze-thaw method.The wild type Arabidopsis thaliana was planted and the seeds were harvested.The wild-type Arabidopsis thaliana plants were genetically transformed by the inoculum method mediated by Agrobacterium.The seeds of the T₀ generation were harvested,and the resistant plants were obtained by antibiotic screening.