Cloning Seeds Germination Key Genes PlCYP707As In Paeonia Lactiflora Pall. And Constructing Plant Expression Vector

Paeonia lactiflora is a perennial herbaceous flower of the family Paeoniaceae.which are traditional Chinese famous flowers.Paeonia suffruticosa,the king of flowers,Paeonia lactiflora can be comparable with it.Paeonia lactiflora are widely applied in landscape gardening.It has formed unique and hypocotyl double dormancy characteristic in the process of the long-term system evolution.It brings a lot of inconvenience in new breeding work and it is very bad for the industrialization development of Paeonia lactiflora.In this study,hybrid seeds is used as experimental material.Selecting four key seed developmental periods as the samples for the transcriptome sequencing analysis.Selecting the key control genes PICYP707A1 and PICYP707A2 in the process of Paeonia lactiflora seed germination.Analysising expression patterns and cloning the two key regulatory genes.Through bioinformatics technology further analysis and forecast the structure and function of genes.The two genes of plant expression vector are constructed so that it can provide the basis for functional identification with agrobacterium-mediated transformation of Arabidopsis thaliana.Test results of this study are as follows:1.Analysising the transcriptome data,researching abscisic acid metabolic pathways of upstream regulatory genes and locking the target gene.On the basis of the transcriptome sequencing CDS sequence obtained,designing quantitative primer and using semi-quantitative RT-PCR and real-time quantitative PCR analysis PICYP707A1 and PICYP707A2 gene expression,the results show that PICYP707A1 gene has increased significantly in PIB and PDA periods,PlCYP707A2 gene content increase gradually in the process of Paeonia lactiflora seed germination.In general,PICYP707A1 and PICYP707A2 gene expression rise in the process of Paeonia lactiflora seed germination play a significant role in to break dormancy.2.On the basis of the transcriptome sequencing the CDS sequence obtained,designing homologous cloning primers,using PCR amplification techniques to get the PICYP707A1 genes cDNA sequences,full length is 1 407 bp,encoding 468 amino acids and PlCYP707A2 genes cDNA sequences,full length is 1 392 bp,encoding 463 amino acids.At the same time,taking PICYP707A1 and PICYP707A2 gene cDNA sequences as probe to compare with GenBank databases,obtaining the homologous amino acid sequence between different species and constructing the phylogenetic tree.Using a variety of online softwares for PICYP707A1 and PlCYP707A2 genes to forecast and analyze the primary,secondary and tertiary structures of proteins,subcellular localization and so on.3.Using pBI121 plant expression vector and recombinant DNA technology to construct the two genes expression vectors,there are named as pBI-PlCYP707A1 and pBI-PlCYP707A2,then transform to Agrobacterium tumefaciens GV3101,to provide the basis for subsequent functional verification with agrobacterium-mediated transformation of Arabidopsis thaliana.