Molecule Clonging And Functional Study Of SsPIN1 In Sinningia Speciosa

The auxin efflux carrier PIN1(PIN-FORMED1)regulates polar auxin transport in plants and plays important roles in plant growth and development,especially in the initiation and arrangement of leaf primordia.In this study,the full-length cDNA of SsPIN1 gene in Sinningia speciosa was obtained via homologous cloning and RACE technology according to the published genomic data such as Arabidopsis thaliana and Antirrhinum majus L.And then,characteristics of the gene sequence were analyzed via bioinformatics.The tissue specific expression pattern of the gene and the response of it to different hormone treatments were analyzed via real-time quantitative PCR.Moreover,the subcellular localization of SsPIN1 protein and its alterations in polarity localization caused via the phosphorylation site mutation were detected by laser confocal microscopy.At last,the SsPIN1 gene was transformed into Nicotiana tabacum cv.Petit Havana SR-1 by leaf disc method.The main results are as follows:1.The full-length cDNA of SsPIN1 gene was 1770 bp,which encodes a protein of 589 amino acids with a molecular weight of 64772.92 Daltons and an isoelectric point of 8.074.Phylogenetic analysis showed that the SsPIN1 protein of S.speciosa was most closely related to AmPIN1 a of Antirrhinum majus L.Further sequence alignment revealed that the SsPIN1 protein consists of a conserved carboxy terminus and amino terminus and a long hydrophilic ring(HL)containing about 300 amino acids.Subcellular localization prediction suggested that SsPIN1 was localized to the plasma membrane(PM).The transmembrane domain prediction showed that the SsPIN1 protein has five transmembrane domains at the carboxy terminus and four transmembrane domains at the amino terminus.All the predications suggested that SsPIN1 belongs to the plant transport vector family(IRRO14024)of the membrane transport protein family(IPROO4776).2.The tissue specific expression pattern of SsPIN1 gene revealed that the gene was expressed in all detected tissues during the flowering stage of S.speciosa.The expression level was highest in the petiole of young leaves,followed by petals and axillary buds.After treatment with the exogenous NAA,the expression level of SsPIN1 gene increased first and then decreased with the time processing.After treatment with exogenous BA or GA,the expression level of SsPIN1 gene increased with time processing.3.The subcellular localization of SsPIN1 protein had been observed on the plasma membrane first via laser confocal microscopy.Further,phosphorylation site(T228,T249 and T287)of SsPIN1 were mutated as single,double or triple mutant.The mutated SsPIN1 protein is no longer localized on the PM but within the cell.Interestingly,the single point mutation has mild effect on the polarity localization of SsPIN1,while the triple has the greatest effect.This indicates that the phosphorylation site mutation affects the polar localization of SsPIN1 with a cumulative effect.4.The pBI111L-SsPIN1 overexpression vector was constructed based on the in fusion method.The SsPIN1 gene was then introduced into N.tabacum by Agrobacterium mediated leaf disc transformation and screened with 100 mg/L Kan to obtain resistant plants.The resistant plants displayed several alterations during growth compared with wild type,such as slow growth and inhibition of root development.PCR detection suggested that the nptII gene was ectopic expressed in all detected resistant plants.Further analysis by real-time quantitative PCR suggested that the expression level of SsPIN1 gene was significantly increased in resistant plants compared with wild type.