Cloning And Transgenic Analysis Of AmPAN1a Regulating Phyllotaxic Development In Antirrhinum Majus

Antirrhinum majus has belonged to Scrophulariales and Scrophulariaceae.Antirrhinum majus is herbaceous perennid,it is widely used ornamental plant in garden,having a wide variety and ecological niche,playing an important role in the market of cut flowers in the world,it is one of the excellent plants for studying the change of plant phyllotaxis.The development of plant phyllotaxis is mainly developed from leaf primordium growth point.At present,it has been found that auxin and cytokinin played the important role in the development of plant phyllotaxis.PIN family proteins mainly control the transport of auxin in plants.The research of PIN family can help us to clarify some mechanism of plant phyllotaxis development.In this study,the Am PIN1 a gene of Antirrhinum majus was cloned and constructed into the expression vector.The wild-type Arabidopsis was infected with the method of dipped flower,then transgenic Arabidopsis seeds harvested.The phenotype of the transgenic Arabidopsis was observed and treated with exogenous IAA to studied on the function of Am PIN1 a gene in Antirrhinum majus.The results is following as below:Firstly,Antirrhinum majus is the research material,through the tender Antirrhinum majus shoot apical meristem transcriptome library analysis in this experiment,founding that in the same period the expression level of PIN1 gene between the branches of opposite phyllotaxis and the branches of spiral phyllotaxis were quite different.Using NCBI the homologous PIN1 a gene of Antirrhinum majus was searched by Blast,and corresponding primers were designed according to the searched sequence,and Am PIN1 a gene was cloned from Antirrhinum majus by PCR.Secondly,the expression patterns of Am PIN1 a gene was analyzed by real-time quantitative PCR.The results showed that Am PIN1 a was found in different organs of Antirrhinum majus in different growth periods,and the expression levels of Am PIN1 a in seedling stage were:stem>root>leaf,adulthood stage expression amount: stem>leaf>flower>root.Thirdly,the full-length CDS of Am PIN1 a gene is 1836 bp,which has high homology with PIN gene of other species.It encodes a protein of 611 amino acids with a relative molecular mass of 66.78 k D and isoelectric point of 8.95.The encoded protein is a hydrophilic protein.The amino acid sequence analysis showed that the protein contains two Mem_trans superfamily domains.Fourthly,the over-expression vector p CAMBIA1301::35S-Am PIN1 a was constructed and transformed into Agrobacterium tumefaciens strain GV1301 by electroporation.The wild-type Arabidopsis was infested with the method of dipped flower and the homozygous transgenic Arabidopsis was obtained after several generations of selection.Fifthly,the observed result showed that in 0-12 days the main root of Am PIN1 a transgenic Arabidopsis was slightly shorter than wild-type Arabidopsis,lateral root's number was significantly increased compared wild-type Arabidopsis with Am PIN1 a transgenic Arabidopsis.After transplanted about 24 days Am PIN1 a transgenic Arabidopsis leaves surpassed the wild-type,and the difference was significant.The inflorescence axis germinated late but developed rapidly.Sixthly,under exogenous IAA treatment,Arabidopsis over-expressing Am PIN1 a gene was found to be more susceptible to IAA stimulation than wild-type Arabidopsis,suggesting that this gene may play a significant role in the auxin transport.