Cloning And Functional Analysis Of AmPIN1c Gene In Snapdragon

Antirrhinum is a plant species of the family Plantaginaceae genus antirrhinum,which belongs to the family Scrophulariaceae.It is named for its flower like goldfish and belongs to perennial herbaceous plant.In addition to extensive landscape applications,antirrhinum is also a classic model crop in scientific research.Its phyllotaxy will change during the growth stage,so it is very suitable for studying the regulation mechanism of phyllotaxy changes.Considering the important role of auxin polar translocation on the phyllotaxy development and the difference in the expression of different phyllotaxy genes in antirrhinum at the same period,the PIN1 c gene of clonal auxin efflux carrier was selected to study the function of Am PIN1 c gene.The following main results were obtained:1.Analyze the transcriptome library of meristem at the top bud of the antirrhinum,select the PIN1 gene varied greatly in different phyllotaxy growth points,homologous search for antirrhinum homologous PIN1 c gene on NCBI website and design primers.Extracted antirrhinum RNA and cloned the Am PIN1 c gene by RT-PCR.2.Am PIN1 c is 1689 bp in size and has high homology with PIN gene of many species,the gene encoding protein formula of Am PIN1 c is C2749H4306N712O772S24,the amino acid number is 553,the molecular weight is 60452.18,and the theoretical p I is 9.14,it is a stable protein,the hydrophilicity is 0.246,there are eight transmembrane overlaps,four potential phosphorylation sites.Auxin efflux carrier was mainly located in the endoplasmic reticulum,up to 55.6%.3.Use fluorescent quantitative PCR technology to analyze the Am PIN1 c gene expression in various organs during the growth cycle of antirrhinum,and sequenced it:Matured stem > Matured root > Matured leaf > Matured flower > Caulicle > Radicle > Spire.4.The overexpression vector was constructed by double enzyme digestion and homologous recombination,and was transformed into Escherichia coli and Agrobacterium by freeze-thaw method,and then transformed into Arabidopsis thaliana by flower dipping method.5.The Arabidopsis thaliana transgenic with Am PIN1 c gene grows slowly in the early stage,and the flower bud differentiation and bolting are late;The leaves increase and become larger,and the lateral roots and branches increase;The opposite phenotype was found in the upper part of the plant,confirming that the Am PIN1 c gene has a certain effect on plant growth and phyllotaxy development.6.With the increase of IAA concentration in the culture medium,the Arabidopsis thaliana transgenic with Am PIN1 c gene is more sensitive than WT,the growth advantage is more obvious,the taproot is longer,the lateral roots are increased,and the leaf area is also increased.