Isolation And Functional Analysis Of Auxin Efflux Carrier Gene TaPIN1 In Wheat(Triticum Aestivum L.)

Plant hormone auxins play an important role in the regulation of branch production and root development.In this study,the evolution,structure and expression pattern of TaPIN1 gene were analyzed,and its biological function was preliminarily studied.The main results are as follows:Using Arabidopsis thaliana AtPIN1 gene sequences as query to blastp in wheat EnsemblPlants database,we found wheat A,B and D genomes contain AtPIN1 orthologous genes.Among them,the chromosome 6A has 1 copy,the chromosome 6B has four copies,and the chromosome 6D has 1 copy.Sequence homology between each copy is greater than 77%.Evolutionary tree analysis showed that TaPIN1 was closely related to OsPIN1 b in rice.TaPIN1 was located in the cell membrane of Arabidopsis thaliana.Transmembrane helical prediction revealed a typical N-terminal transmembrane conserved domain,and a variable domain composed of a middle transmembrane cytoplasmic region and a C-terminal transmembrane segment.qRT-PCR results showed that TaPIN1 gene was mainly expressed in young leaves,roots,stem apex of single stage and double stages,spikelet primordia and piriform embryos of wheat;In situ hybridization results showed that TaPIN1 gene was mainly expressed in young leaves and lateral roots,lateral roots primordia and stem apex of single ridge stage of wheat.In order to analyze whether the expression of TaPIN1 gene was induced by auxin,we treated wheat root tips germinated for 5 days used IAA and 2,4-D,and results showed that the expression of TaPIN1 in root tips was changed with the treatment time and concentration.In order to determine the function of TaPIN1 gene in wheat,pUbi::TaPIN1-RNAi vector,pUbi::TaPIN1A over-expression vector and pTaPIN1::gTaPIN1A-GFP fusion expression vector were constructed.Genetic transformation mediated by Agrobacterium-mediated was carried out using the immature embryos of wheat lines CB037 and Fielder as explants.T2 generation positive transgenic lines of pUbi::TaPIN1-RNAi vector have been obtained,and T0 generation positive plants with TaPIN1 A over-expression vector and GFP fusion expression vector have also been obtained.Preliminary results showed that the expression of TaPIN1 gene in positive transgenic lines of TaPIN1-RNAi was significantly decreased.The number of secondary roots of transgenic strains was significantly reduced.According to the phenotypic analysis at seedling stage of positive transgenic lines,the number of tillers was increased and the angle of tiller was increased obviously.In conclusion,evolutionary tree analysis,subcellular localization and spatiotemporal expression patterns of TaPIN1 gene were analyzed in this study.TaPIN1-RNAi vector,over-expression vector and GFP fusion protein vector were constructed and wheat genetic transformation was performed.The result showed that the expression level of TaPIN1 gene was decreased by RNAi.Furthermore,the occurrence of lateral roots was inhibited,and both tiller number and tillering angle were increased.The results provide new data for the evaluation of TaPIN1 function in wheat development.