Cloning And Validation Of RhDFR And TaDFR As Morphologic Marker Genes For The Screening Of Transgenic Plants

Marker genes are essential parts of transgenic techniques,including antibiotic genes,reporter genes,physiological,biochemical and morphological marker genes.Among them,morphological marker genes emerged as promising markers due to the visibility,low background noise and low interference to host organisms.Wheat is one of the major staple crops in the world,but the development of transgenic techniques in wheat largely lags behind other major cereal crops.Currently,many transgenic wheat plants have been obtained through tissue culture-based transformation technology,such as gene gun and Agrobacterium-mediated methods,but drawbacks of this technology e.g.genotype dependency,intensive labor,time consuming and somaclonal variation,have impeded its extensive application.In this study,RhDFR-1 gene and TaDFR A-1 gene were cloned and evaluated as morphological marker genes and non-tissue-culture transgenic methods had been tried to establish a high efficient platform for wheat transformation.In this study,the coding sequence of putative Rh DFR-1 and TaDFR A-1 was cloned by RT-PCR?reverse transcription-polymerase chain reaction?.Bioinformatics analysis showed that there are 4 bases differences between RhDFR-1 and reference gene?ROZD4R,GI:1945120?.This new allelic RhDFR gene encoded a protein with 349 amino acids.Domain analysis showed RhDFR-1 contained domain FR_SDR_e,and belonged to NADB-Rossmann family.Bioinformatics analysis demonstrated Ta DFR A-1 gene is a new allelic gene of TaDFR A and contained similar sequence feature to RhDFR-1.Phylogenetic analysis revealed RhDFR-1 shared high similiarity with dicot plants,while TaDFR A-1 has much close evolutionary distance with monocot plants.To validate the functions of RhDFR-1 and TaDFR A-1 in anthocyanin synthesis,p806-303-RhDFR-1 and pGwb6-TaDFR A-1 were constructed using Gateway technoloty.The fusion protein sGFP-TaDFR A-1 of vector p Gwb6-TaDFR A-1 was localized on the chloroplast in the subcelluar level.The transgenic Arabidopsis thaliana harbored RhDFR-1and TaDFR A-1 gene were obtained by Agrobacterium-mediated floral-dip method.The results showed that the hypocotyl,the abaxial side of the cotyledon and the base of the inflorescence branches had red-brown pigment accumulation compared to those of wild-type plants.And GFP reporter protein can be detected in the relevant parts.Meanwhile,"cutting the wheat stem tip and vacuum infiltration"method was developed with the modification on"piercing the stem tip and vacuum infiltration"method.And a screening program were optimized by soaking the albino T₀ seedlings to the preliminary screen,then use PCR to select T₁ generation plants to eliminate chimeric T₀ transformants.A large number of PCR positive T₁ plants were obtained by this modified transformation method.The screening of T₀ generation resulted in 3.72%survival rate,while the rate of the T₀ plants with seeds harvested is only 1.57%.And 85.71%of the T₀ generation plants that can harvest seeds are got PCR positive T₁ plants,which produced 24.03%positive rate in T₁ generation.The PCR validation of T₂ plants of Liaochun17 varieties reached a positive rate 61.04%.Taken all data together,the modified non-tissue culture transgenic method in wheat can be operated easily and will be efficient way to obtain transgenic wheat.