Ac/Ds Transposon Tagging System Controlled By Egg-Specific Promoter EC1.2 And Mutant Screening Analysis

Wheat is the most important grain crop in our country.Increasing the yield of wheat is beneficial to solve the food security problem in our country.However,wheat belongs to allohexaploid（AABBDD）,which has a large genome,and lacks efficient genetic transformation method.The Brachypodium distachyon has a series of advantages as a model plant,and is closely related to gramineous crops such as wheat.Therefore,the study of functional genomics of Brachypodium distachyon can promote the study of functional genomics of wheat.Ac/Ds transposon system is a common method for building mutant libraries in plants.However,conventional Ac/Ds transposon systems generally use constitutive promoters to initiate the transcription of Ac gene.The disadvantage is that there are both germinal transposition and somatic transposition in plants,which cannot be inherited to the next generation and will interfere with the mapping of inserted genes.This study uses Arabidopsis egg-specific promoter EC1.2 to initiate the transcription of Ac gene,so that transposition behavior occurs specifically in the egg cells,reducing the difficulty of gene mapping.The main findings are as follows:（1）In this study,a binary Ac/Ds transposon vector p XSY1 was constructed for Brachypodium distachyon.The Ac gene in the p XSY1 vector was initiated by the Arabidopsis egg-specific promoter EC1.2.The Ac gene was modified to lose its transposition ability.Three EDS-PCR primers P1,P2 and P3 could be used to verify the transposition of Ds transposons.（2）In this study,a total of 143 transgenic lines were obtained by the Agrobacterium transformation method.EDS-PCR showed that somatic cell transposition occurred in 4（2.78%）transgenic lines.The q PCR experiments showed that Ac gene was highly expressed in egg cell and slightly expression in other cells.（3）In this study,GFP and RFP fluorescent proteins were used to label Ac and Ds elements,respectively.GFP and RFP were expressed in Brachypodium callus and plants with strong fluorescence signal and high accuracy,which could replace tedious PCR experiments.（4）A total of 98 T₀transgenic lines with more than 30 seeds were selected,totaling 4290strains.Determine whether the plants meet the 3:1 separation ratio by the chi-square test.The results showed that 76（77.6%）lines met the 3:1 separation ratio,and these independent insertion lines were planted and screened for enriched mutants.（5）Ds only plants can be enriched in T1 generation population,which proves that the Ac/Ds system used in this study can produce heritable germinal transposition.The results showed that the Ds only plants screened in the T₁generation varied between 0 and 13.3%with a average screening frequency of 2.83%.（6）Different from the traditional Ac/Ds system,which only mapped the genes of Ds line,this study also mapped the genes of Ac/Ds line.It have shown that the Ac/Ds system used in this study can reduce the interference of gene mapping by somatic transposition.（7）The Ac/Ds system used in this study can be screened for mutants with plant height,leaf shape,spike shape,germination rate,flowering time and fertility.（8）In this study,gene mapping and fluorescence quantification experiments were performed on a mutant with abnormal spike spacing,and the mapping results showed that the Ds element was inserted into the intron of the candidate gene,and the expression of the candidate gene decreased significantly.In this study,complement vector of candidate genes were constructed to investigate the relationship between gene and phenotype.In conclusion,this study constructed an Ac/Ds transposon vector with egg-specific promoter,and labeled Ac and Ds with GFP and RFP fluorescent proteins,respectively.The vector can play a role in Brachypodium distachyon,producing heritable germinal transposition and reducing the occurrence of somatic transposition.GFP and RFP fluorescent proteins can play a role in replacing the tedious PCR experiments.The Ac/Ds system used in this study can screen mutants and make gene mapping relatively simple,which greatly improves the screening efficiency of mutants.