Editing Of Wheat TaEPFL1 Gene Mediated By CRISPR/Cas9 System

The TaEPFL1 gene belongs to the EPIDERMAL PATTERNING FACTOR-LIKE?EPFL?family.The EPFL family is a group of plant-specific secreted peptides,which were ligands for ERECTA?ER?-family,regulates a series of plant growth and development processes.To figure out how the TaEPFL1 gene contributes to the plant growth and development,we used the mature wheat seed of Chinese spring,and employed CRISPR/Cas9 technology to specifically edit the exon region to produce targeted knockout plants.The normal function of the gene was elucidated by studying the mutant gene.In this study,we investigated the effects of seed sterilization methods,dicamba concentration,the amino acid composition,Agrobacterium and mature embryo co-culture time on callus induction and growth.And the influence of Agrobacterium and callus co-culture time on plant differentiation and growth.The results are as follows:1.The mature embryos are not contaminated when the 75%alcohol soaking time is controlled at 180s and the 30%hydrogen peroxide sterilization time is more than 15s,or when the 30%hydrogen peroxide disinfection time is controlled at 180s and the 75%alcohol soaking time is more than 15s.The optimum concentration of dicamba to induce callus of mature embryos was 2.5 mg·L^-1.Glycine,aspartic acid,proline and leucine are essential amino acids in callus induce.Orthogonal experiments showed that the best amino acid formula for Chinese spring culture was glycine 60.0mg·L^-1,aspartic acid 2.0 mg·L^-1,proline 2.0 mg·L-¹,leucine 0.1 mg·L^-1.With this culture protocol,the ratio of callus induction reached 97%. When mature embryos and Agrobacterium were co-cultured for more than 10hours,the ratio of callus induction decreased.It was also found that callus were more susceptible to infection and necrosis when water was not removed.3.We successfully obtained a transgenic plant,which an amino acid codon was converted from AAC to AGC,and the encoded amino acid was converted from asparagine?Asn,N?to serine?Ser,S?,with the optimized tissue culture and genetic transformation system.We found that the SAM synthase gene has a lower expression level in transgene g transgenic plant than in non-transgenic plants.The ACC synthase gene and ACC oxidase genes had similar expression pattern with wild type.Transgenic plant showed a range of stunted phenotypes with short internodes,less seed,small and chlorotic leaves,compared with wild type?non-transgenic plants?.This study optimized the callus-induced culture system of wheat mature embryos,and provided a reference for studying the physiological process of wheat amino acid metabolism.It was found that TaEPFL1 gene has an interaction with ethylene metabolism pathway,which will provide a scientific basis of further study the molecular mechanism of the EPFL family on wheat growth and development.