T-DNA Insertion Mutant Library Construction Of Brachypodium Distachyon And Identification Of Rust Resistance

Wheat stripe rust caused by Puccinia striiformis f.sp.tritici heavily threatens the food security in China.Rapid identification of key genes in interaction between wheat stripe rust fungus and host plants can provide theoretical basis and genetic resources for genetic improvement of resistance to stripe rust,and which is great significance long-term prevention and control of wheat stripe rust.However,due to the huge genome with high frequency of repetitive sequences and the low transformation efficiency of wheat,study on the function of wheat resistant related genes against Pst has been largely restricted.Hence utilizing the model plant to rapidly study and reveal the mechanism of host-rust fungus interaction become very meaningful.In this study,Brachypodium distachyon,the closest Gramineae related to wheat,was used as a receptor material to create a Bd21-3 ecotype T-DNA inserting mutant library using Agrobacterium-mediated genetic transformation system.And the resistance of the mutants to Puccinia brachypodii was tested.The research may provide a basis for excavating the rust resistance related gene or the non-host resistance gene.The main results are as follows:1.Optimization of Brachypodium distachyon genetic transformation systemBased on the existing genetic transformation system of Brachypodium distachyon,the culture time of the embryo in the culture medium and the screening concentration of hygromycin B were optimized.The young embryos were picked under a microscope and cultured in the callus induction medium for three weeks.Then pale yellow callus of good quality were transferred to a new medium for three-week subculture.After that the embryogenic callus were transferred to fresh medium for another 0ne-week subculturing.In order to improve the screening efficiency of Hygromycin B antagonistic callus,different concentration of antibiotics were tested.The results showed that the best screening effect of Hygromycin B on the callus of Brachypodium distachyon was 40 mg/L.2.Creation of T-DNA insertion mutant materialThe Agrobacterium-mediated genetic transformation system was used to transfer the p JJ2 LB vector into the callus,and by inserting T-DNA into the callus to obtain the T-DNA insertion mutants.Here,1739 regenerated seedlings were obtained.The seedlings with good growth and leaf stretch were selected for DNA extraction and PCR detection.The results showed that 97 of 279 seedlings were identified as positive plants with a positive rate of approximately 35%.3.Identification of mutant phenotypesThe positive T0 generation mutants were inoculated with Puccinia brachypodii for resistance identification,and the phenotypic identification statistics were based on the 9-level classification standard.The results showed that among the 43 positive mutant plants,29 showed significant improvement in disease resistance to Puccinia brachypodii compared with wild type,6 of which showed high resistance,23 strains were medium resistant,and the remaining 14 is the same as the wild type.