Optimization Of Genetic Transformation System And Editing Of Genes In Centromere Region Of Brachypodium Distachyon

Centromere is a special chromosomal structural region in eukaryote,which participates in assembling kinetochore proteins to ensure the right movement and separation of chromosomes.It has been thought to be the garbage sequence during the mitosis and meiosis formerly.However,recent studies have shown that centromere is not only involved in plant chromosome pairing,isolation and maintenance of chromosome structure stability,but also owns genes with transcriptional activity.Bd21,as a monocotyledonous model plant of gramineous,its genomic sequencing has been completed,and its research on tissue culture and genetic transformation has already been established relatively mature.Therefore,Bd21 was selected as research material,and its genetic transformation system was established and optimized,and the function of centromere regional gene was carried out.Combining the ChIP-Seq data and RNA-Seq data,the active Bd21 centromere genes were obtained through bio-informatic analysis.Reasonable target sequence was designed to construct CRISPR-Cas9 vectors based on the CDS sequence.As the genetic transformation system was constructed and optimized by Bd21mature seeds,the active genes of the centromere region of Bd21 were edited with CRISPR-Cas9 technology.The main findings of this topic were as follows:?1?The effect of different hormones and seed treatment methods on the induction and regeneration of Bd21 callus was studied.The results showed that 2,4-D combined with shear treatment of Bd21 seeds induced callus had the best effect.FPX was superior to KT in the regeneration of callus.It had the same survival rate of photosynthetic autotrophic rooting as the rooting culture.Photosynthetic autotrophic rooting saved more time.?2?In the process of optimizing Agrobacterium-mediated genetic transformation system of Bd21,the optimum conditions of genetic transformation were explored through gradient contrast experiments such as concentration and treatment of Agrobacterium,infection time,pH value of co-culture medium and callus age.The results showed that the highest infection efficiency was 94.5%when the OD₆₀₀ was 0.6,the 6weeks¹0 weeks callus were infected for 15 minutes,and cultured on co-culture medium containing AS and pH 5.8 for 2 days.The transformation efficiency of positive seedlings was 52.6%.?3?Bd21 callus was induced by cutting seeds,GA3 and ABA hormones and seeds with different storage time.The time of induce callus indicated that Bd21 had physiological after-ripening characteristics.The storage time need for completing physiological ripening of Bd21 seeds was similar to that of wheat seeds.It indicated that Bd21 and wheat had close genetic relationship on biological evolutionary branches.?4?The centromere genes with transcriptional activity were screened by combining ChIP-Seq data with RNA-Seq data.The validation of transcriptional centromere genes was carried out by qRT-PCR.The result showed that the region of centromere has transcriptional activity genes as the same as chromatin.The genes with transcriptional activity of centromere were specifically expressed in different tissues.?5?Seventy transformed seedlings were obtained,including one Bd6g06147,eight Bd2g29350,thirty-five Bd1g40980,seven Bd1g41115and nineteen pCambia1391 transformed seedlings.