The CRISPR/Cas9 System Application In Arabidopsis EIF2 Alpha Gene Silencing And Optimization In Maize

In recent years,with the rapid development of biotechnology,more and more species genome sequencing work is complete,one after another after gene function research as the main task of the post-genomic era,constantly move forward in the field at the same time,has been a rapid development of related technologies.Among them,the genome editing techniques that decide a dot,can provide gene function research knockout mutant materials,is an ideal gene function research methods.Genome fixed-point editing techniques include:Zinc finger nuclease(ZFNs),Transcription activator-like effector nuclease(TALENs)and CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats/CRISPR associated system 9).Zinc finger nuclease(ZFNs)is the first gene editing techniques be designated and developed,because ZFNs design and selection is very difficult,and often miss effect will cause the cells,these factors make its application is restricted.Later,gene editing techniques to develop a new technology,namely the transcriptional activation effect factor nucleic acid enzymes(TALENs),any gumming point of sequence can be obtained in theory,but the system assembly and screening needs a lot of sequencing,cost is higher.At present,the technology of the gene editing CRISPR/Cas9 fixed-point editor as a new genome technology gradually be applied to animals and plants gene knockout,gene operation efficiency is high,the method is simple,the system in greatly reduced off-target effects at the same time also can guarantee the efficient and specific locus of cutting.The above three methods lead to gene activate their own injury after double chain fracture repair mechanisms:homologous recombination repair and non homologous end connection repair,mediated gene sequence of insertions and deletions or replacement,cause genetic mutations.This research mainly through the CRISPR/Cas9 gene editing technology,gene in arabidopsis and maize for editing,and get the mutant gene,and then to study gene function.At the same time,build and optimize the CRISPR/Cas9 expression vector of corn,for the corn of genetically modified(gm)and further the study of gene function.In arabidopsis,this study used the CRISPR/Cas9 technology for the purpose gene eIF2 alpha family two members of fixed-point knockout.Two members of the family were designed in accordance with the principle of PAM specific gRNA(Guided RNA),sequence length of 20 bp,respectively,to identify at2g40290,at5g05470 two genes specific targeted point,we will both gRNA named 2 g Ⅱ-gRNA and 5 gⅢ-gRNA,.To two members of the family of gene sequence analysis,the results showed that there was a highly conservative homologous sequences of 20 bp length,conform to the 3 ’end NGG structure,with the sequence as a target gene identification sequence design gRNA,we named it gⅠ-gRNA,in order to achieve the same plant and knock out the purpose of the two genes in the body.But no C or G at 5’end,trial operation in the process of synthesis gRNA primers artificial added a bases at the 5’ end G,can guarantee the stability of gRNA.The synthetic gRNA primers with Cas9 carrier and 1300 carrier connection,in turn by 3 complete CRISPR/Cas9 system.Obtained by agrobacterium mediated infection inflorescence method into arabidopsis thaliana plants,use of antibiotics and genotype screening qualification to gⅠ-gRNA,2 g Ⅱ-gRNA,5 g Ⅲ-gRNA positive mutants.In maize,this study of CRISPR/Cas9 original carrier promoter and terminator for transgenic corn is optimized,ZmU3 promoter start gRNA expression,and Cas9 promoter and terminator by original pAtUBQ and tAtUBQ replaced pOsUBQ and OCS,in order to improve the expression in maize.Target genes to select HLH and BRD two leaf angle related genes,and for the design of gRNA and CRISPR/Cas9 carrier construction.In addition,in order to enable the carrier to fusion of multiple genes gRNA,we polyclonal site optimized transformation of carrier,added PmeI,PacI,SmaI,XmaI and SwaI five restriction enzymes for splice site recognition.Can effectively use two different specific to a CRISPR/Cas9 gRNA fusion system,provide gene editing efficiency,shortening the time of mutant screening.In addition,we also optimized the 35 S promoter and OCS terminator lead Cas9 protein translation,AtU6 start gRNA expression vector,is expected to be used for the transformation of wheat.