Selection And Cultivation Of Maize Strains With Gene Editing Gene Cas9 Background Expression And Design And Preliminary Test Of High-Throughput Conversion AIDS For Maize Stem Tips

Gene editing technology refers to the technology that can change the target gene sequence directionally,so as to carry out the knockout and insertion of specific DNA fragments.Compared with ZFNs and TALENs technologies,CRISPR/Cas9 technology is a new generation of genome editing technology,with the advantages of simple operation,simple structure,high cutting efficiency,multiple target sites,etc.It is therefore rapidly accepted and applied in gene function studies and geneticimprovement of important traits.In this dissertation,a total of 235 independent transgenic maize lines carrieing the pSCZ3-Cas9expression vector were obtained..Through genetic analysis and real-time qPCR analysis,single-copy inserted lines were screened and the expression level of Cas9 gene was evaluated.Single-copy transgenic lines with high Cas9 expression level were selected as the background material or high-throughput gene editing.In order to establish an efficient maize transgenic system,this dissertation designed a set of auxiliary equipment for high-throughput maize stem tip transformation,and carried out a preliminary test.The main results of this dissertation are as follows:?1?Construction of gene editing expression vector in maizeUsing the plasmid pSCZ3 with 2x35S strong promoter and Bar gene screening marker provided by our laboratory as the skeleton,Cas9 gene was cloned from the rice gene editing vector pP1C.1 and inserted into the pSCZ3 vector skeleton by enzyme digestion and ligation to construct a binary expression vector pSCZ3-Cas9 suitable for mass screening of maize transgenes.?2?Generation and genetic analysis of maize transgenic linesThe successfully constructed pSCZ3-Cas9 vector was delivered to Beijing bomei xingao biotechnology company for large-scale genetic transformation,and a total of 235independent transgenic lines were obtained.For the transgenic T₀ strains delivered,After PCR verificationof T0 T₀ plants and T₁ lines,xx single-copy inserted Cas9 tansgenic lines were selected according to Mendel's law of heredity.?3?Expression analysis of Cas9 gene in transgenic linesCas9 gene expression analysis was carried out in 23 transgenic lines using RT-qPCR.The results showed that the expression level of Cas9 gene was significantly different in different lines,with 55 to 350 folds variation in high expression lines and low expression lines.Comparative analysis of Cas9 gene expression level in four T₁ lines showed that the expression level could be stably inherited.?4?Design and test of high-flux maize shoot tip transformation equipmentIn this dissertation,we designed a set of assisting equipment which can be used for high-throughput maize shoot tip transformation.By using this equipment,the operating efficiency for mazie transformation could be improved by 5-8 times.The equipment is also cost effective with goodpotential to be widely accepted.As plant receptors were grown in the soil throughout the experiment,and no aceptic environment is required,the transformation process does't need MS medium.Possible contamination during the current transformation process could be avoided,and the requirements of the transformation environment is reduced.During the operation,only the shoot tips of the plant will be damaged,which reduced the mechanical damage to the whole plant during the current operation systems.It is also,beneficial to the growth of the transformed plant.