| Maize(Zea mays L)is one of the world’s most important crops.The development of genome sequencing technology reveals numerous information of maize gene,however the function of maize genes still requires deeper researches.Transcription factor is a class of proteins that regulates gene transcription and plays an important role in plant growth and development.Mutants are the basic materials for gene function research.CRISPR/Cas9 system can produce gene mutations with high efficiency.The generation of large-scale transcription factor mutants at the whole-genome level is of great value for both study of functional genomics and genetic improvement of maize.Based on the previous researches,this study created a sgRNA database by selecting the sgRNA at the whole genome level of maize,and selected the genes of different transcription factor families as target genes to construct a mutant library of maize transcription factor family.The main results are as follows:1.At the maize genome-wide level,all potential sgRNA from the genomic,exon,CDS,c DNA,and gene datasets were reproducibly and specifically screened using the PERL program to obtain specificity driven by the U6 or U3 promoter.The sgRNA database is divided into three categories: class0,class1,and class3.The library is predicted by sgRNA targeting gene distribution,and class0 is the key research object.2.Based on the existing RNA polymerase class III promoter,151 maize-targeted transcription factors were selected from class0 of the sgRNA database,and brief information was recorded.Through literature search,summarizing the results of previous studies,select 13 of these genes,as the focus of follow-up transcription factor function research;3.The target fragment was added based on CPB-Cas9 vector,and a total of 151 functional deletion vectors targeting maize transcription factors were successfully constructed.The maize inbred line KN5585 was used as a transforming receptor,and 13 of them were stably inherited by Agrobacterium-mediated transformation.Eighty T0 positive plants were obtained by Bar speed test paper.After identification of mutant types.As a result37 strains contained 13 mutant vector types,37 strains were analyzed for gene mutation types,and the mutation types were recorded.The results showed that one plant can insert multiple vectors at the same time,and the mutation is mainly caused by base deletion and insertion cause.The corn kernels were identified by fluorescence,and the positive seeds were found to be red by the fluorescent light source;... |
