Study On Screening Technology Of Transcription Factor Target Gene Mediated By Base Editing Technology

Transcription factors play an important role in the regulation of gene expression by binding to specific sequences on the DNA near its target gene to regulate transcription initiation to ensure the suitable and accurate expression of the gene at the appropriate time.Therefore,the functional analysis of transcription factors is an important link in the study of the growth and development of organisms and the interaction mechanism with the environment.Transcription factors are regulated by upstream genes and also have a certain influence on the expression of downstream genes.The regulatory network is complicated.At present,many screening methods for identification of transcription factor’s target genes have been developed,but some methods have some shortcomings such as fussy and low efficiency.If a rapid,efficient and accurate tool for screening transcription factor target genes can be explored,it will bring greater convenience to the research work of transcription factors.This paper focuses on exploring a transcription factor’s target gene screening technology mediated by gene editing technology.In this study,two new base editing enzymes(adenine base editing enzyme ABE7.10 and cytosine base editing enzyme BE3)were mainly used for the experimental study of transcription factor target genes.These two base editing enzymes are capable of efficiently and specifically altering one base on the gene of interest to another base in a programmable manner.Through these two base editing enzymes and the transcription factors used in this study,we constructed two base editing systems,correspondingly ABE7.10 base editing system(pOX-ABE7.10,pOX-ABE7.10-SPL9,pOX-ABE7.10-SPL14)and BE3 base editing system(pOX-BE3,pOX-BE3-SPL9,pOX-BE3-SPL14).Where pOX-ABE7.10 and pOX-BE3 were negative controls,pOX-ABE7.10-SPL14 and pOX-BE3-SPL14 were reference controls as the target genes of OsSPL14 is known.In addition,in this study we used two different expression systems to construct materials that screened for transcription factor(OsSPL9)and its target genes.One is the method of stable transformation.The transgenic lines of the above six plasmids were constructed by agrobacterium-mediated genetic transformation.The whole genome sequencing was performed by extracting the DNA of the leaf tissue,and the sequencing results were compared and analyzed.For screening of downstream target genes,the other method used was transient transformation.The same six plasmids were co-transformed into rice protoplasts with GFP plasmid,and the protoplasts were observed by laser confocal microscopy.The whole genome DNA was subjected to resequencing analysis,and the sequencing results were compared to the analysis of the target genes downstream of the transcription factor.The two expressions were mutually validated,increasing the reliability of the experiment.By analyzing the frequency of two base mutations in the genome(A·T to G·C and C·G to T·A),we revealed the editing incidence of the editing enzymes ABE7.10 and BE3,analyzed certain specificity,and also verified the credibility of the editing effects of these two editing enzymes.By comparing the analysis results of plant samples and protoplast samples of pOX-BE3-SPL14 with the target genes of reported in the literature,it was found that 19 genes in the plant sample of pOX-BE3-SPL14 were the reported target genes of OsSPL14 that can be found,and 74 genes in the protoplast sample pOX-BE3-SPL14 can be found in the reported target genes of OsSPL14.This result demonstrated that we can use the editing enzyme BE3 to screen transcription factors.This attempt to identify downstream target genes is feasible.By comparing the results of the plant samples(protoplast samples)pOX-ABE7.10-SPL9 with the results of the plant samples(protoplast samples)pOX-BE3-SPL9,we again confirmed that the results of the editing enzyme ABE7.10 screening were reliable.In this study,we screened 2714 possible OsSPL9 target genes by editing enzyme BE3,and screened 5400 possible OsSPL9 target genes by editing enzyme ABE7.10.The COG functional classification analysis found that most of the selected genes were ’replication,recombination and repair genes’,followed by ’general function prediction genes’,’transcription related genes’,’signal transduction mechanism genes’,etc.GO functional analysis found that most of these target genes act on rice metabolic process,followed by biological processes such as cellular metabolic process and biological regulation process.This series of results demonstrates that the technique for screening transcription factor target genes by editing enzymes is credible,but whether the screened genes are really downstream targets of OsSPL9 requires further validation.