Study On Foxtail Millet SiFMBP Gene Expression Regulation By Genome Editing Technology

Foxtail millet contains a variety of dietary fibers,polyphenols,antioxidants and other biologically active molecules.Peroxidase of foxtail millet bran(FMBP),a kind of bioactive molecule in the bran,which has good anti-colon cancer and anti-atherosclerosis effects.While the content of FMBP protein in foxtail millet bran is low.Therefore,increasing the content of FMBP protein in foxtail millet can not only promote the development of anti-colon cancer drugs,but also contribute to the reuse of bran resources.Genome editing tools based on CRISPR/Cas9 technology can not only modify the function of target genes,but also achieve precise regulation of the expression of target genes.There are two main approaches for the increase of target gene expression.One is to activate gene expression through an activator that targets the promoter of DNA,and the other is through the editing of the important components in the promoter region of the target gene.This study intends to increase the content of FMBP protein in foxtail millet through the above two ways.The main results are as follows:(1)Through the mutation of the upstream open reading frame(uORF)of the SiFMBP gene,the translation of the main coding frame of SiFMBP is promoted.First,through dual luciferase reporter system,we verified that editing the uORF of the SiFMBP gene can increase the expression of the downstream open reading frame.Therefore,two sgRNAs were designed for the uORF region of the SiFMBP gene,and a CRISPR/Cas9 knock-out vector contains sgRNA frame was constructed to transform the protoplasts for transient expression.The mutation efficiency was detacted up to 19.2%.Among them,small-scale(1-5bp)deletions accounted for 84%,2% were 29-80 bp insertions.According to the sequencing results,the inserted sequence was part of the knockout vector.Finally,by Agrobacterium-mediated transformation,we obtained 6 transgenic positive plants through screening and identification of mutants.In which,the mutation efficiency reached up to 100%,and one of them was a homozygous mutation.(2)Use CRISPR/dCas9-VP64 andCRISPR/dCas9-VPR site-specific activation systems to target the region near the transcription start site(TSS)of the millet SiFMBP gene to activate SiFMBP gene expression.First,we designed single-guide RNA(sgRNA)targeted the area around the TSS upstream of SiFMBP,and connected them to an expression vector containing the CRISPR/dCas9-VP64 andCRISPR/dCas9-VPR,respectively.In order to verify its activation effect,we transformed our constructions into protoplasts through PEG,then extract RNA for relative quantitative analysis.The results show that the VP64 activation system can increase the expression of SiFMBP by 3.5 times,and the VPR activation system can increase by 13.5 times.For the SiFMBP gene,the optimal activation target of the CRISPR/dCas9-VP64 system is located 170 bp upstream of the transcription start site,and the optimal activation target of the CRISPR/dCas9-VPR system is located 84 bp upstream of the transcription start site.The activation efficiency guadually decreased with the distance from the optimal target site.In short,we successfully applied the uORF editing andCRISPR activation system to the study of millet gene regulation,and realized robust activation of the SiFMBP gene.Our results provide a reference for future gene expression research in foxtail millet,and will contribute to the development of anti-colon cancer and anti-atherosclerotic drugs.