DNA Interference In Komagataella Phaffii GS115 And Escherichia Coli

DNA interference（DNAi）is a phenomenon in which gene expression is specifically suppressed in organisms by one fragment of DNA homologous to the target gene.Studies of DNAi are very limited,and its molecular mechanism is still not clear.Therefore,the phenomenon of DNAi was explored in Komagataella phaffii GS115 and Escherichia coli to clarify the characteristics and mechanisms of DNAi,with the housekeeping genes of Actin in yeast and MreB in bacterium as the target genes.Five single-stranded DNA（ssDNA）fragments of 21 nt were designed according to the sequence of Actin,synthesized and transformed into yeast cells by electroporation.The mortality of transformed cells was significantly increased,indicating that the DNAi phenomenon exists in K.phaffii GS115.However,the introduction of ssDNAs of MreB,which is functionally similar to eukaryotic Actin,into E.coli did not cause any ill effect,indicating that there might be no DNAi action for the MreB gene in E.coli.After different types of nucleotide fragments were individually introduced into yeast,it was found that the double-stranded DNA（dsDNA）fragments had a significantly higher interference effect than did ssDNA and similar interference effects to those of dsRNA.There was no significant difference in the interference effect among dsDNAs with different CG%,but the dsDNA length positively correlated with interference effects.The number and distribution of mismatched bases in the dsDNA affected the interference efficiency.When the mismatched bases were consecutive in the dsDNA,the effect of DNAi was decreased as the number of mismatched bases was increased.The DNAi effect caused by dsDNA with nonconsecutive mismatches was weaker than that caused by dsDNA with the same number of consecutive mismatches.Transcriptomic sequencing analyses of yeast cells treated by ssDNA and dsDNA of Actin,dsDNA of EGFP and water revealed that the most significant effect was observed 3 h after treatment.Combined with the qPCR results,it was found that the expression of Actin in dsDNA-treated yeast cells was decreased 3 h after treatment but did not show significant differences compared to the other treatment groups.The expression level were significantly increased in dsDNA-treated yeast cells for RNase Ⅲ,one of the important elements of RNAi;viral defense factor PAS_chr1-3₀079;and the translation initiation factors;the expression of the transcription factors and ubiquitin-related genes was decreased.In summary,the phenomenon of DNAi was first demonstrated in yeast,and the relationship between the DNAi efficiency and the sequence type,GC%,length,or the number and distribution of mismatched bases of the DNA fragment was clarified,and genes involved in the DNAi process were identified.Our results indicate a new direction for future research on the DNAi phenomenon in other species and provide new ideas for exploring the mechanism of DNAi.