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Background: Histone modification is one of the key epigenetic regulation mechanisms which plays a critical role in various biological processes.In Saccharomyces cerevisiae,the histones H3 and H4 gene are encoded by two copy,named HHT1-HHF1 and HHT2-HHF2.Specific sites of histone acetylation,methylation and other modifications have a important biological significance to eukaryotic DNA replication.Methods: We used the method of two-step substitution.First,we used gene knockout technology to replace HHT1-HHF1 gene on Chromosome Ⅱ with NatMX4 gene,then we knock out HHT2-HHF2 gene on ChromosomeⅩⅣ with the hhtS-HHFS mutant gene.Results: Histone H3K14A mutant strain in yeast BY4741 were obtained successfully.