Functional Study Of MYB108 Transcription Factor In Arabidopsis Thaliana

The MYB transcription factors in Arabidopsis thaliana are divided into four categories according to the R repeat sequence,MYB108 belong to the R2R3-MYB transcription factor.The bos1 mutants studied in this paper was obtained T-DNA insertion,because it's particularly sensitive to Botrytis known as botrytis-susceptible1(bos1).The current study on bos1 mutant suggests that the sensitivity of Botrytis and the phenotype of cell death after mechanical injury are due to the deletion of BOS1 gene.T-DNA insertion in the 5'-UTR region of the MYB108 leads to gene high expression on bos1 mutants,which makes the conclusion that bos1 mutants are recessive functional deletion mutants doubtful.Because of the lack of exon insertion mutants to verify the function of MYB108 genes,we explored the following:1.We constructed CRISPR knockout vectors and used CRISPR knockout and transgenic techniques to obtain homozygous mutant seeds with successful knockout,named myb108-crispr(BOS1-Knockout).The phenotypic analysis of myb108-crispr mutants showed that myb108-crispr mutants showed no similar lesion with bos1 after being stained by botrytis.The phenotypic analysis of cell death after mechanical injury showed that myb108-crispr mutants did not appear cell death around injury sites after mechanical injury.Thus,myb108-crispr mutants are not equal to bos1 mutants,MYB108 are not involved in regulating Botrytis resistance and cell death.2.To explore whether the high expression of MYB108 gene in the bos1 mutant was the real cause of cell death after mechanical injury,we first constructed the overexpression vector of BOS1 gene.After mechanical injury,the spread range of p35S::BOS1 dead cells was not significantly different from that of the wild type,indicating that the 35 S promoter driven MYB108 could not cause cell death.We found the MAS promoter sequence on the T-DNA of the bos1 mutant by resequencing it.Activation of gene expression by MAS was induced by mechanical damage.We constructed the transgenic plant of p MAS::BOS1,and the results showed that the cell death phenomenon of the positive plant of p MAS::BOS1 after injury,indicating that the cell death of the bos1 mutant was caused by the MAS promoter.We knocked out MYB108 in bos1 using CRISPR-cas9,and the results showed that frameshift mutation of MYB108 in bos1 eliminated the cell death phenotype in bos1.These results suggest that the MAS promoter promotes the accumulation of MYB108 and initiates cell death.3.To explore the function of MYB108 genes,we selected Na Cl and ABA treatments according to the bioassay map of MYB108 genes on the website.The results showed that MYB108 single gene deletion had no effect on salt stress response.On the contrary,the tolerance of overexpressed plants to salt stress decreased.ABA treatment results showed MYB108 negative regulation ABA response pathway.Transcriptome sequencing results showed that BOS1 gene may regulate cell death through ABA pathway.In addition,MYB108 is also involved in the regulation of flowering period and seed fertility,the mechanism of which remains unclear.