| There are more than 150 chemical modifications on RNA.N6-Methyladenosine(m6A)is currently the most common and abundant chemical modification.In mRNA,m6A is mainly distributed in stop codons and 3’-UTR attachments,and there is also a small amount of enrichment at 5’-UTR.There are two main types of m6A distribution motifs:RRACH(R=G/A,H=U/A/C,and U>A>C)and URUAY(R=A/G,Y=C or U).The former is highly conserved in animals and plants,and the latter is highly conserved only in plants.The m6A modification is involved in regulating biological processes such as Pre-mRNA processing,alternative splicing,nuclear export,mRNA stability and translation.It plays a vital role in plant growth,leaf morphogenesis,and epidermal hair morphology.m6A modification is a dynamic and reversible regulation process.The relevant enzymes of m6A mainly include:m6A methyltransferase(Writer),m6A recognition enzyme(Reader)and m6A demethylase(Eraser).In Arabidopsis thaliana,m6A methyltransferase MTA,MTB,and FIP37 form a complex together that mediates the establishment of m6A modification.The m6A recognition enzymes are mainly represented by the ECT family,in which ECT2,ECT3 and ECT4 jointly regulate the regulation of epidermal hair and leaf morphology of Arabidopsis.m6A demethylase is a kind of protein family containing ALKB domain.The m6A demethylase family in Arabidops is contains five gene members:ALKBH9A,ALKBH9B,ALKBH9C,ALKBH10A and ALKBH10B.ALKBH9B is mainly involved in the virus defense of Arabidopsis,and ALKBH10B is involved in the regulation of Arabidopsis flower morphogenesis.Although ALKBH9B and ALKBH10B have been reported,we still do not know much about the mechanism of action of m6A demethylase in plants.In order to further explore the function of m6A demethylase in Arabidopsis and rice,we performed homologous gene search for m6A demethylase in Arabidopsis and rice at the whole genome level.Identify homozygous mutants based on the found Arabidopsis and rice m6A demethylase genes.In Arabidopsis thaliana.we isolated and identified T-DNA insertion hoimozygous mutants of the m6A demethylase gene family,and constructed mutli-gene mutants.We observed the growth of Arabidopsis single mutants and multiple mutants during the vegetative growth stage,and found that there was no obvious difference in traits between the mutants and the wild type.By analyzing the expression pattern of m6A demethylase in Arabidopsis thaliana,we found AT1G14710 gene is expressed in the whole plant during the vegetative growth period of Arabidopsis,and expressed at both ends of the flower and pod during the reproductive growth period.AT2G48080 gene,is expressed in roots,shoot meristems and anthers.AT4G36090,is only expressed in anthers.We did not find AT1G48980 expression.Dot-blot was performed on the mutants with deletion of the AT1G14710 gene,and we found the deletion of the gene resulted in an increase in the level of m6A modification of RINA,indicating that AT1G14710 is involved in regulating the accumulation level of m6A modification.In rice,we used CRISPR-Cas9 technology to create mutants of the m6A demethylase gene.After testing at the DNA level and transcriptome level,a total of 3 homozygous materials were identified.Observing the growth phenotype of the rice m6A demethylase CRISPR homozygous mutants,it was found that compared with Nipponbare,the phenotype of the mutant showed no obvious phenotypic changes.By Dot-blot detection,it was found that the m6A level of m6A demethylase gene LOC_Os05g33310,LOC_Os03g13560 and LOC_Os10g02760 mutant mRNA in rice was not significantly different from that of control Nipponbare.In summary,this study identified and constructed mutants of the Arabidopsis and rice m6A demethylase genes,and preliminary analysis of the expression patterns and functions of some genes.The AT1G14710 gene was found to be involved in regulating the accumulation level of m6A modification in Arabidopsis.This study provides a theoretical reference for the subsequent functional research of m6A demethylase in plants. |
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