Analysis Of SlYTH Gene Family And Biological Function Of SlYTH1 Gene In Tomato

N6-methyladenosine（m⁶A）is the most abundant and common modification on eukaryotic m RNA.m⁶A plays a key regulatory role in a variety of life processes by regulating m RNA stability,translation efficiency,and pre-m RNA splicing.The m RNA m⁶A reader can specifically recognize and bind to the m⁶A site of m RNA,mediate m⁶A to regulate m RNA,and thereby determine the fate of the target m RNA modified by m⁶A.Therefore,the study of readers is of great significance to reveal the molecular regulation mechanism of m RNA m⁶A.The currently confirmed reader is the YTH protein containing the conserved domain of YTH（YT521B homology）.However,so far there are relatively few studies on YTH protein in plants,and the understanding of its function is still lacking.As an important model plant for studying the regulation of fruit development and ripening,the study of YTH protein in tomato has not been reported yet.This study systematically analyzed the SlYTH gene family in tomato through bioinformatics technology,clarified the expression characteristics of each gene.Furthermore,SlYTH1 was knocked out by gene editing technology,and the biological function of SlYTH1 was identified.The main findings are as follows:（1）Nine SlYTH genes were obtained from the tomato genome database by using homologous comparison technology;PCR amplification and sequencing verification found that the true sequences of SlYTH1 and SlYTH6 were 45 bp more and 4 bp less than their predicted sequences,respectively.It can still encode a complete YTH protein;Phylogenetic tree analysis shows that the 9 members of the SlYTH gene family can be divided into two subfamilies,YTHDF and YTHDC.（2）The results of bioinformatics analysis showed that the gene structure of SlYTH family members is quite different,segmental replication is the main evolutionary mode;SlYTH protein has highly conserved domains and similar spatial structure;SlYTH family genes’promoter contains multiple hormone response elements and abiotic stress response elements.（3）The analysis of the expression pattern of SlYTH family genes showed that in different tissues of tomato,the SlYTH gene is highly expressed in flowers and fruits;In different parts of the flower development period,five SlYTH genes showed similar dynamic expression patterns,and have the highest expression in the stamens of 4 dpa.The dynamic expression patterns of the remaining four SlYTH genes are different;During the development and maturation of the fruit,the SlYTH family genes show different dynamic expression patterns.Most of the SlYTH genes have the highest expression level during the development period,and have the lower expression level during the fruit ripening period.Treatment with exogenous hormones found that most of the SlYTH family genes respond to the hormones contained on their promoters.（4）Knock out the SlYTH1 in tomato by CRISPR/Cas9 gene editing technology to obtain homozygous mutant plants.Phenotypic analysis found that knocking out the SlYTH1 gene would affect seed germination,delay plant growth and development,and make the fruit appear long and flat.（5）Phytohormone content determination found that the GA₃ content of the SlYTH1knockout plant was lower than that of the wild type;The q RT PCR test found that the expression level of related genes in the GAs synthesis pathway was changed,and the exogenous GA₃ treatment could partially restore the phenotypic defect of the SlYTH1knockout line.It indicates that the SlYTH1 gene may affect the biosynthesis of GAs,reduce the content of GA₃,delay plant growth and development.In addition,the results of quantitative PCR showed that the expression level of fruit shape-related genes in the fruit of the SlYTH1 knockout plants changed,indicating that the knockout of the SlYTH1 gene would affect the shape of the fruit.（6）Transcriptome sequencing analysis showed that there are large number of differential expressions of genes between SlYTH1 knockout plants and wild-type plants;Through GO and KEGG enrichment analysis,it is found that most of the differentially expressed genes are enriched in pathways such as carbon metabolism.It shows that SlYTH1 may play an important role in these pathways.The main conclusions of this study are as follows:There are 9 SlYTH genes in tomato,which can be divided into YTHDF/DC subfamily,the YTH domain is highly conserved,the protein structure is highly similar,and the promoter contains a variety of hormone response elements and abiotic stress elements.The SlYTH family genes have different expression patterns in different tissues and stages in tomato.The SlYTH1 gene may reduce the gibberellin content by affecting the gibberellin synthesis pathway,thereby delaying the growth and development of plants.In addition,knocking out the tomato SlYTH1 gene will also change the shape of the fruit.