Screening And Identification The Expression Of Salt-related RNA M~6A Modifying Enzymes In Sugar Beet

Soil salinization has become a global problem.In China,saline-alkali land and sugar beet production area coincide highly,which puts forward higher requirements for sugar beet salt tolerance.As a major sugar-producing crop,salt tolerance of sugar beet has always been the focus of agricultural research.m~6A modification has become a focus of transcriptome research in recent years.It has been proved to be involved in the differentiation,growth and development of plant cells and response to abiotic stress.It includes three modifying enzymes:methyltransferase,demethylase and binding protein.At present,studies on plant m~6A modification are focused on Arabidopsis thaliana,the model organism,and studies on sugar beet have not been carried out.Therefore,it is of great significance to explore the function of RNA methylation modification in the salt stress response process of sugar beet for the breeding of new salt-tolerant varieties.In this paper,bioinformatics method and molecular biology method were combined to screen and identify the sugar beet m~6A modification enzyme gene and analyze the expression level.The specific results are as follows:According to the amino acid sequences of five m~6A methyltransferases in Arabidopsis,the corresponding five homologous proteins were obtained from Blastp in beet genome-wide database.Domain identification showed that they all contained the conserved domain of the corresponding family,two proteins belonged to the same family,and the gene structure(exon/intron distribution)was similar.Subcellular localization showed that all of them were located in the nucleus,which was consistent with Arabidopsis methyltransferase localization.Arabidopsis m~6A demethylases include ALKBH9B/10B and belong to the ALKBfamily.According to the common conserved domain of 14 ALKB family proteins in Arabidopsis,10 ALKB proteins in sugar beet were screened out.10 sugar beet proteins were used as candidate m~6A demethylase for biochemistry analysis.The phylogenetic tree analysis of 24 ALKB proteins was carried out.All the proteins were divided into 5 groups.The exon/intron number,distribution and mode distribution of the same proteins were similar.Among them,there were three homologous genes with ALKBH9B/10B:njrf,eskg and uxaj,which might be potential demethylases in sugar beet.The subcellular localization of the proteins predicted that most of the proteins were located in the nucleus,while a few proteins were located in the cytoplasm and extracellular,which might be related to the different roles played in the process of transcription regulation.Arabidopsis m~6A binding proteins include ECT2-4 and CPSF30,both belonging to the YTH family.According to the conserved domain shared by YTH family,6 YTH proteins were screened out from the whole genome of beet.The mean isoelectric point was 6.72.Protein was predicted to be nuclear localization and might be involved in the process of RNA nuclear modification.The phylogenetic tree analysis showed that tirq(homologous ECT2-4)and mced(homologous CPSF30)were potential m~6A binding proteins in sugar beet.The sugar beet salt-tolerant strain"O"68"was treated with 300 m M sodium chloride solution.The expression levels of 21 modified enzyme genes in sugar beet were detected at different time(0.5,1,2,3,6,12,24 h).Compared with the control,with the increase of salt treatment time,beet leaves gradually wilted in 0-6 h and then recovered gradually,and no significant changes were observed in root system,indicating that beet had a response to salt stress.The expression levels of all the genes were significantly changed under salt treatment,most of which were significantly changed at 1 h and 24 h.4 potential m~6A modifiers eskg,uxaj,tirq and mced were significantly upregulated at 6 h,which suggested that they played an important role in salt stress response.The genes encoding methyltransferase and demethylase were initially down-regulated,then up-regulated and down-regulated,while the binding protein genes were generally up-regulated.Genes arcy(encoding methyltransferase),swwm and uxaj(encoding demetylase)and mced(encoding binding protein)with high expression and obvious expression differences were selected for cloning and submitted to Gen Bank,and the access numbers were obtained as follows:MZ358115,MZ385116,MZ358117 and MZ385118.By double enzyme digestion,it was linked to the expression vector p CAMBIA-4×Myc-MCS-3×FLAG and the silencing vector p TRV 2,and transformed into Agrobacterium.The engineered bacterium can be used for subsequent functional verification experiments.