| Cucumber green mottle mosaic virus（CGMMV）belongs to the Tobamovirus of plant Virgaviridae and is mainly transmitted through seeds.It is one of the most important diseases that endanger the safety of watermelon production and also one of the most important viruses that infect Cucurbitd crops.In recent years,it has broken out many times in China,which not only poses a serious threat to the production of Cucurbitd crops,but also brings huge economic losses to human production.At present,there is no resistant cultivar and effective control method for the virus disease in field.As one of the means of RNA methylation,6-methyladenine（m6A）modification plays an important role in animal and plant defense against virus invasion.In this study,a yeast c DNA library was constructed using CGMMV watermelon pathogenic material in the early stage.A watermelon protein ClMTB interacting with MP was obtained by yeast two-hybrid screening using CGMMV motor protein（MP）as bait.It was found that it was a homologous protein of m6A methyltransferase METTL14/MTB,which contained the specific MT-A70domain of m6A methyltransferase,and its expression was inhibited during CGMMV infection.In this paper,the disease system of CGMMV-Benn’s smoke interaction is taken as the research object,and the following four aspects are carried out:（1）The interaction between CGMMV movement protein MP and watermelon ClMTB protein was verified by yeast two-hybridization and Bi FC.（2）The watermelon methyltransferase gene ClMTB was identified by bioinformatics methods from the aspects of protein structure,gene structure,conserved motif,evolutionary analysis and spatiotemporal expression pattern.（3）Through the construction of overexpression and knockout of ClMTB transgenic materials,the inoculation identification analysis and stress resistance analysis of the transgenic materials inoculated with CGMMV were carried out to compare the effects of ClMTB gene expression changes on the plants infected with CGMMV and the response to drought stress.（4）Transcriptome sequencing was performed on the inoculated CGMMV materials to analyze the virus induced by CGMMV infection and the target genes of abnormal modification of watermelon m6A,so as to clarify the mechanism of m6A modification mediated by ClMTB-MP interaction in CGMMV infection.the results of the study are as following:1)The yeast two-hybrid and Bi FC vector of ClMTB and MP were successfully constructed.ClMTB and MP proteins were detected for self-activation and toxicity,respectively.The self-activation test showed that MP protein had no self-activation ability,while ClMTB protein had self-activation ability.Finally,the yeast two-hybrid technique was used to conduct point-to-point verification of movement protein MP and ClMTB.Preliminary verification results showed that there was an interaction between movement protein MP and ClMTB.2)Through the identification,evolution and protein structure analysis of ClMTB gene,it was found that ClMTB gene contains five intron structures,and contains a conserved MT-A70 domain at its C-terminal.The MTB protein domain sequences from seven species,namely Arabidopsis thaliana,tomato,grape,cabbage,rice,maize and watermelon,were used for multi-sequence alignmations.The MT-A70domain of these species was quite conserved.Multiple sequence alignment and phylogenetic tree construction of these species revealed that the MT-A70 proteins in these species were divided into three subgroups.In addition,we extracted RNA from watermelon roots,stems,leaves,male flowers,female flowers and fruits to analyze ClMTB gene expression pattern of space and time in the different parts watermelon using q RT-PCR.The results found that ClMTB are expressed in the different parts of watermelon,but highest expression in female flowers,followed by the blade,and lowest expression in roots and fruits.3)The ClMTB overexpression and CRISPR/Cas9 knockout expression vectors were successfully constructed.In addition,ClMTB overexpression and CRISPR/Cas9 knockout expression vectors were successfully constructed to transform tobacco and watermelon,and transgenic tobacco lines and watermelon bud lines were obtained.At present,transgenic tobacco lines have received T1 generation seeds,which laid a solid foundation for later phenotypic observation and other related experiments.4)Transcriptome sequencing was performed on overexpressed tobacco materials after inoculation.The results showed that the expression of 840 genes in WT significantly changed after CGMMV infection,and they were mainly concentrated in the signaling pathways such as ribosome,photosynthesis,plant hormones,and plant-pathogen interaction.However,after OE-ClMTB was infected with CGMMV,the number of differentially expressed genes decreased.After the infection of WT and OE-ClMTB by CGMMV,some KEGG enrichment signal pathways are different,which is probably the main reason for the difference in their resistance to CGMMV.Compared with WT,after CGMMV infection of OE-ClMTB plants,the number of affected genes expressed in photosynthesis,ribosome,hormone signaling,autophagy,pathogen interaction and other signaling pathways decreased.In conclusion,overexpression of ClMTB gene resulted in a significant decrease in the number of disease-resistant genes responding to CGMMV infection,which also indicated that ClMTB was responsible for regulating CGMMV resistance in tobacco plants.In conclusion,this study studied the function and regulatory mechanism of watermelon ClMTB protein interacted with CGMMV movement protein,and selected over-expressed tobacco materials for functional verification.The function of watermelon ClMTB protein was preliminarily verified,and the test results showed that ClMTB negatively regulates tobacco CGMMV resistance. |
