Cloning Of M6A Methyl-modifying Enzyme Genes And Their Effects On Viral Infection In Potato And Tomato

m6A methylation refers to the modification of the 6th nitrogen atom(N)of Adenine,which has become one of the research hotspots in the field of life science.Some studies have shown that m6 A methylation plays a regulatory role in the replication of human and plant viruses.The previous study found that the host endogenous gene m6 A methylated significantly changed after the potato Y virus(PVY)infected the tobacco.In this thesis,the m6 A methylase homologous genes of potato and tomato were cloned and identified,and their characteristics and conserved domains were analyzed.The effect of m6 A methylase homologous gene on infection of PVY virus and other potato viruses was studied by Agrobacterium-mediated transient expression.The main research results are as follows:1.By searching the m6 A methylase sequence in the NCBI gene bank and designing specific primers,the m6 A methylase gene homology was cloned from the leaf tissue of potato variety Huangxin 2R6 and named PO＿MTA70.The homologous gene of tomato m6 A methylase,named TO＿MTA70,was cloned after RNA was extracted from the leaf tissue of Tomato Herb No.4 and reverse-transcribed into c DNA.The full length of PO＿MTA70 and TO＿MTA70 genes is 2720 nt,including a 2217 nt coding region,encoding a protein containing 738 amino acids.The molecular weight of PO＿MTA70 is 81.4 k D and the isoelectric point is 6.37.The molecular weight of TO＿MTA70 protein is 81.38 k D and the isoelectric point is 6.37.The amino acid sequences of PO＿MTA70 and TO＿MTA70 contain several highly hydrophilic regions,which are presumed to be hydrophilic proteins.The online analysis tool(Novo Pro)was used to analyze the secondary structure of methylase and construct the tertiary structure model.The results further verified that PO＿MTA70 and TO＿MTA70 contain m6 A methylase gene conserved domains.2.The obtained methylase genes PO＿MTA70 and TO＿MTA70 were cloned into the plant expression vector p MDC32 through the gateway recombinant system,and the potato and tomato m6 A methylase gene expression vectors p MDC-PO＿MTA70 and p MDC-TO＿MTA70 were constructed successfully.Expression vectors p MDC-PO＿MTA70 and p MDC-TO＿MTA70 were transformed into Agrobacterium C58C1 and injected into Ben’s tobacco.The effect of m6 A methylase on virus infection was studied by friction inoculation of PVY virus after 3 days.The results showed that the accumulation of PVY virus in p MDC-PO＿MTA70 and p MDCTO＿MTA70 plants was significantly lower than that in the control group.These results indicated that potato PO＿MTA70 and tomato TO＿MTA70 both had certain inhibitory effects on PVY virus infection.3.To further study the effects of potato and tomato m6 A methylase on the other two potato Y viruses,turnip Mosaic virus(Tu MV)and tobacco pulse Mottle virus(TVMV).The expression vectors p MDC-PO＿MTA70 and p MDC-TO＿MTA70 were mixed with the infectious cloning vector of Tu MV-GFP,respectively,and then injected into Ben’s tobacco.5 days later,virus infection symptoms were observed and virus accumulation was detected.Results There was no significant difference in TVMV accumulation between p MDC-PO＿MTA70 and p MDCTO＿MTA70 co-injected with TVMV and control.Tu MV virus accumulation was significantly increased in p MDC-PO＿MTA70 and p MDC-TO＿MTA70 plants co-injected with Tu MV.These results indicated that potato PO＿MTA70 and tomato TO＿MTA70 both promoted Tu MV virus infection to a certain extent,but had no significant effect on TVMV virus infection.