Clone And Functional Analysis Of ECT7 In Arabidopsis

N~6-methyladenosine(m~6A)is widely present in the RNA among all higher eukaryotes.m~6A modification regulates various RNA metabolism steps,including RNA transcription,translation and degradation.The realization of multiple biological functions of m~6A requires"m~6A reader"that specifically recognizes m~6A in various tissues and stages.ECT(Evolutionary Conserved C-Terminal Region)proteins,which has 11members,were reported as the m~6A reader proteins in Arabidopsis.In animals,m~6A-binding proteins have been found to play important roles in multiple developmental processes.However,only few studies of ECT family gene have been reported in plants.The function and molecular mechanism of plant ECT protein are still unclear.In this thesis,ECT7 gene was cloned and functional analyzed,especially the role in the response to low phosphate condition.The T-DNA insertion mutant ect7-4 showed the inhibit primary root under low phosphate condition,while it showed the normal phenotype on the normal phosphate medium.The full-length ECT7 gene was cloned into an over-expression vector that driven by the 35S promoter and fused with YFP tag,ECT7-YFP.Then ECT7-YFP was transformed into Col-0 and ect7-4mutant plants.ECT7 localized in both nucleus and cytosol.The over-expression of ECT7 protein repressed the ect7-4 phenotype under low phosphate.ECT7 plays a negative role in the primary root elongation under low phosphate condition.In order to further analyze the molecular mechanism of ECT7 in plant growth and development.We performed immunoprecipitation followed by mass spectrometry(IP-MS)analysis,and identified 34 interacting proteins of ECT7.Six RNA-binding proteins were found as ECT7 interacting proteins,including its homolog ECT8(EVOLUTIONARILY CONSERVED C-TERMINAL REGION 8)?APUM3(PUMILIO 3)?ATRH53(PUTATIVE MITOCHONDRIAL RNA HELICASE 2)?AT5G61970?CID11(CTC-INTERACTING DOMAIN 11)and AT2G45810,which may be involved in the m~6A recognizing or binding.In addition,we identified four phosphorylation modification sites and 3 O-linked?-N-acetyl glucosamine(O-Glc NAc)modification sites on ECT7.Using yeast two-hybrid,ECT7 were confirmed to physically interact with both two O-Glc NAc glycosyltransferase(OGT)protein homologs in Arabidopsis,SPY(SPINDLY)and SEC(SECRET AGENT).Furthermore,the ECT7 protein was stabilized by co-expression of SEC protein.In summary,ECT7 participates in the low phosphate response,by regulating the primary root elongation.This thesis lays a foundation for further study the regulate mechanism of m~6A modification in Arabidopsis roots elongation and regulating roles under low phosphate condition.