The Role Of Citrus Neutral Ceramidase Gene NCER2 In Arbuscular Mycorrhizal Symbiosis

Root hair of citrus is sparse,mainly relying on the formation of arbuscular mycorrhizal symbiosis to absorb water and mineral nutrients.The arbuscular mycorrhizal symbiosis can also improve the resistance of citrus under biotic and abiotic stresses.Therefore,it is of great significance to study the molecular mechanism of arbuscular mycorrhizal symbiotic formation of citrus.In the early transcriptome study,the gene CsNCER2 from Poncirus trifoliata which is the common rootstock for citrus,was significantly up-regulated after inoculated with arbuscular mycorrhizal fungi,indicating that the gene may be involved in arbuscular mycorrhizal symbiosis.The aim of this study is to investigate the expression and function of the CsNCER2 gene and its Medicago homologous gene MtNCER2 during arbuscular mycorrhizal symbiosis through bioinformatics,promoter-GUS,RNAi and other techniques,to lay the foundation for the next step of citrus mycorrhiza function research.The research results of the thesis are as follows:1.Phylogenetic analysis showed that CsNCER2 gene and other homologous genes of arbuscular mycorrhizal plant species such as Medicago were gathered in the same clade,and there were no non-mycorrhizal plant species on this clade.It indicated that the special clade for arbuscular mycorrhizal plants may be related to mycorrhizal symbiosis.The alignment of NCER amino acid sequence from different plant species indicated that the hexapeptide functional domain of GDVSPN of NCER in plant was evolutionally conserved.However,the serine site of this conserved domain has changed in some of the NCER genes in the specific clade for arbuscular mycorrhizal plant.We speculated that the change of conserved site may affect the function of NCER,which was distinguished fromthe genes of non-specific branch,and thus may play a role in arbuscular mycorrhizal symbiosis.2.Analysis of pre-laboratory transcriptome data showed that the expression level of CsNCER2 gene was significantly up-regulated after inoculated with arbuscular mycorrhizal fungi,while the other two homologous genes expression level did not change significantly.And qRT-PCR result indicated that the homologous gene MtNCER2 in Medicago was also significantly up-regulated after the inoculation with arbuscular mycorrhizal fungi.In this study,the promoter-GUS expression analysis of MtNCER2 was performed using the established transient transformation system.The result showed that the GUS activity was not expressed when arbuscular mycorrhizal fungi were not inoculated.After inoculated with arbuscular mycorrhizal fungi,GUS activity was detected in the root cortical cells containing arbuscules and was tissue-specific.3.In this study,the RNAi functional analysis of MtNCER2 was performed using the established transient transformation system.The qRT-PCR assay showed that the expression of MtNCER2 in the interfering seedlings was decreased by about 75%compared with the control group,and the marker genes MtPT4,MtEXO70 I and RiEF were significantly down-regulated,which were related to mycorrhizal infection and arbuscular formation and development.These suggested that the RNAi efficiency of MtNCER2 was high,and the interference could affect hyphae growth and arbuscule formation.Further observations and statistics on symbiosis indicated that the interference of MtNCER2 gene resulted in a decrease in the infection rate(F%),but did not affect the growth of hyphae in the roots and the development of arbuscule morphology.We hypothesized that MtNCER2 function in the early stage of symbiosis formation and participates in the attachment or invasion of extracellular hyphae on the root surface.