Screening Of Symbiotic Genes Of Arbuscular Mycorrhizal Fungi And Amorpha Fruticosa By Suppression Subtractive Hybridization

Arbuscular mycorrhizal fungi (AMF) are widely distributed and can form mutualism with terrestrial plants. Through two-way transfer of resources, the partners overcome all kinds of biotic (pathogens and herbivores) and abiotic (e.g., drought) stresses. The symbiosis between AMF and plants is a fine molecular regulation process, from pre-symbiotic signaling dialogue to plant stress defense and cell morphological remodeling in symbiosis stage, every step involve many biological molecular. Different from traditional mode plants, Lotus japonicus and Medicago sativa which are herbs, in this study, Amorpha fruticosa as woody leguminosae plant who may harbors slightly different symbiosis mechanism was selected as the host plant, at transcriptional level to investigate the molecular mechanisms underlay the symbiosis between AMF and A. fruticosa.Plants of A. fruticosa inoculated with Glomus mosseae (syn Funneliformis mossea) or without were pot cultured, a recently developed simple, low cost, low toxicity acetate ink staining method was used for detecting the colonization status of A. fruticosa root system by G mosseae. It was found that the staining effects on A. fruticosa roots of this method was no less than the classic acid fuchsin staining method, arbuscular structure could be observed under microscope at magnitude of 400 fold. Using Phillip and Hayman staining method, fungal vesicles in roots were found to develop into endogenesis spores which clearly stained.Afterward, suppression subtractive hybridization (SSH) method was employed to screen the differential expressed genes in A. fruticosa roots induced by AMF during the colonization process. Totally,47 unigenes were obtained,35 of which could be aligned with known function genes in GenBank through Blast. According to GO classified information,Blast annotation and related literatures, the 35 unigenes were assigned into eight categories including stress and defense (24%), metabolism (17%), energy (14%), protein synthesis (11%), signaling (11%), cellular structre/ cytoskeleton (11%), transcription related (6%), protein folding and degradation related (6%). Among them, stress and defense occupied the largest fraction, implying stress and defense related genes and metabolism related genes play an important role in the symbiosis process between A. fruticosa and AMF. Meanwhile, the eleven unknown sequences were predicted regard to their encoding primary amino acid sequence, physical and chemical properties and secondary structure of the predicted peptides. The majority of these unknown sequence encoding peptides displayed unstable under in vive condition, subalkalic, contained different percents of alpha helix, β-pleated sheet, turn and coil. Some of them might locate in cellular membrane, while many might function in cytoplasm.The colonization of AMF on A. fruticosa is a system activity involving many biological processes, all aspects of the body function unit work together to realize the final goal of mycorrhiza formation between AMF and A. fruticosa, facilitating the exchange of nutrition.