| Forest carbon and nitrogen cycle,tree nutrition,and ecological adaptation are inseparable from soil microbial activities.Among them,mycorrhizal fungi can promote the growth of trees and form a"World Wide Web"of communication between trees through a huge network of hyphae.Therefore,an in-depth study on the formation and development of ectomycorrhiza is an important basic scientific issue to be explored.It is generally believed that mycorrhizal fungi can obtain carbon source through plant hosts while converting the nutrients（especially nitrogen and phosphorus）difficult for plants to absorb in the soil into those absorbable and supplying them to plants.However,the molecular mechanism of nitrogen transport remains unclear by ectomycorrhizal fungi to plant hosts.In this study,the ectomycorrhizal fungus-Laccaria bicolor was selected as the research object,and four key enzymes in the nitrogen metabolism pathway of fungi were selected:nitrate reductase（LbNR）,glutamine synthase（LbGS）,glutamate synthase（LbGAT）,and glutamate dehydrogenase（LbGDH）.Used Agrobacterium-mediated transformation means and RNA interference（RNAi）technology to obtain fungal transformants were screened.Subsequently,the fungal transformants were cultured on different nitrogen sources to observe the mycelial growth and branching of fungal transformants.On this basis,fungal transformants and poplar seedlings were co-cultured,and the mycorrhizal formation rate and mycorrhizal morphology were statistically analyzed.Finally,the effects of nitrogen metabolism-related genes in L.bicolor on fungal-plant mycorrhizal symbionts were elaborated.The following results are obtained in this study:1.RNAi transformants of ectomycorrhizal fungi with 80%interference efficiency were successfully screened,including nitrate reductase（LbNR）,glutamine synthetase（LbGS）,glutamate synthetase（LbGAT）and glutamate dehydrogenase（LbGDH）;2.It was found that under the same culture conditions,compared with wild-type and empty vector fungi,the RNAi transformants of key genes LbGS and LbNR in the process of fungal nitrogen metabolism had smaller biomass and slower growth.The mycelia of fungi growing on the medium with NO3-as the sole nitrogen source were thin,the mycelia on the edge of liquid culture biosphere were sparse,and the mycelia growing state with NH4NO3 was the best nitrogen source;3.It was found that the ectomycorrhizal formation was significantly reduced by LbGS RNAi and LbNR RNAi fungal transformants of L.bicolor.The results of cross section showed that the development of bacterial sheath and hastellar network formed by the fungal transformants LbGS RNAi and LbNR RNAi were poor,and the fungal transformants LbGS RNAi and LbNR RNAi affected the formation of ectomycorrhizal and hastellar network.In summary,in this study,the function of genes involved in nitrogen metabolism pathway in mycorrhizal fungi was studied,and the effects of key genes in fungal nitrogen metabolism on the formation of symbiotic plants and mycorrhizal fungi in mycorrhizal fungi-plant symbiosis were expounded.This study is helpful to understand the metabolic pathways of nitrogen transported by soil microorganisms to forest host,thus providing a theoretical basis for the study of forest ecological carbon and nitrogen cycle,and providing a theoretical basis for the use of microbial agents to improve forest breeding,forest protection and ecological environment restoration. |
