| Vaccinium is one of the most popular emerging fruits.The distinctive feature of its fruit relative to many other commercially-available fruits is that it contains a wide range of compounds demonstrated to have health-protective properties,such as anthocyanins and flavonoids.Vaccinium belongs to the family Ericaceae.It doesn't have large main roots,its roots are fibrous and underdeveloped,and it has poor ability to absorb water and nutrients in the soil.However,in the natural state,basically all the Vaccinium can coexist with mycorrhizal fungi,forming ericoid mcorrhiza(ERM),and ERM can enhance the nutrient absorption and stress resistance of Vaccinium.The formation of mycorrhiza is a complex dynamic process.In this study,the dynamic changes,colonization characteristic and microscopic state of the roots of Vaccinium infected by mycorrhizal fungi were analyzed,and the expression of miRNAs in Vacciniu,rm was analyzed after the invasion of mycorrhizal fungi.The results of this study are as follows:The stem segments of Vaccinium uliginosun were used as experimental materials,and the Vaccinium seedlings were obtained by tissue culture.The trypsin blue staining method was used to determine the infection rate of mycorrhizal fungi in the roots.The infection rates of the inoculated mycorrhizal fungi after two weeks and three weeks were 28%and 72%,respectively.The results showed that the mycorrhizal fungi of Vaccinium could be initially colonized for two weeks.The infection rate of mycorrhizal fungi for young root,mature root,and old root,of Vaccinium was 37%,72%and 100%,respectively.The sterile tissue culture seedlings of Vaccinium were used as materials to inoculate mycorrhizal fungi.The colonization of fungi in Vaccinium roots was examined by scanning electron microscope.The results indicated that the mycorrhizal fungi formed a typical mycelium structure,and the hyphae showed a honeycomb structure.After inoculation of mycorrhizal fungi for one week,two weeks and tliree weeks,in situ hybridization was used to detect the eolonization of mycorrhizal fungi in hair roots.The results showed that mycorrhizal fungi mainly infect epidermal cells and cortical cells.The stem-loop primers of 52 miRNAs were designed,and the miRNA was detected by RT-PCR,and then the expression level was detected by real time PCR.The results demonstrated that the expression levels of miR157a-5p,miR535d and miR7726a-3p were significantly increased after inoculation with mycorrhizal fungi for three weeks.The tissue localization analysis of the three miRNAs by in situ hybridization showed that three miRNAs didn't show any positive signal after one week of inoculation and the control group,while after two weeks of inoculation a few positive signals were observed;the positive signal is mainly located near the epidermal cells.By compared the results between real time PCR and in situ hybridization,it concluded that miR157a-5p,miR5335d and miR7726a-3p were miRNAs related to symbiotic.Protoplasts of Vaccinium hair root cells were prepared,the protoplasts originating from uninoculated hair root cells,inoculated hair root cells containing mycelium,and inoculated hair root cells without mycelium,were obtained,respectively.After inoculated three weeks,the expression of miRNA were detected by real time PCR.The results showed that the expression levels of miR157a-5p,miR5335d and miR7726a-3p were higher in the protoplasts containing mycelium than protoplasts without mycelium.One week,two weeks and three weeks after inoculation of mycorrhizal fungi,the protoplasts of Vaccinium hair root cells were prepared.The expression of miRNA was analyzed by real time PCR,and the results showed that the trend of miR157a-5p,miR5335d,miR7726a-3p expression was consistent with the trend of hairy root tissue. |
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