Preliminary Functional Analysis Of ENT3 And PUP3 In The Course Of Symbiosis Between Plant And Piriformospora Indica

Our research team found that the gene CyENT3 and CyPUP3 were specifically induced by mycorrhizal fungi.CyENT3 can effectively transport nucleosides,while CyPUP3 may can transport purine bases or its derivatives by crossing membrane.Piriformospora indica,a beneficial endophytic fungus,have a wide range of host.It can colonize in the Cruciferae and interact with Arabidopsis which without mycorrhiza.We found that Piriformospora indica produced auxin and cytokinin during the colonization,which promoted root growth and differentiated multiple lateral roots.Piriformospora indica can form typical mycorrhiza with Cymbidium.It can also induce the expression of CyENT3 and CyPUP3 genes.So,what is the expression pattern of the AtENT3 and AtPUP3 induced by the Piriformospora indica,and will AtENT3 and AtPUP3 affects the invasion of the Piriformospora indica in the Arabidopsis root?In order to further explore the functions of CyENT3 and CyPUP3,and their important functions in the formation of mycorrhizal and regulation of symbiosis,this paper mainly carried out the following there aspects: The first one is to verify whether CyENT3 has the function of transferring Uridine on 5-FU toxicity plate,and whether CyENT3 can promoting the symbiotic process of Arabidopsis and Piriformospora indica.The seconed one is firstly to obtain double mutants by hybridization,then studying AtENT3 and AtPUP3's functions in the symbiosis of Arabidopsis and Piriformospora indica.Their homologous gene are CyENT3 and CyPUP3.The last one is studying whether the AtENT3 and AtPUP3 can response to exogenous purine hormone.The main research results are as follows:?1?The T3 generation of CyENT3 overexpressed plants were obtained.The plant can grow normally on the 20?M 5-FU toxic uracil tablet,but the plant is weaker than that of WT.This indicates that CyENT3 have the function of transferring uridine.?2?The result of qPCR shows that the T-DNA insertion homozygote atpup3?SALK₀61408?is AtPUP3 deletion mutant.atent3?CS3729?is point mutant.It has been identified that it is a AtENT3 deletion mutant by sequencing technology and 5-FU toxic uracil tablet.By studying the root systems of Arabidopsis thaliana,we found that the mutation AtPUP3 is beneficial for the development of lateral roots.By studying phenotype characterization,we found that atent3 is smaller and shorter than WT.?3?Two kinds of Arabidopsis double mutated homozygous chimeras were obtained from their parents of atent3?CS3729?and atpup3?SALK₀61408?.The results of Real-time PCR showed that the expression level of AtENT3 in the double mutant homozygous plants wasn't down-regulated,which close to the expression level of its parent atent3?CS3729?.However,we has verified that the gene function of AtENT3 has disappeared on 5-FU toxicity plate.The AtPUP3 gene expression has loss.?4?In differnent culture medium,the Colonization of Piriformospora indica in Arabidopsis thaliana and its symbiotic effect on host plants are viarant.In LW01 culture medium,we find that the Piriformospora indica could enter the root through the epidermis and produce few chlamydospores.It also can be observed that Piriformospora indica promotes the growth and development of the lateral roots and increase the biomass accumulation.In PNM culture medium,the Piriformospora indica could also enter the root through the epidermis,but it produces a large number of chlamydospores,and the community gathered in the root segment in the cells.But the Mycorrhizal effect is stronger than that in PNM culture medium.?5?In LW01 culture medium,by analyzing the number of lateral roots,fresh weight of shoot and root,we find that the mycorrhizal effects of different materials are viarant.For atpup3 and atpup3/ atent3,their fresh weight of plant and number of lateral roots' s MGR value is lower than WT,while atent3 is the same as WT,which says that AtPUP3 plays a positive regulatory role in the process of symbiosis.?6?By applying different concentrations of 6-BA and ZT,it was found that with the increase concentration of 6-BA and ZT,the seedlings were inhibited more obvious.The inhibition effect of 6-BA was more obvious than that of ZT,which directly led to the death of the seedlings?under the concentration of 5?M and 20?M?.From the root and shoot responses of seedlings,we can speculate that AtENT3 and AtPUP3 may participate in the process of transportating purine hormone from root to shoot.Their lack of functionality inhibites the shoot growth.