Study On The Function Of Pleurotus Pulmonarius Metal Tolerance Protein Gene PpMTPs In Response To Manganese Stress

The mycelium of Pleurotus pulmonarius(Fr.)Quél was found to be enriched with more than 10,000 mg/kg of manganese through screening,and is a potential manganese hyperenriched macrofungus.The two key candidate genes,which are homologous to CDF(cation diffusion facilitator)/MTP(metal tolerance protein)metal transport protein genes,were screened by RNA-seq technique before and after Mn stress treatment,and were speculated to be related to Mn uptake and transport,and were named as PpMTP1 and PpMTP2,respectively.Our laboratory completed the cloning and sequence validation of PpMTP1 in the previous stage,and partially analyzed its protein structure and physicochemical properties,expression pattern under manganese stress treatment of the gene,subcellular localization and function;this study will continue to identify the functions of PpMTP1 and PpMTP2 in detail.The concrete findings of the study are as follows:1.The PpMTP2 gene was cloned from the transcriptome sequence and its sequence was verified by sequencing.Bioinformatics analysis revealed that PpMTP2 is a protein containing 400 amino acids,containing cation efflux domain and zinc transporter protein dimer,as well as five transmembrane structural domains,which is consistent with the classical features of the CDF/MTP family with 4-6 transmembrane structural domains.Using the Neighbor-Joining method to construct a phylogenetic tree of the MTP family,it was found that PpMTP1 and PpMTP2 both clustered to Mn-MTP sub-family,indicating that PpMTP1 and PpMTP2 are both manganese transporter proteins.2.The response pattern of PpMTP2 to manganese stress treatment was analyzed.The expression of PpMTP2 showed two peaks at 6 h and 5 d with increasing duration of manganese treatment;The expression of PpMTP2 was 31.6 times higher than that of the control group at 6 h and 8.2 times higher than that of the control group at 5 d.The experimental results were consistent with the transcriptomic data,indicating that PpMTP2 is an early responder gene.The expression of PpMTP2 increased and then decreased with the increase of Mn treatment concentration,and reached the highest peak at 600 mg/kg.3.PpMTP2-GFP fusion protein expression vector was constructed for transient expression in tobacco leaves and transformation of Arabidopsis thaliana protoplasts,PpMTP2 was preliminary determined to be localized at the plasma membrane.4.Transformed yeast mutant strains(Δpmr1,Δcot1,Δcup2,Δzrc1,Δycf1,Δccc1,Δsmf1)and PpMTP2 were found to specifically enhance the tolerance to Mn in theΔpmr1 mutant strain by functional complementation experiments.The manganese content in yeast cells was measured,and PpMTP2 performed the function of transporting excess Mn outside the yeast cells under high Mn conditions.5.PpMTP1 and PpMTP2 heterologous expression of Arabidopsis thaliana mutants revealed a significant increase in biomass and root length of the transgenic Arabidopsis thaliana strain compared to the mutant in response to Mn stress.6.The sensitivity experiments of Pleurotus pulmonarius to cefotaxime and hygromycin were conducted,and the concentration of cefotaxime was determined to be 300 μg/m L and the concentration of hygromycin was 100 μg/m L.PpMTP1 and PpMTP2 overexpression vectors were constructed for genetic transformation of Pleurotus pulmonarius,and transformants with hygromycin resistance were obtained by screening,but they were found to be false positive after PCR,and no positive transformants were screened.In summary,PpMTP1 and PpMTP2 have important roles in maintaining manganese homeostasis and tolerance,and the detoxification processes involved in PpMTP1 and PpMTP2 are important components of plant manganese detoxification mechanisms,providing a basis for a comprehensive understanding of the phylogeny and function of CDF/MTP family proteins.However,the subcellular localization of PpMTP1 and PpMTP2 in Pleurotus pulmonarius and the specific direction of manganese transport are yet to be further identified by establishing the genetic transformation system of Pleurotus pulmonarius and obtaining homologous transformants of Pleurotus pulmonarius.