Analysis Of Manganese Accumulation Characteristics In Pleurotus Pulmonarius And Identification Of The Associated Genes

Remediating soil contaminated with heavy metals using macrofungi is a novel approach in environmental remediation.The key issue in bring this approach into large-scale practical application lies in the success of screening out a series of ideal hyperaccumulators of heavy metals.To remediate heavy metals contaminated soil using macrofungi and even create genetic modified macrofungal hyperaccumulators,the mechanism and the associated genes involved in heavy metals absorption and accumulation in vivo of hyperaccumulation macrofungi must be elucidated.A number of easily cultivated macrofungi were collected in our laboratory,and their tolerance and accumulation of manganese stress were compared.It was found that the mycelium of Pleurotus pulmonarius（trade name:Xiuzhen Gu）had the strong ability to tolerate and accumulate Mn.In this study,the tolerance and accumulation characteristics of P.pulmonarius to Mn were analyzed.RNA-seq was used to study the response of mycelium transcription level to manganese stress,to identify the differentially expressed genes in mycelium under manganese stress,to identify the main physiological processes involved in manganese transport,and to screen genes related to manganese stress.The key candidate gene PpMTP1 related to manganese stress was cloned and analyzed.The main results of this study are as follows:1.When the concentration of Mn²⁺in the medium was≤100mg/L,the mycelial of P.pulmonarius LD1 growth was not significantly inhibited.The content of manganese in mycelium was as high as 27260 mg/kg（dry weight）,reaching the level of hyperaccumulation.The manganese content in the mycelium of the other four strains（GY18,HZ3,XY Xiu 1,and SM）was respectively 20582 mg/kg,15176 mg/kg,19179 mg/kg,and 23952 mg/kg,when the mycelium growth was not significantly inhibited,which all reached the hyperaccumulation level.It is indicated that the P.pulmonarius may be a manganese hyperaccumulation macrofungi.The content of manganese in the fruiting body did not reach the hyperaccumulation level.With the extension of Mn²⁺treatment time,the content of manganese in the mycelium of P.pulmonarius increased,reached the highest（37510 mg/kg）at 72 hours,decreased and tended to be stable after 72 hours.2.Transcriptome analyses were carried out on manganese stress（100 mg/L manganese treatment for 6 h,12 h,5 d）and the control（0 mg/L manganese treatment）.A total of 30 GB data was obtained.The data volume of each sample was more than2.03 GB,Q30≥88.96%,and the mapping rate with reference genome ranged from83.18%to 86.80%.The differentially expressed genes under manganese stress were mainly enrichment on amino acid metabolism,fatty acid metabolism,organic acid metabolism,secondary metabolite synthesis and the other metabolic pathway.Among them,amino sugar and nucleotide sugar metabolism and fatty acid degradation deserve attention.Fifteen genes that may be related to the response to manganese stress and have relatively high expression were screened,including gene families such as CDF/MTP,NRAMP,ZIP,P-TYPE ATPASE and genes related to peroxidase,superoxide dismutase and laccase.3.After manganese stress,the expression of transcript gene-EYR40＿008542 was significantly up-regulated,and the expression was relatively high,which may be related to the absorption and transport of manganese.Bioinformatics analysis showed that gene-EYR40＿008542 conformed to the characteristics of CDF/MTP family,so it was temporarily named PpMTP1 and cloned successfully.The q RT-PCR was used to detect the response of PpMTP1 to manganese stress treatment.It was found that under the condition of manganese treatment（100 mg/L）,the expression of PpMTP1increased at first and then decreased,and then increased and then decreased.There were two peaks at 6 h and 5 d,which were 81 times and 34 times of that of the control,respectively,and remained at a level with no significant difference after 10 days.Transient expression in Arabidopsis protoplasts and tobacco leaf cells showed that PpMTP1 was located on the plasma membrane.The results of functional complementary experiment of yeast mutant showed that PpMTP1 could restore the manganese tolerance of manganese mutantΔpmr1.The above experimental results show that PpMTP1 may be a gene for Mn²⁺transport located on the plasma membrane.Above all,a potential manganese hyperaccumulation macrofungus,P.pulmonarius,was screened,and the characteristics of manganese accumulation in its mycelium were studied.At the transcriptional level,the molecular mechanism of P.pulmonarius mycelia responding to high manganese stress was analyzed,and the function of the key candidate gene PpMTP1 in response to manganese stress was preliminarily verified.Besides,the identified genes responsible for Mn hyperaccumulation would aid the creation of genetic modified macrofungal hyperaccumulator for bioremediation of heavy metals contaminated soils.