A Preliminary Study On Adaptability Of Volvariella Volvacea And Flammulina Velutipes Under Low-temperature Stress

Volvarila volvacea is a typical hygrophilous and thermophilic grass-saprophytic fungus.Its delicious taste and abundant nutrition make people like it deeply.However,when it is stored at 0-10℃,the sporocarp will be softening and under autopepsia,thus,lose its commodity value.Therefore,the problem of V.volvacea autolysis at low temperature is a key constraint for its commercialized production.V volvacea unfrigostable molecular mechanism and frigostable V.volvacea varietal breeding have become its research hotspot.This assay selected typical frigostable edible mushrooms as control,take morphological structure observation and several physiology and biochemistry measurement of V.volvacea and Flammulina velutipes under low temperature stress.Based on our laboratory V.volvacea,F.velutipes genome and transcriptome data,a pretty comprehensive and systemic research on low temperature response related fatty acid dehydrogenase synthetic pathway was carried out.Analysis for synthetic pathway related genes and coding protein by using bioinformatics way was performed.V.volvacea and F.velutipes expression quantity under low-temperature stress was detected by using Real-Time Quantitative PCR(qRT-PCR).And research on fatty acid dehydrogenase synthetic pathway key genes was taken by using RNA interference and overexpression.The main research results are as follows:(1)Using temperature and time combination screening,the final lethal low temperature and time combination is 0℃,48 h,F.velutipes in the control grows normally,and morphologic observation was taken on each bacterial colony after that.We found that bacterial colony color and morphology have no obvious difference.Then,typan blue(live cell monochromate solution)staining for F.velutipes and V.volvacea hypha cellular was taken,which were growing under normal temperature or low temperature stress.Microscopic observation indicated that under low temperature stress hypha cells were dyed as light blue,none of them were stained under normal temperature.Scanning electron microscope was used for ultrastructure observation for normal temperature or low temperature stress F.velutipes and V.volvacea,as a result,the straw mushroom hypha growth under low temperature stress present as "dry and collapse" shape,incomplete fracture.Straw mushroom,needle mushroom growth under normal temperature and needle mushroom growth under low temperature appeared satiation and full.(2)measurement was takenfor electrical conductivity and nucleic acid seepage of under low temperature stress(0℃ for 0 h、6 h、12 h、24 h、36 h、48 h),and theresults showed that with the increase of time,conductivity and nucleic acid exudation of V.volvacea mycelia have also increased,and the largest increase is in the 6th h.The electrical conductivity and nucleic acid exudation of the mycelium of F.velutipes were small,and some of the strains were slightly higher.It was determined that the contents of 35 fatty acids in mycelium of V.volvacea and F.velutipes treated with low temperature stress at 0℃ for 48 hours,and the results showed that the unsaturated fatty acids content of mycelium of partial V.volvacea under low temperature stress was higher than mycelium grown under normal temperature.The control group of F.velutipes has the same trend.There was no significant difference in the content of unsaturated fatty acids between the two treatments of partial F.velutipes,slightly fluctuating and irregular.(3)Bioinformatics analysis of correlative genes and corresponding proteins of Fatty acid dehydrogenase synthesis pathway was carried out in V.volvacea and F.velutipes.The encoded protein are mostly hydrophilic proteins,which are stable in the cell.The results of the conserved domain analysis showed that all proteins contain membrane fatty acid dehydrogenase domains(membrane-FADs),which was very conservative.Transmembrane structure analysis showed that most proteins have multiple transmembrane structures;however,VvFAD7 and VvFAD8 proteins have no transmembrane structure.The modification of phosphorylation is ubiquitous in the pathway,and there is no signal peptide.(4)The expression of 15 key genes in the fatty acid dehydrogenase synthesis pathway of V.volvacea and F.velutipes was detected by low temperature stress.The results showed that the expression level of most genes in the treatment group was lower than that in the control group,and a few genes were unstable in different treatments of different strains,and the trends were slightly different.(5)pBHg-BCAl-gpd was used as the original expression vector to construct RNAi and overexpression vector of fatty acid desaturase gene FvFAD6,Named pBHg-FvFAD6-RNAi and pBHg-FvFAD6-OE.The transformation of F.velutipes mononuclear strain L11 was achieved by Agrobacterium tumefaciens,and 6 positive transformants were successfully obtained,of which 2 were overexpressed transformants and 4 were RNAi transformants.Compared with the wild type strain L11,the expression level of the two RNAi strains was significantly lower than the wild-type strain L11,the expression level of an over-expression strains was up regulated than the wild-type strain L11,which is consistent with the expected results.The results showed that under low temperature stress,the ratio of un saturated fatty acids was decreased in the mycelium cell membrane of V.volvacea.It was also observed that under low temperature,fluidity of the cell membrane is reduced resulted in cell ruptured,and the contents of cel ls were infiltrated,leading to the death of mycelium.In addition,RNA int erference and overexpression of two fatty acid dehydrogenase genes(8602 and 797)mutants of F.velutipes strain L11,also provided a good materi al for the subsequent study at low temperature stress adaptation mechanis ms in F.velutipes.