Heat Tolerance Analysis Of The Mycelium Of A Wild Lepista Sordida Obtained From Chongqing

The Lepista sordida is a kind of high-quality wild edible which has a great development prospect mushroom.The production of Lepista sordida is still come from wild filed and is not enough to the market demand.At present,the cultivation of Lepista sordida mushroom is in the stage of small-scale domestication.In the process of cultivation,when faced with high temperature stress,the growth of Hypha stops or even dies,which greatly affects the cultivation and production of mushroom.Therefore,how to control the heat tolerance of hyphae is of great significance to the mushroom production.Heat shock transcription factor（HSF）is an important member of the gene transcription activation signal transduction pathway in organisms under heat shock or other stress conditions,directly initiating the downstream heat shock protein gene expression.It plays an important role in regulating the expression of HSP gene,transmitting information of stress,especially heat stress,and improving plant stress tolerance.The expression of heat shock protein（HSP）induced by heat stress was positively correlated with the heat tolerance.Hence,HSF and HSP could be used as an important factor to study the heat tolerance of Lepista sordida.In this paper,the morphological characteristics,ITS molecular identification and mycelium growth conditions of a wild edible mushroom obtained from Chongqing were systematically analyzed.The expression pattern of HSFs and HSPs,on the molecular biological level,which may affect the heat tolerance of the mushroom were also analyzed under heat stress.The function of HSPs which may related to the tolerance of heat stress were analyzed.The main results are as follows:1.A wild mushroom strain was collected from Chongqing Chengkou area and the was identified both in morphology and molecular biology.According to the morphological observation of fruit body and mycelium,combined with ITS sequence analysis,the strain was identified as Lepista sordida.The results showed that the optimum temperature for the mycelium growth was 24℃,the optimum nitrogen source was inorganic nitrogen ammonium chloride,and the optimum C/N Ratio was between 30:1and 40:1.The PDA medium containing 1000 mg/L K₂HPO₄ in inorganic salt was the best for the mycelium growth of Lepista sordida.In addition,it was found that adding vitamin had little effect on the mycelium growth rate,but adding different concentration of vitamin had some effect on the mycelium growth.The optimum medium for the mycelium growth of Lepista sordida based on PDA was 46 g PDA powder,0.568 g NH₄CL,330 mg K₂HPO₄,120 mg vitamin B₁₂,45 mg vitamin B2 and 1000 m Ldistilled water At p H 7 the mycelium grows best.The research on the temperature test of Fragaria variegata was found that the optimum growth temperature of Lepista sordida was 24℃,the mycelium of Lepista sordida could grow normally at 20℃-28℃,and the mycelium under 30℃was grown under high temperature stress Slowly,stop growing at a high temperature of 34℃.2.Bioinformatics analysis the LsHSFS and LsHSPs family proteins of Lepista sordida mainly included construction of phylogenetic tree,conservative domain,gene structure,basic physical and chemical properties prediction,secondary structure and subcellular localization.The homology of the related proteins was verified by phylogenetic analysis.The results showed that the domains of LsHSP100 family and LsHSP60 family were more conserved.The LsHSP90 family proteins are unstable proteins with 25 introns while the of HSP70 family which content the largest number only have 14 introns.The subcellular localization showed that the HSFs family proteins were mainly located in the nucleus and the LsHSPs family proteins were located in the Cytoplasm,endoplasmic reticulum and mitochondria,respectively.3.Heat shock protein and heat shock transcription factor related gene expression analysis under temperature treatment combined with the growth of Lepista sordida mycelium under different temperature treatment.The wild Lepista sordida mycelium from Chongqing was treated with different temperature and the expression levels of heat shock transcription factors and heat shock proteins which may be involved in the regulation of heat stress were analysed by q RT-PCR,12 genes were induced by the heat stress.The Hsfs family genes LsHsf-1 and LsHsf-3 were induced by heat stress and LsHsf-1 had short response time to heat stress.The expression of HSPs protein family genes LsHsp88,LsHsp78-2 and LsHsp70-8 stopped responding to the heat stress after48 hours.The expression of LsHsp104,LsHsp90-1,LsHsp78-1,LsHsp70-1,LsHsp70-2,LsHsp70-6 and LsHsp60-1 increased significantly under the heat stress treatment.And the LsHsp104 and LsHsp70-1 respond to heat stress immediately and can be synthesized in large quantities in short time.Among the Hsps genes,the expression of LsHsp70-1 is obviously induced by high temperature.4.The CDS sequence of LsHsp70-1 gene was amplified and fusion in the p ET32a（+）heterologous expression vector.Then the recombinant plasmid was transferred into E.coli BL21,and the heterologous expression of the fusion protein was induced by IPTG.The results of survival rate of E.coli under heat stress showed that the survival rate of E.coli containing the recombinant plasmid was significantly higher than that of the Control Group.The results indicated that the heterologous expression of LsHSP70-1could enhance the heat stress tolerance and the survival rate of E.coli.It suggested that LsHSP70-1 could enhance the tolerance to heat stress.