| Auricularia heimuer is a widely cultivated large fungus with both edible and medicinal value.In recent years,the scale of A.heimuer industry has been expanding,and the continuous implementation of natural forest protection project has limited the source of wood chips to a certain extent,resulting in a surge in production costs.There are still more cellulose and other substances in the spent mushroom substrate of A.heimuer,and the utilization rate of matrix is low,and the cellulolytic enzyme is one of the important factors affecting the utilization rate of matrix.In view of this,in this paper,glucose,microcrystal cellulose and wood chips were used as the only carbon sources to culture the mycelium of A.heimuer.Based on transcriptome sequencing,genes related to cellulose degradation of A.heimuer were excavated,and a key endoglucanase gene Aa-eg was overexpressed,thus improving the biological efficiency of A.heimuer.The main results are as follows:1、Transcriptome analysis of A.heimuer mycelium cultured with different carbon sources and mining of genes related to cellulose degradationGlucose（Glc）,microcrystalline cellulose（MCC）,and wood chips（WD）were used as the only carbon sources to culture the mycelium of A.heimuer for 10 d.It was found that the number of mycelia with Glc as the carbon source was small,the diameter was large and the surface was smooth,and the mycelium biomass is 0.393 g/100 m L;when MCC is used as a carbon source,the mycelial flora is small in diameter and evenly distributed,and the mycelium biomass is 0.648 g/100 m L;when WD were used as carbon source,the number of mycelial balls was the least and the mycelium biomass is 0.149 g/100 m L.The cellulase activity of the fermentation broth was further measured,and the endoglucanase activity was most affected by the carbon source,when MCC and WD were used as the carbon source,the enzyme activities were 6.10±0.79 U/m L and 2.35±0.23,respectively,which were significantly higher than Glc.Collect the mycelia of A.heimuer cultured from different carbon sources for transcriptome sequencing.By stating the difference gene,it was found that in Glc-vs-MCC,there were 5175differentially expressed genes,3207 genes up-regulated,and 1968 genes down-regulated.In Glc-vs-WD,there were 6441 differentially expressed genes,3739 genes up-regulated,and2702 genes down-regulated.KEGG enrichment analysis was performed on the differentially expressed genes shared by Glc-vs-MCC and Glc-vs-WD,and a total of 109 metabolic pathways were annotated,of which 23 differentially expressed genes were enriched in the starch and sucrose metabolism related to cellulose degradation,through further annotated and screened in the Carbohydrate-Active en ZYmes（CAZy）database,and finally obtained 8candidate genes involved in cellulose degradation,including 2 endoglucanase genes,3exoglucanase genes and 3β-glucosidase genes.2、Cloning and prokaryotic expression of cellulase gene of A.heimuerThe highly expressed genes in mycelia with wood chips as carbon source,including endoglucanase gene Aa-eg,exoglucanase gene Aa-cbh andβ-glucosidase gene Aa-bgl,were cloned by RT-PCR.Among them,the total length of the CDS of Aa-eg is 1242 bp,encoding413 amino acids,the molecular weight of the protein is 43.59 k Da,the theoretical isoelectric point is 4.42,and the molecular formula is C1949H2946N508O599S16,which is a hydrophobic protein,the protein does not have a transmembrane structure,there is a signal peptide shear site VGA-QQ between the 19th and 20th amino acids,and there is a conserved domain of the 5th family of glycosyl hydrolase;the CDS of Aa-cbh is 1551 bp in full length,encoding 516 amino acids,the molecular weight of the protein is 54.44 k Da,the theoretical isoelectric point is 5.24,and the molecular formula is C2338H3596N650O790S31.It is a hydrophilic protein,the protein does not have a transmembrane structure,there is a signal peptide cleavage site VRG-QQ between the 18th and 19th amino acids,which has the conserved domain of glycoside hydrolase family 7;the CDS of Aa-bgl is 2583 bp in full length,encoding 860 amino acids,the protein molecular weight is 93.97 k Da,the theoretical isoelectric point is 5.49,and the molecular formula is C4171H6561N1175O1260S20,which is a hydrophilic protein,the protein has neither a transmembrane structure nor a signal peptide,and has a conserved domain of glycoside hydrolase family 3.The proteins was successfully expressed in E.coli under the induction of Isopropylβ-D-Thiogalactopyranoside（IPTG）,and sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE）analysis found that the expression of Aa-eg and Aa-cbh proteins did not change significantly with induction time,while the expression of Aa-bgl protein gradually increased with the increase of induction time.3、Analysis of the expression characteristics of cellulase gene in different growth and development periods of A.heimuerThe mycelium cultured in liquid culture,mycelium in the cultivation substrate,primordium,fruiting body during differentiation and mature fruiting body were used as materials,respectively.The q RT-PCR method was used to detect gene transcription levels in different materials.It was found that the change of Aa-eg and Aa-cbh gene expression was similar,the expression in mycelium and fruiting bodies was low,and the expression reached the highest in the primordial period,the expression of Aa-eg was 8.41 times that of the hyphae of 6 d in liquid culture,and the expression of Aa-cbh was 8.65 times that of mycelium in liquid culture for 6 d,while the expression level of Aa-bgl during the whole growth and development period was first reduced and then gradually increased,and the highest expression was in mature fruiting bodies,which was 6.69 times that of hyphae in liquid culture for 6 d.4、Establishment of the genetic transformation system of A.heimuerThe Gpd promoter was cloned with the genomic DNA of A.heimuer to replace the two CAMV 35S promoters in the binary vector p CAMBIA 1301,and the vector p CAMBIA 1301-Gpd-Gpd suitable for genetic transformation of A.heimuer was obtained,which was transferred to Agrobacterium EHA105.Agrobacterium carrying p CAMBIA 1301-Gpd-Gpd is incubated overnight in 1 m L of LB liquid medium（Rif 25μg/m L,Kan 50μg/m L）at 28℃200rpm,then all of them were transferred to MM medium（Kan 50μg/m L）and expanded to OD6000.6 to 0.8,6000 rpm centrifuged to collect the organism and resuspend with IM medium（As200μM）to OD600 0.2 to 0.3,25℃induction for 4 h,with the homogenized mycelium of A.heimuer as the receptor,after 60 h of incubation at 25℃,cover with a layer of PDA selective medium（Cef 400μg/m L,Hyg 14μg/m L）for initial sieve and re-sieve with a plate with hygromycin concentration of 16μg/m L.The germinated hyphae were re-inoculated into screening medium after 5 subcultures in antibiotic-free plates,and positive transformants were obtained by PCR identification,finally the genetic transformation system of A.heimuer was successfully established.5、Acquisition and biological characteristics of overexpressed Aa-eg transformantsThe Aa-eg overexpression vector p CAMBIA 1301-Gpd-Gpd-Aa-eg was constructed,and the established transformation system was applied to overexpress it in A.heimuer,and finally 4positive transformants were obtained.The average growth rate of transformant 7 was found to be higher than that of the wild type,but the difference was not significant,and the other 3transformants were significantly lower than the wild type;after 7 days of liquid culture,the activity of endoglucanase in the fermentation broth and the transcription level of Aa-eg in the mycelium were detected,and transformant 7 is higher than wild-type.Fruiting bodies are obtained through cultivation,it was found that the hyphae of transformant 7 had the strongest feeding ability,and its average biological efficiency was significantly improved（P<0.05）,which was 8.26%higher than that of wild-type.Therefore,this paper is of great significance to reveal the degradation law of cellulose in A.heimuer,and also provides a new idea and method for molecular directed breeding of A.heimuer. |
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