Research And Application For Mushroom Proteins

Part I:Transcriptome and proteome analysis for mycelium and fruiting body of C. militarisCordyceps sinensis, a traditional Chinese medicinal fungus, is the complex of stroma which parasitic on the Hepialidae insect larvae and the larvae corpse. It has been used as an herbal drug because of its antitumor and immunomodulatory activities. The natural Cordyceps is scarce and expensive, the resources are exhausted. Cordyceps militaris is another medicinal fungus containing insect and fungus. The active ingredients are similar to those in Cordyceps sinensis. The artificial cultured C militaris are available and can substitute the natural Cordyceps. But, except the cordycepin, cordycepic acids and polysaccharide, there is not enough study on other medicinal ingredients especially the proteins. In this study, we performed systematic molecular research on the different developmental stages of artificial cultured C. militaris at transcriptional and translational levels in order to discover more active proteins/peptides.We performed high-throughput Illumina sequencing to the mycelium and fruiting body of C. militaris and analyzed the transcriptome data. All the clean reads were mapped to the known C. militaris genome database and sequencing randomness assessment and genes'coverage calculation were performed. The results showed that nearly90%of clean reads can be mapped to the genome, the distribution of reads is homogeneous in C. militaris genes and the gene coverage is higer than90%. These results indicated the good quality of our sequencing and mapping data.738alternative splicing events in total and nearly3000novel transcripts in each sample were predicted, which enriched the C. militaris database. Different gene expression analysis showed that2,113genes were up-regulated in mycelium while599genes up-regulated in fruiting body. Gene functional annotation ?GO, KEGG? were performed to these different expressed genes ?DEGs? and the result of which suggested that intracellular nucleotide binding and metabolism, transcription regulation are more active in mycelium while signal transduction and carbohydrate metabolism in fruiting body. These conclusions were related to the growth needs of C. militaris. In addition, the putative cordycepin metabolism pathway was studied in different stages. We found that the metabolism is more active in mycelium stage, which provided data support to the production optimization of cordycepin.One-dimensional gel electrophoresis and nESI-LC-MS/MS were combined to produce the proteome data of C. militaris.359and214proteins were identified from mycelium and fruiting body, respectively. Gene functional annotation ?GO, KEGG, COG? results showed the similar conclusions to the transcriptome data. Moreover, some noteworthy proteins such us lectin, superoxide dismutase and proteins involved in cordycepin metabolism pathway were analyzed, which provide data information for further C. militaris protein study.According to the proteome data of C. militaris, we cloned three genes from the mycelium or fruiting body and performed prokaryotic expression. They are RicinB-related lectin ?CCM03832?, ConA-like lectin ?CCM03227? and Superoxide dismutase ?CCM₀4979?. Biological activity studies showed that RicinB-related lectin ?CCM₀3832? has certain clotting activity, RicinB-related lectin ?CCM₀3832? and ConA-like lectin ?CCM₀3227? can inhibit the proliferation of HepG2and all of three can stimulate the proliferation of mouse spleen lymphocytes.In addition, we purified three proteins CM-1, CM-2and CM-3from fruiting body of C. militaris using protein seperation technologies. The gene sequences had been found by MS and the bological activity study is ongoing.Part II:Application of Agrocybe aegerita lectin AAL-2on the O-GlcNAcylated protein/peptide enrichment and the detection methods studyO-GlcNAc modification is a very important posttanslational modification, which has crosstalk with other modifications such us phosphorylation, ubiquitination and methylation to regulate the cellular process and perticipate in the disease development. However, the enrichment and detection of the O-GlcNAc modification is the bottleneck problem in this field.Our research team purified a novel lectin AAL-2from A. aegerita which had been reported that it has extremly high affinity and specifility to GlcNAc. This suggested that AAL-2is much better than wheat germ agglutinin ?WGA? for the O-GlcNAc modification enrichment.In this study, we firstly used AAL-2to enrich the O-GlcNAc modified peptide from the simple standard peptides sample and a positive O-GlcNAcylated protein. The positive peptides had been identified by MALDI-MS, thus AAL-2can effectively enrich O-GlcNAc modified from simple samples. Moreover, we used different chromatographic methods to enrich O-GlcNAc modified proteins/peptides:For complex peptide samples ?peptides digested from B16-BL6cytoplasmic protein, peptides digested from diabetic rat liver proteins?. LC-MS/MS results showed that O-GlcNAc modified peptides are in the flow through tail. For complex protein sample ?normal and diabetic rat liver proteins?, the O-GlcNAc modified proteins were proved in the elution by western and LC-MS/MS ?CID?. Lastly, LC-MS/MS ?ETD? was utilized to identify some O-GlcNAc modified proteins and sites. In B16-BL6cytoplasm sample,79O-GlcNAcylated proteins ?73in the tail of flow through fraction,9in the elution fraction,3can be detected in both fractions? and totally123O-GlcNAcylation sites ?111in the tail of flow through fraction,13in the elution fraction,1can be detected in both fractions? were detected. In the diabetic rat liver,99O-GlcNAcylated proteins and totally119O-GlcNAcylation sites were detected. In the normal rat liver,66O-GlcNAcylated proteins and totally82O-GlcNAcylation sites were detected. These results need our further verification. We initially established the method of enrichment of O-GlcNAc modification by AAL-2.