Identification and characterization of differentially expressed genes in shiitake mushroom (Xianggu) Lentinula edodes.

摘要

In this study, gene expression profiling of Lentinula edodes at different developmental stages of dikaryotic mycelium and mature fruit bodies before and during spore formation were determined by using high-throughput technologies: LongSAGE, cDNA microarray, and random cDNA sequencing. L. edodes has been studied at the molecular level, most of which focused on the gene expression associated with fruit body initiation or primordium formation but few focused on dikaryotic mycelium or mature fruit bodies especially for sporulation. Those identified genes were mostly isolated using some time-consuming or expensive approaches such as EST, RAP-PCR, and cDNA-RDA. To advance our knowledge in biological systems of L. edodes, we have developed a comprehensive transcriptome analysis approach on different developmental stages of L. edodes.; I improved the LongSAGE technique by increasing the size of concatemer for sequencing and used this modified LongSAGE technique to generate two LongSAGE libraries. About 4000 and 7000 LongSAGE tags were obtained from mature fruit bodies before and during spore formation. These LongSAGE libraries were compared to identify genes that are expressed for sporulation. Expressions of genes relevant to sporulation were expected to increase and products of some genes would be stored in basidiospores for the use during germination. LongSAGE tags were also compared with SAGE tags obtained from other developmental stages such as dikaryotic mycelium and primordium to analyze the transcription profiles of some interesting genes at different stages. However, most LongSAGE tags did not match our gene database that was generated from dikaryotic mycelium and primordium. Random cDNA sequencing (454 Life Science) of mature fruit bodies was thus performed to produce about 7000 contigs to enrich our gene database. They were annotated with NCBI BLASTX search and matched with the LongSAGE tags. Identified LongSAGE tags were greatly increased by the sequencing results. Top 100 contigs with highest number of reads were analyzed and provided a snapshot in the gene expression profile of mature fruit bodies. From dikaryotic mycelium and primordium to mature fruit bodies, expression of structural proteins, hydrophobins, were greatly decreased and expression of riboflavin aldehyde forming enzyme, histones, proteins for cell defense and ubiquitin-mediated pathway were highly expressed. The results from LongSAGE and random cDNA sequencing showed totally different gene expression profiles of mature fruit bodies comparing to those of dikaryotic mycelium and primordium.; cDNA microarray was used to screen genes from dikaryotic mycelium of different strains of L. edodes. These genes may be relevant to two characteristics: faster mycelial growth rate and more fruit body number. By analysis with TM4 software package, genes from the microarray clones were clustered and some interesting genes were identified for further study. More genes may be needed in preparation of producing more fruit bodies than mycelial growth. Genes important for other developmental stages expressed lower to conserve energy for growth in strains with faster mycelial growth rate.; Quantitative Real-time PCR was used to confirm the results of LongSAGE and in situ RNA-RNA hybridization was used to localize the most abundant gene, riboflavin aldehyde forming enzyme in both LongSAGE libraries in mature fruit bodies of L. edodes.; In this study, LongSAGE, cDNA microarray, and random cDNA sequencing with analysis with bioinformatic tools provided important data in studying the gene expression profiles of dikaryotic mycelium and mature fruit bodies before and during spore formation. These data advanced our knowledge in the biology of L. edodes.