De Novo Analysis of Wolfiporia cocos Transcriptome to Reveal the Differentially Expressed Carbohydrate-Active Enzymes (CAZymes) Genes During the Early Stage of Sclerotial Growth

Abstract

The sclerotium of Wolfiporia cocos has been used as an edible mushroom and/or a traditional herbal medicine for centuries. W. cocos sclerotial formation is dependent on parasitism of the wood of Pinus species. Currently, the sclerotial development mechanisms of W. cocos remain largely unknown and the lack of pine resources limit the commercial production. The CAZymes (carbohydrate-active enzymes) play important roles in degradation of the plant cell wall to provide carbohydrates for fungal growth, development, and reproduction. In this study, the transcript profiles from W. cocos mycelium and 2-months-old sclerotium, the early stage of sclerotial growth, were specially analyzed using de novo sequencing technology. A total of 142,428,180 high-quality reads of mycelium and 70,594,319 high-quality reads of 2-months-old sclerotium were obtained. Additionally, differentially expressed genes from the W. cocos mycelium and 2-months-old sclerotium stages were analyzed, resulting in identification of 69 CAZymes genes which were significantly up-regulated during the early stage of sclerotial growth compared to that of in mycelium stage, and more than half of them belonged to glycosyl hydrolases (GHs) family, indicating the importance of W. cocos GHs family for degrading the pine woods. And qRT-PCR was further used to confirm the expression pattern of these up-regulated CAZymes genes. Our results will provide comprehensive CAZymes genes expression information during W. cocos sclerotial growth at the transcriptional level and will lay a foundation for functional genes studies in this fungus. In addition, our study will also facilitate the efficient use of limited pine resources, which is significant for promoting steady development of Chinese W. cocos industry.

Keywords: CAZymes; Wolfiporia cocos; differentially expressed genes; sclerotial development; transcriptome.

FIGURE 1

The mycelium and sclerotia of W. cocos used for De Novo Sequencing. (A) Colony morphology W. cocos mycelium grown on PDA for 7 days at 25°C. (B) W. cocos sclerotium of 2-months-old, scale bar = 5 cm. (C) The total RNA isolated from mycelium and 2-months-old sclerotium.

FIGURE 2

Assembled unigenes length distribution of W. cocos transcriptome from mycelium and 2-months-old sclerotium.

FIGURE 3

The number of unigenes for mycelium and sclerotium from different two data sets. Mycelium_P0 and Sclerotia_P1 were from the data set sequence in this project and Mycelium_W0, Sclerotia_W1 were from Shu et al. (2013).

FIGURE 4

Distribution of the homology search of expressed sequence tags against the nr database.

FIGURE 5

Gene transcription profile between mycelium and sclerotial stages. Scatter plot of total unigenes from the W. cocos transcriptome. The data was normalized as RPKM values and represented on a log₁₀ scale. Red areas represent up-regulated unigenes in sclerotia, green areas represent down-regulated unigenes in the sclerotia and the blue areas represent no significant expression difference unigenes between mycelium and sclerotia. (A) Sclerotia_P1 and Mycelium_P0 were from the data set sequence in this project. (B) Sclerotia_W1 and Mycelium_W0 were from Shu et al. (2013). (C) Differentially expressed unigenes in both data sets.

FIGURE 6

qRT-PCR validation of differentially expressed CAZymes genes. The relative expression of target genes in mycelium stage was set as level 1. Expression of W. cocos alpha-tubulin gene was used to normalize different samples. Bars represent means and standard deviations (three replications).