Comparative Analysis of the Characteristics of Triterpenoid Transcriptome from Different Strains of Wolfiporia cocos

Abstract

The dried sclerotia of Wolfiporia cocos (Schwein.) Ryvarden & Gilb., a traditional Chinese medicine, has triterpenoid as its main active component. Breeding high-yield triterpenoid in W. cocos is an important research topic at present. We screened out two monosporal strains from the same W. cocos 5.78, high-yielding DZAC-Wp-H-29 (H) and low-yielding DZAC-Wp-L-123 (L), and cultured mycelia for 17 days, 34 days, and 51 days, respectively. Transcriptome analysis results showed that triterpenoid synthesis is closely related to gene expression in triterpenoid synthesis pathways (hydroxymethyl glutaryl-CoA reductase (HMGCR), farnesyl diphosphate synthase (FDPS), 4-hydroxybenzoate polyprenyltransferase (COQ2), C-8 sterol isomerase (ERG2), sterol O-acyltransferase (ACAT), tyrosine aminotransferase (TAT), torulene dioxygenase (CAO2), and sterol-4alpha-carboxylate 3-dehydrogenase (erg26)), and is limited by the expression of enzyme M20 combined with domain protein peptide (Pm20d2), aryl-alcohol dehydrogenase (norA), ISWI chromatin-remodeling complex ATPase ISW2, GroES-like protein (adh), cytochrome P450 (ftmP450-1), and unknown proteins unigene0001029 and unigene0011374. In addition, maintaining high triterpenoid accumulation in W. cocos may require a stable membrane structure, so the accumulation ability may be related to the high synthesis ability of sterols. The low accumulation of triterpenoid in W. cocos may be due to the products of key enzymes increasing flow to other pathways.

Analysis of weighted gene co-expression network of triterpenoid-related transcriptome characteristics from different strains of Wolfiporia cocos

“…The filtrate was mixed with 1000 mL distilled water with 20 g glucose, 1 g KH 2 PO 4 , 0.5 g MgSO 4 ·7H 2 O, 10 mg VB 1 , and 18 g agar at natural pH. Mycelia were cultured for 17, 34, and 51 d at 25 °C in the dark, quickly frozen in liquid nitrogen, then, stored in a refrigerator at − 80°C 26 .…”

“…The mixture was bathed in 70 °C water for 20 min, then removed from the water bath and cooled to room temperature, and 5 mL of anhydrous ethanol was added and mixed in. Then, 200 μL of mixed liquor was taken for absorbance measurement at 560 nm for 10–25 min; the reference substance was oleanolic acid 26 .…”

“…The RNA integrity number (RIN) values (> 8.0) of these samples were evaluated by Agilent 2100 Bioanalyzer (Santa Clara, CA, USA). The purity, concentration, and integrity of total RNA samples were qualified through testing and evaluation, then the samples were prepared for use 26 .…”

“…The cDNA fragments were purified with 1.8× Agencourt AMPure XP Beads and end-repaired, and poly (A) was added and ligated to Illumina sequence adapters. The ligation products were size-selected by agarose gel electrophoresis, PCR-amplified, and sequenced using Illumina HiSeqTM 4000 by Gene Denovo Biotechnology Co. (Guangzhou, China) 26 .…”

“…All RT-qPCR experiments were repeated three times, with three technical repeats for each experiment. Expression levels of candidate genes were determined using the 2 −∆∆ C t method 26 . Expression levels were normalized against the reference gene pab1 (unigene0013050).…”