## 3 Results
### Diterpenoid Biosynthesis Pathway  
_H. coralloides_ produces erinacines, a class of cystahane diterpenoid compounds. The direct precursor of diterpenoid biosynthesis is GGPP, formed by the condensation of the common precursors IPP and DMAPP and giving rise to larger structural units, including GPP(C10), PPP(C15) and GGPP(C20). Diterpenoid synthases catalyze the synthesis of diterpenoid compounds from GGPP via the mevalonate (MEP) pathway. Different types of diterpenoid synthase are responsible for the synthesis of a variety of diterpenoid compounds. Diterpenoid synthase (ditps), diterpenoid cyclase, and cytochrome p450 are the key diterpenoid synthase enzymes in fungi. To identify genes involved in diterpenoid biosynthesis in _H. coralloides_, the GGPPS sequence of the basidiomycetes species _Clitopilus pusseckerianus_ was used to search for homologous amino acid sequences in the _H. coralloides_ genome [41; 42]. After identifying a gene (_Her011461_) encoding GGPPS, the sequences located 20 kb upstream and downstream of _Her011461_ were subjected to BLAST analysis. Ten genes, forming diterpene biosynthesis gene clusters, were identified (Table 6). \begin{table}
\begin{tabular}{l l l l l l} \hline \hline
**Gene ID** & **Function** & **Chromosome** & **Log2 Ratio** & **Log2 Ratio** & **Log2 Ratio** & **Log2 Ratio** \\  & & & MK-1/MK-2 & DK/FB-A & DK/FB-B & DK/FB-C \\ \hline _Her005768_ & 4-diphosphocytidyl-2-C- & Chr05 & 1.00 & 1.83 & 1.50 & 2.01 \\ _Her008335_ & FDPS/GGPPS & Chr08 & 1.27 & 2.14 & 1.73 & 1.97 \\ _Her011461_ & FDPS/GGPPS & Chr13 & 1.93 & 1.47 & 2.30 & 2.55 \\ _Her010901_ & ubiA/COQ2 & Chr12 & -0.02 & 2.92 & 2.59 & 2.71 \\ _Her009959_ & ubiA/COQ2 & Chr11 & 0.51 & 0.01 & 2.82 & 1.31 \\ _Her001132_ & ubiF/COQ7 & Chr01 & 0.14 & 1.46 & 1.21 & 1.53 \\ _Her006816_ & TAT/ARO8 & Chr07 & -0.26 & 0.12 & 0.93 & 1.27 \\ \hline \hline \end{tabular} MK-1 and MK-2: mononuclear mycelium; DK: dikaryotic mycelium; FB-A: primitive stage; FB-B: fruiting body stalk; FB-C: fruiting body spines. Orange indicates a significantly up-regulated gene; gray indicates no significant difference in expression.  
\end{table} 
Table 5: Genes and enzymes of the terpenoid backbone biosynthesis pathway. The predicted gene cluster for diterpenoid biosynthesis was located on chromosome 13 and included one GGPPS, three cytochrome p450, two NAD(P) binding proteins, one UDP glycoproteins, one diterpene cyclase, one ABC transporter, and one ubiA pentenyltransferase. Comparison of the MK-1 and MK-2 strains showed that the levels of expression of genes encoding NADP binding protein (_Her011458_), diterpene cyclase (_Her011463_), UbiA preyltransferase (_Her011463_), NAD binding protein (_Her011465_), and UDP-glycostransferase (_Her011467_) were significantly higher in MK-2 than in MK-1, suggesting that diterpenoids may be more abundant in MK-2 than in MK-1. The levels of expression of three cytochrome P450 (_Her011457_, _Her011459_, and _Her011466_) encoding genes did not differ significantly in FB-A, FB-B, FB-C, and DK. In contrast, the levels of expression of genes encoding geranylgeranyl pyrophosphate synthase (_Her011461_), NAD binding protein (_Her011465_) and UDP-glycostransferase (_Her011467_) were significantly higher in DK than in FB-A, FB-B, and FB-C, further indicating that diterpenoid content may be higher in DK than in FB-A, FB-B, and FB-C. Similar results were observed in previous studies. Because fungi contain many types of diterpenoids and the extraction methods are difficult, differences in diterpenoid contents among strains were verified by assaying the amounts of erinacine, a representative diterpenoid in \(H\). _colloides_. The contents of erinacine in DK were 2.62-, 1.23-, and 3.6-fold higher than in FB-A, FB-B, and FB-C, respectively, and 1.5-fold higher in MK-2 than in MK-1 (Figure 6).