| 摘要: | 當真菌處於光照環境，會抑制其有性生殖路徑，轉往無性生殖，且二次代謝減少；回復至黑暗環境，則又轉往有性生殖路徑，同時二次代謝較旺盛。這些代謝與型態學上之調控機轉，均與velvet protein (VeA)、velvet-like protein (VelB) 及轉錄因子 LaeA 有關。目前已知的 VelB、VeA、LaeA 皆發現於子囊菌門 (Ascomycota)，本實驗選用台灣特有種的樟芝(Antrodia cinnamomea) 屬於擔子菌門Basidiomycota)，至今尚未分離出上述基因。由於我們在先前研究中發現樟芝亦有光照與否影響基因表現的現象，於是本實驗乃以照光/黑暗等環境因子處理，並針對已發表之子囊菌VelB、VeA、LaeA 基因高度保留區域設計退化性引子，進行聚合酶鏈鎖反應以選殖出樟芝 VeA 部分 cDNA 片段與 LaeA 部分 cDNA 片段，VelB 基因片段則尚未分離出。發現樟芝固態培養菌絲體在黑暗環境下其 VeA 及 LaeA基因表現量比光照環境來得高，而液態培養菌絲體則是在以葡萄糖為碳源之M25G 培養基培養 7 天，VeA 基因表現量大於 14 天；在以果糖為碳源之M25F 培養基培養 7 天及 14 天基因表現量相近。而 LaeA 基因在 M25G 培養 7 天及 14 天基因表現量相近，在 M25F 培養 14 天基因表現量大於 7 天。本研究為首次於擔子菌分離出 VeA 及 LaeA 基因，且根據實驗結果可知，以不同的環境因子處理樟芝菌絲體引發 VeA 及 LaeA 基因表現變化，同時推論此變化很有可能是參與下游二次代謝物生物合成的調控關鍵。
In fungi, the influence of light for the morphological development was found that ght can reduce and delay the asexual development; whereas in dark, fungal development is directed towards the sexual stage. Furthermore, secondary metabolism is also closely related to light/dark and the heterotrimeric velvet complex, VelB (velvet-like protein)/VeA (velvet protein)/LaeA, is involved in the light-responding developmental regulation and the control of secondary metabolism. Many studies indicated that the VelB, VeA, LaeA genes were isolated from Ascomycota. In this study, we are interested in these genes in a kind of Basidiomycota, Antrodia cinnamomea, a specific species in Taiwan. VelB, VeA, and LaeA genes are still unknown in Basidiomycota. However, in our previous studies, we found that the changes in the gene expression in Antrodia cinnamomea by light/dark and heat stress. According to the highly conserved regions of the cDNA sequences of VelB, VeA, and LaeA genes in Ascomycota, we designed the degenerated primers for the amplification of Antrodia cinnamomea VeA, VelB, and LaeA genes by polymerase chain reaction (PCR). The partial cDNA sequences of Antrodia cinnamomea VeA (AcVeA) and Antrodia cinnamomea LaeA (AcLaeA) were isolated. On the other hand, we found that the mRNA levels of AcVeA and AcLaeA in solid cultured mycelia are higher in dark than in light. In liquid cultured myceli, the expression of AcVeA is increased after 7 days by using glucose as carbon source, and the expression of AcLaeA is increased after 14 days by using fructose as carbon source. The induction of AcVeA and AcLaeA expression are also observed under heat stress environment. This study is the first to isolate VeA and LaeA genes in Basidiomycota. Our results suggest that the expression of VeA and LaeA genes are affected by different environmental factors, and maybe involved in the regulation of secondary metabolism in Antrodia cinnamomea. |
