Medicinal Plant Research 2025, Vol.15, No.2, 51-61 http://hortherbpublisher.com/index.php/mpr 54 3.2 Regulatory mechanisms of triterpenoid biosynthesis Transcription factors play an important role in the regulation of triterpenoid biosynthesis in G. lucidum. For example, the transcription factor GlbHLH5 was found to be a key regulatory factor that responds to the induction of methyl jasmonate (MeJA) and plays an important role in the synthesis of triterpenoids (Xu et al., 2022a). Its role in regulating the expression of key enzyme genes was verified by gene overexpression and silencing experiments, thereby increasing the accumulation of triterpenes. Xu et al. (2022b) conducted a transcriptome analysis of transcription factors in G. lucidumunder MeJA induction and found a total of 103 differentially expressed transcription factors, including members of 22 families such as C2H2, HTH, MADS and HMG. The study further showed that the expression of these transcription factors is closely related to the content of triterpenoids in G. lucidum. In addition, Meng et al. (2021) studied the function of the MADS-box transcription factor GlMADS1 in G. lucidumand found that after the gene was silenced by RNA interference technology, the content of ganoderic acid and flavonoids in G. lucidum increased, indicating that GlMADS1 may act as a negative regulatory factor to inhibit the accumulation of these metabolites. Epigenetic changes also help control how triterpenoids are made. The activity of key genes in this process can be influenced by signals from the environment or other molecules (Bai et al., 2018). Research has shown that sodium acetate can increase the amount of ganoderic acid, a type of triterpenoid, by turning on important genes like hmgs, fps, and sqs that help make it (Meng et al., 2019). Sodium acetate also affects how sodium and calcium ions move in cells by acting through the calmodulin and calcineurin pathways. This helps boost ganoderic acid production evenmore. 3.3 Challenges in identifying genetic targets Finding genes that can directly improve the efficiency of triterpenoid synthesis is easy to say but difficult to do. The problem is that the entire biosynthesis process is not completed by a few genes alone, but a very complex network system involving many genes and regulatory levels, and there are cross-influences between each other (Aminfar et al., 2019; Paramasivan et al., 2022). Take the relationship between carbon metabolism and triterpenoid synthesis as an example. Under conditions of limited nitrogen supply, the integration between the two involves multiple metabolic pathways. This complex "collaboration model" makes it difficult for us to see at a glance which gene is really worthy of priority transformation (Lian et al., 2020). In addition to the complex mechanism itself, higher fungi such as G. lucidum are also not easy to manipulate genetically. They have a slow growth cycle and a genetic system that is not easy to manipulate. It takes a long time to do a mutation experiment. In order to circumvent this problem, researchers have come up with a way to put some target genes into a system such as Saccharomyces cerevisiae for heterologous expression (Wang et al., 2018). Although this method speeds up the progress of the experiment, it also has shortcomings - the yeast environment is different from the metabolic background of G. lucidumitself, and it is often impossible to truly restore the regulation of genes in the original background. This still brings certain obstacles to understanding the true function of genes. 4 Current Approaches to Genetic Modification inGanoderma lucidum 4.1 Traditional methods and their limitations The breeding of G. lucidum actually started a long time ago, but the initial methods were more traditional. Excellent varieties such as "Hunong 5" were selected through conventional hybrid breeding. This variety has higher content of polysaccharides and triterpenoids, and has stronger medicinal value (Chao et al., 2018). In addition to breeding, some treatment methods have also been tried, such as spraying salicylic acid (SA) during the fruiting stage of G. lucidum. The results showed that this treatment not only significantly increased the content of triterpenoids, but also activated the expression of some related synthetic genes (Ye et al., 2018).
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