Medicinal Plant Research 2025, Vol.15, No.2, 51-61 http://hortherbpublisher.com/index.php/mpr 55 But the issue is that these traditional methods are often guided by natural mutations or external stimuli, and the operation process is quite slow. If you want to select a good strain with stable performance, you may have to cultivate several generations, and each generation must be carefully selected, which takes a lot of time and manpower. Moreover, sometimes the effect is not stable, and it is difficult to accurately control the improvement of the target trait (Ye et al., 2018; Zhou et al., 2021). 4.2 CRISPR/Cas9-based gene editing inGanoderma lucidum Compared with traditional methods, the emergence of CRISPR/Cas9 has undoubtedly accelerated the genetic modification of G. lucidum. This technology, in the final analysis, can precisely destroy or modify specific DNA sites. For example, the cytochrome P450 monooxygenase (CYP450) gene is closely related to the synthesis of ganoderic acid. Once it is knocked out, the content of ganoderic acid immediately drops a lot, which shows that this editing system can indeed work (Wang et al., 2020). Although CRISPR is very effective, it is not without its challenges in higher fungi such as G. lucidum. The biggest problem is that its homologous recombination efficiency is too low. This results in a low success rate when you want to insert or replace a gene at a specified location. To solve this problem, Tu et al. (2021) tried to suppress the non-homologous end joining (NHEJ) repair mechanism and found that doing so could improve the efficiency of gene insertion and replacement. Tan et al. (2023) further simplified the CRISPR system into a ribonucleoprotein (RNP) form and delivered it directly into G. lucidumcells. This method does not require any carriers. The pure "protein + RNA" combination achieved 100% editing efficiency on the selective culture medium right away. More importantly, they successfully targeted multiple genes related to triterpenoid synthesis. This achievement means that if G. lucidumstrains with high triterpenoid production are to be cultivated, this type of RNP system is likely to become a key technical support. 4.3 RNA interference and other gene silencing techniques RNA interference (RNAi) is not really a "new technology", but it still has unique value in the study of G. lucidum. It is a biological process that inhibits gene expression by neutralizing target mRNA molecules. dsRNA is cut into small interfering RNA (siRNA) by Dicer enzyme, and then these siRNAs carry RNA-induced silencing complex (RISC) to accurately identify and cut off the target mRNA, thereby effectively silencing the gene. RNAi technology has been used to silence certain key genes in G. lucidum, thereby enhancing the synthesis of triterpenoid compounds. For instance, Wang et al. (2018) conducted an interesting experiment: instead of directly enhancing the genes for triterpenoid synthesis, they chose to silence some metabolic pathway genes that "grab resources". The results showed that this could significantly increase the production of ganoderic acid, proving that this is an "indirect acceleration" strategy. Lu et al. (2020) also selected the LAG1 gene, which is closely related to lipid synthesis. After being interfered with by RNAi, lipid metabolism was weakened, and resources were freed up for triterpenoid synthesis, resulting in an increase in the accumulation of ganoderic acid. RNAi has also been used to silence P450 enzyme genes, such as CYP5150L8, which plays an important role in the multi-step biotransformation of ganoderic acid. Wang et al. (2020) confirmed this by silencing this gene. After silencing this gene, the production of ganoderic acid was greatly reduced, which basically confirms that CYP5150L8 is the key to the synthesis of ganoderic acid. Now there are many studies focusing on the upstream regulatory level, such as the role of signal molecules. Ye et al. (2018) found that signal substances such as calcium ions and salicylic acid can actually indirectly increase the level of triterpene synthesis by regulating the expression of a series of metabolic genes. This shows that the factors affecting triterpene synthesis are far more than just the genes in the synthesis pathway, and the entire signal transduction network is also playing a role behind the scenes.
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