Content of paclitaxel and isolation, purification, and species identification of mycorrhizal fungi in southern Chinese yew
Mycorrhiza is a symbiotic relationship between soil fungi and plant roots. As a beneficial microorganism that can establish mutualistic symbiosis with the vast majority of plant roots, research on mycorrhizal fungi mainly focuses on their specific morphological structure and functions. Mycorrhizal fungal mycelium infects the roots of host plants that have not yet become cork like, forming a fungal sheath on the surface of the root tip, a Hartian network in the root cortex, and extended hyphae outside the roots. It has characteristic structures such as promoting plant growth and development, enhancing plant stress resistance and disease resistance, improving plant absorption and utilization of mineral elements (especially N and P), and can also expand plant root absorption area, improve water use efficiency, promote the absorption and utilization of mineral nutrients, improve carbon cycle in the body, and affect primary and secondary metabolism of plants by forming a dense mycelial network between rhizosphere soil and root cortex cells. Mycorrhizal fungi form close symbiotic relationships with most terrestrial plants, and different fungi can invade individual root segments simultaneously. In addition to the biological and physiological ecological effects mentioned above, they also affect and participate in secondary metabolism of plant roots, but there are few related research reports.
Taxus chinensis var. mairei is a national first-class protected plant and a variety of Taxus distributed in China. It belongs to the Quaternary glacial relic species and is mainly distributed south of the Yangtze River Basin. The plants of the Taxus genus are currently recognized as endangered natural rare anti-cancer plants in the world. Their roots, stems, and leaves all contain natural anti-cancer medicinal ingredients such as paclitaxel and its precursor 10-DAB Ⅲ, as well as taxane compounds. Therefore, in order to seek new medicinal sources, there are many research reports on endophytic fungi of southern Chinese yew, but there are very few research reports on mycorrhizal fungi of southern Chinese yew. When Zhang Xiangyu et al. studied the distribution of paclitaxel content in various organs and tissues of southern Chinese yew plants, they found that the highest content of paclitaxel was in the fibrous roots, which was 0.101%. Later, Gong Xueyuan et al. further studied and found that there were some mycorrhizal fungi in the fibrous roots. After isolation and cultivation, 21 mycorrhizal fungi were obtained from the mycorrhizal tissues. Through liquid fermentation pure culture, it was found that 4 of them produced 10-DAB III but did not produce paclitaxel, and 6 produced paclitaxel but did not produce 10-DAB III. However, the species identification of 21 mycorrhizal fungi was not carried out. After dissecting the mycorrhizal fungi in this study, a red structural area was found in the central region (see Figure 1), with thin-walled cells outside. Dissecting and peeling off the red structural area revealed that it was the original root tissue. After testing and research, it was found that the paclitaxel content was 0.209%, which was 0.065% higher than that of the fibrous root. This study also conducted mutual cultivation between mycorrhizal fungi of southern Chinese yew and its stem callus tissue, and found that the paclitaxel content in the callus tissue increased significantly after the interaction. The mycorrhizal arbuscular mycorrhizal sheath is the only key anatomical structure added by mycorrhizal fungi relative to ordinary fibrous roots. Therefore, the mycorrhizal fungi in the arbuscular mycorrhizal sheath should be the key factor in the high accumulation of paclitaxel in the original root tissue of mycorrhizal fungi. In order to reveal the correlation between the two, this study restarted the research on the isolation and purification of mycorrhizal fungi in southern Chinese yew using tissue culture method, and planned to use morphological observation and molecular biology techniques to identify the species of this mycorrhizal fungus, in order to obtain information on the correlation mechanism. Compared with the research reports in relevant literature, it was found that there were significant differences between the species diversity of mycorrhizal fungi in the root tissues of southern Chinese yew and the endogenous fungal biodiversity in other organ tissues of southern Chinese yew plants, indicating that the specificity of mycorrhizal fungi species in southern Chinese yew may There is a correlation between them, The research on the diversity of organisms involved in the biosynthesis of paclitaxel and the interaction between mycorrhizal fungi and plants to produce secondary metabolites is of great scientific significance.

 

There are 11 species of Taxus plants worldwide, including 1 variety and 1 hybrid. Among them, 9 species of Taxus endophytic fungi have the ability to produce paclitaxel. Li et al. co cultured paclitaxel producing endophytic fungal culture medium with suspension cultured cells of Taxus cuspidata, and the results showed that it not only increased the release rate of paclitaxel, but also did not cause significant damage to the cell membrane of Taxus cuspidata, indicating that the endophytic fungal fermentation medium may have the functional activity of activating enzymes related to active transportation of paclitaxel. There are also studies showing that co culture of hazelnut tree cells and strains is effective, with fungal inoculation and co culture time being important factors in achieving maximum paclitaxel production in this co culture system. However, there has been no clear report on the biological relationship between endophytic fungi and Taxus chinensis cells in the biosynthesis of paclitaxel. Gong et al. reported that southern Taxus chinensis mycorrhizal fungi have the ability to produce paclitaxel and 10-DAB Ⅲ, but rapidly decay and disappear after subculture. Therefore, the correlation mechanism between mycorrhizal fungi and higher paclitaxel content in mycorrhizal primitive root tissues still has value for further research.
There are monographs that classify the Chinese yew plant as an endophytic mycorrhizal plant and point out that the southern Chinese yew can form arbuscular vesicle mycorrhizal fungi with arbuscular mycorrhizal fungi. The morphological diversity of spores is not sufficient to reveal the actual diversity of fungi in ecosystems. The application of molecular biology techniques in mycorrhizal fungi research can significantly increase their taxonomic diversity. Studies have shown that genetic diversity is high within populations and even within individual spores. In addition, there is evidence to suggest that differences in the composition of mycorrhizal fungal communities may have varying impacts on plants and potentially play a role in determining plant diversity, ecosystem diversity, and productivity. The results of this study showed that the mycorrhizal fungi of southern Chinese yew have a different microbial community than endophytic fungi in other organ tissues. The original root tissue has a much higher content of paclitaxel than ordinary roots and other organ tissues, and the interaction between callus tissue and mycorrhizal fungi can increase its paclitaxel content. Therefore, the synergistic mechanism between mycorrhizal fungi and the root cells of southern Chinese yew in the biosynthesis of paclitaxel is an important direction worth studying. This has significant scientific significance and application value for the development and utilization of mycorrhizal fungi and the efficient green production of natural organic functional active compounds.