Bioscience Evidence 2024, Vol.14, No.5, 206-217 http://bioscipublisher.com/index.php/be 208 characterized for their roles in converting linoleic acid to ALA. These genes were found to be highly expressed in tissues with high ALA content (Li et al., 2021). Additionally, the genome-wide analysis of the fatty acid desaturase gene family in E. ulmoides has identified key genes like EU0103017, which is highly expressed and significantly contributes to ALA biosynthesis. 3.2 Molecular markers associated with ALA biosynthesis Single nucleotide polymorphism (SNP) markers have been identified and linked to ALA traits in E. ulmoides. These markers are crucial for understanding the genetic variation associated with ALA content and can be used in marker-assisted selection to enhance ALA levels in breeding programs. The identification of SNP markers in genes involved in the fatty acid biosynthesis pathway, such as those coding for desaturase enzymes, has been a significant advancement. Quantitative trait loci (QTL) mapping has been employed to identify genomic regions associated with ALA biosynthesis. This approach helps in pinpointing specific loci that contribute to the variation in ALA content among different E. ulmoides varieties. For instance, transcriptome analysis has facilitated the mapping of QTLs linked to the expression of key enzymes in the ALA biosynthetic pathway, providing a framework for future genetic studies (Xue et al., 2018 ). 3.3 Genetic modification strategies to enhance ALA content CRISPR/Cas9 technology offers a powerful tool for the precise editing of genes involved in ALA biosynthesis. By targeting and modifying specific genes, such as those encoding FAD enzymes, it is possible to enhance the ALA content in E. ulmoides. This approach has the potential to create high-ALA varieties by knocking out negative regulators or enhancing the expression of positive regulators in the ALA biosynthetic pathway. Overexpression and gene silencing techniques have been employed to manipulate the expression of genes involved in ALA biosynthesis. For example, overexpressing genes like FAD3 in model organisms such as Arabidopsis thaliana has been shown to significantly increase ALA content (Figure 1) (Duan et al., 2021). Similarly, gene silencing techniques can be used to downregulate genes that negatively impact ALA accumulation, thereby enhancing the overall ALA content in E. ulmoides seeds. In conclusion, the genetic basis of α-linolenic acid biosynthesis in Eucommia ulmoides is being unraveled through high-throughput sequencing, functional annotation, and the identification of molecular markers. Genetic modification strategies, CRISPR/Cas9 and overexpression techniques, are expected to increase ALA content and successfully establish transgenic technology and CRISPR/Cas9 technology (Zhao et al., 2009; Wang et al., 2023), paving the way for the development of high-nutritional and medicinal value superior Eucommia species. Duan et al. (2021) found that overexpression of the PfFAD3.1 gene under the control of the CaMV35S promoter in Arabidopsis thaliana significantly altered both the expression levels of the transgene and the fatty acid (FA) composition in seeds. The transgenic plants exhibited marked increases in specific fatty acids, particularly C18:3, which is consistent with the role of PfFAD3.1 in modulating FA desaturation pathways. Quantitative RT-PCR analysis revealed that the transgenic seeds had significantly higher PfFAD3.1 expression compared to wild-type controls, confirming the successful integration and expression of the transgene. Additionally, fatty acid profiling demonstrated changes in both saturated and unsaturated FA content, indicating that PfFAD3.1 plays a critical role in the biosynthesis and regulation of FAs in seeds. These findings highlight the potential for PfFAD3.1 to enhance seed oil composition in plants through targeted genetic modification. 4 Role of Environmental Factors in ALA Biosynthesis 4.1 Influence of temperature on ALA production Temperature plays a crucial role in the biosynthesis of α-linolenic acid (ALA) in various organisms, including Eucommia ulmoides. Studies have shown that low temperatures can significantly enhance the accumulation of ALA-rich lipids. For instance, in the microalga Desmodesmus sp., incubation at 5 ℃ resulted in a 1.5-fold increase in lipid content, with ALA constituting 44% of the total fatty acids (Sijil et al., 2019). This suggests that lower temperatures may create an optimal environment for ALA biosynthesis by possibly influencing the activity of enzymes involved in the fatty acid synthesis pathway. Temperature stress, particularly low-temperature stress, has been observed to affect ALA content in various species. In Desmodesmus sp., UV-treated cultures incubated
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