Tree Genetics and Molecular Breeding 2024, Vol.14, No.3, 144-154 http://genbreedpublisher.com/index.php/tgmb 147 physiological indexes under salt stress, highlighting the gene's role in salt tolerance (Guo et al., 2021). Additionally, the NAC13 gene was cloned and functionally characterized in poplar, where overexpression of NAC13 significantly enhanced salt tolerance, while suppression of the gene increased salt sensitivity (Zhang et al., 2019a). These case studies underscore the importance of advanced genomic tools and rigorous selection criteria in identifying and validating salt tolerance genes in poplar. 4 Functional Verification of Identified Genes 4.1 Experimental strategies for gene function testing To verify the function of identified salt tolerance genes in poplar, various experimental strategies were employed. One common approach involved the cloning and overexpression of target genes in transgenic poplar lines. For instance, the NAC13 gene was cloned and overexpressed in Populus alba × P. glandulosa, resulting in enhanced salt tolerance compared to wild-type plants (Zhang et al., 2019a). Similarly, the PeERF1 gene was overexpressed in Populus alba × P. glandulosa, leading to improved growth and physiological characteristics under salt stress (Ge et al., 2022). Another strategy included the use of RNA interference (RNAi) to suppress gene expression, as demonstrated with the PagWRKY75gene, where RNAi lines exhibited increased salt tolerance (Zhao et al., 2019). 4.2 Techniques for gene silencing and overexpression Gene silencing and overexpression techniques are crucial for functional verification. Overexpression constructs typically involve the use of strong promoters such as CaMV35S to drive high levels of gene expression. For example, the NAC13 gene was overexpressed using the CaMV35S promoter, resulting in significant salt tolerance in transgenic poplar (Zhang et al., 2019a). Conversely, gene silencing often employs RNAi or antisense suppression constructs. The PagSAP1 gene was downregulated using RNAi, which increased salt tolerance in poplar by altering ionic homeostasis (Yoon et al., 2018). Additionally, virus-induced gene silencing (VIGS) was used to study the function of FcWRKY40 in salt tolerance, where silenced lines showed increased salt susceptibility (Dai et al., 2018). 4.3 Integration of physiological and molecular data to confirm gene function The integration of physiological and molecular data is essential to confirm the function of identified genes. Physiological assays often include measurements of growth parameters, ion content, and stress-related enzyme activities. For instance, overexpression of PagERF072 in poplar led to increased activities of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT), along with reduced reactive oxygen species (ROS) levels under salt stress (Zhang et al., 2022). Molecular analyses, such as transcriptome profiling, provide insights into the regulatory networks and pathways affected by gene manipulation. Transcriptome analysis of PeERF1 overexpressed lines revealed enrichment of stress response-related genes, supporting its role in salt tolerance (Ge et al., 2022). Similarly, the overexpression of PtGSTF1 in poplar was associated with upregulation of genes involved in ion homeostasis and ROS scavenging, corroborating its function in enhancing salt tolerance (Gao et al., 2022). 5 Case Studies: Functional Analysis of Salt Tolerance Genes 5.1 Detailed examination of specific salt tolerance genes in poplar Several genes have been identified and characterized for their roles in enhancing salt tolerance in poplar species. For instance, the HD-Zip transcription factor family, particularly PsnHDZ63, has been shown to significantly improve salt tolerance when overexpressed in Populus simonii × P. nigra. This gene enhances reactive oxygen species (ROS) scavenging ability, which is crucial for mitigating salt-induced oxidative stress (Guo et al., 2021). Another key gene, PeERF1, identified in Populus euphratica, has been demonstrated to enhance salt tolerance in transgenic Populus alba × Populus glandulosa by regulating stress-related genes and improving antioxidant enzyme activity (Ge et al., 2022). The NAC13 gene, a transcription factor, has also been found to play a vital role in salt stress response. Overexpression of NAC13 in Populus alba × P. glandulosa significantly enhances salt tolerance, while its suppression increases sensitivity to salt stress (Zhang et al., 2019a). Additionally, the PalERF109 gene in Populus alba var. pyramidalis has been shown to enhance salt tolerance by upregulating the high-affinity K+ transporter
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