Triticeae Genomics and Genetics, 2025, Vol.16, No.6, 269-277 http://cropscipublisher.com/index.php/tgg 272 4 Transgenic Construction Strategies for Nitrogen Efficiency Genes 4.1 Selection of candidate genes (e.g., GS1, NRT1.1, NUE1) Candidate genes for improving nitrogen use efficiency (NUE) in wheat are usually selected based on their roles in nitrogen absorption, assimilation and metabolism. Key genes include glutamine synthase isoenzymes, such as GS1 and GS2, which catalyze the assimilation of ammonium into amino acids, thereby enhancing the nitrogen cycle within plants. Nitrate transporter genes, such as NRT1.1, promote the absorption of nitrate in soil and directly affect the efficiency of nitrogen acquisition. Other promising targets include NUE1 and amino acid transporter genes, such as AAP1, which contribute to the transport and reuse of nitrogen. These genes have been proven to have positive effects on nitrogen fertilizer utilization and yield of various crops such as rice and corn. Therefore, they are strong candidate genes for transgenic improvement of wheat (Chen et al., 2020; Lal et al., 2023; Zhu et al., 2025). 4.2 Construction of expression vectors driven by root-specific promoters To achieve targeted improvement in nitrogen fertilizer utilization efficiency, researchers fused selected nitrogen-related genes with root-specific promoters to construct expression vectors, ensuring that transgenic expression is confined to the root tissues where nitrogen absorption occurs. For instance, promoters such as OsNAR2.1 and OsAnt1 have been successfully applied to drive the preferential expression of rice roots, thereby enhancing the efficiency of nitrogen absorption and assimilation. The use of root-specific promoters can minimize the potential negative impact on non-target tissues and optimize resource allocation. Expression vectors typically contain regulatory elements for stabilizing high-level expression and can integrate reporter genes to monitor transgenic activity. This strategy can achieve precise spatial control of gene expression, thereby enhancing the effectiveness of transgenic methods in improving nitrogen fertilizer utilization efficiency (Chen et al., 2017; Sisharmini et al., 2019; Tiong et al., 2021). 4.3 Genetic transformation and regeneration process of transgenic wheat The genetic transformation of wheat is usually carried out by Agrobacterium-mediated method or gene gun method, introducing expression vectors into embryonic callus or immature embryos. After transformation, complete plants were regenerated from the transformed cells using tissue culture techniques and screened on antibiotic or herbicide culture media to identify successful transforms (Han et al., 2025). Molecular analyses were conducted on the regenerated plants, such as PCR, Southern blotting and expression analysis, to confirm the integration and expression of the transgenes. Screen subsequent generations to ensure the genetic stability and heritability of genetically modified organisms. Optimizing transformation efficiency and regeneration schemes is crucial for the production of transgenic wheat lines driven by root-specific promoters with higher nitrogen utilization efficiency (Zhu et al., 2025). 5 Phenotypic and Molecular Analysis of Transgenic Wheat 5.1 Molecular validation (PCR, qRT-PCR, Southern blot) To confirm whether a genetically modified wheat strain is truly successful, one must first start from the molecular level. The most fundamental approach is to use PCR to detect whether transgenic fragments have integrated into the genome. Sometimes, PCR alone is not enough; Southern blot is also needed to see how many copies have been transferred and where they are inserted. qRT-PCR is also indispensable. It can measure the expression levels of these genes in different tissues, especially paying attention to whether the roots have been successfully activated by the promoter. As Zameer et al. (2025) mentioned, many wheat transgenic materials have copy numbers ranging from 1 to 5, can be stably inherited, and follow Mendelian segregation rules. Although these verification methods are conventional, they are crucial for the subsequent assessment of traits. 5.2 Analysis of root structure, nitrogen uptake ability, and related enzyme activities Can any changes be observed in appearance after genetic modification? Phenotypic testing often focuses on the root. Especially when materials expressing nitrogen-related genes are driven by root-specific promoters, it is generally observed that the roots are more developed and the biomass is larger. This kind of phenomenon is often associated with the nitrogen absorption capacity. However, merely looking at the root length is not enough; the
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