Triticeae Genomics and Genetics, 2025, Vol.16, No.2, 79-91 http://cropscipublisher.com/index.php/tgg 74 break down starch. Many pathogenic fungi need to digest starch from seeds to get nutrients when infecting cereal grains. By flooding the system with BDAI, the plant essentially cuts off the fungus’s food supply, slowing or preventing the infection (Li et al., 2021). Detoxification and stress tolerance: Some antifungal genes help the plant by breaking down fungal toxins or by enhancing the plant’s ability to cope with oxidative stress caused by infection. For instance, the Fhb7 enzyme mentioned earlier detoxifies Fusarium toxins, preventing toxin buildup in the plant. Additionally, some genes that boost levels of certain transcription factors or signaling molecules can strengthen the plant’s overall immune readiness, leading to heightened systemic resistance (Martínez-Culebras et al., 2021). These multifaceted modes of action are a big advantage. Unlike a chemical fungicide that might hit a pathogen in just one way, these proteins attack fungi on several fronts at once. This makes it harder for the fungus to develop resistance to the plant’s defenses. 2.3 Successful applications in other crops Using antifungal genes in transgenic breeding has already met with success in many crops, providing a valuable reference for our barley work. In wheat, for example, adding a barley class II chitinase gene resulted in transgenic wheat plants with enhanced resistance to powdery mildew and Fusarium head blight - one of the early success stories in this area. In rice, scientists created transgenic lines that could resist rice blast and sheath blight by inserting a dual-function gene encoding both a chitinase and a glucanase from barley (Yan et al., 2015; Chiu et al., 2022). Antimicrobial peptide genes have also shown great promise in crops. Researchers have introduced insect-derived AMP genes like Metchnikowin into fruit trees and horticultural plants, and found that those transgenic plants had strong field immunity to powdery mildew. Another landmark case involves the Fhb7 gene mentioned earlier: originally, Fhb7 came from an endophytic fungus and made its way into a wild wheat relative via horizontal gene transfer. Chinese scientists cloned Fhb7 and then inserted it into wheat using transgenics. The result was wheat that had broad resistance to Fusarium head blight and crown rot without any yield loss across different wheat varieties. This breakthrough - essentially giving wheat a gene it never had - is seen as a milestone in using transgenes to improve disease resistance in major crops. Similar strategies are being attempted in barley as well. For example, introducing genes that encode antifungal proteins into barley has been tested to combat diseases like powdery mildew and leaf spot, with promising outcomes. There’s also progress with a technique called host-induced gene silencing (HIGS) in cereals: the plant is engineered to produce RNAs that specifically silence critical fungal genes during infection, thereby increasing the plant’s resistance. All these success stories reinforce that adding antifungal protein genes via genetic engineering is both feasible and effective. It gives breeders a new set of tools, especially for diseases where traditional breeding doesn’t have good solutions. It’s expected that applying these proven strategies to barley will yield similar positive results. 3 Construction and Screening of Transgenic Barley 3.1 Gene cloning and vector construction Holásková et al. (2018) selected two target antifungal genes: one was a chitinase gene (nicknamed Chi) from plants, and the other was an antimicrobial peptide gene (nicknamed AMP) from insects. These two genes represent two different types of antifungal proteins - Chi encodes an enzyme that degrades fungal cell walls, and AMP encodes a peptide that can perforate fungal membranes. They first obtained the full-length cDNA sequences of each gene. The Chi gene was cloned from a barley tissue cDNA library (they used PCR to amplify the coding sequence from barley samples), and the AMP gene was chemically synthesized with optimized codons to ensure it would be efficiently translated in barley cells (plants sometimes prefer certain DNA “spellings” for genes, so codon optimization can help boost protein production). Each gene was then inserted into a plant expression vector. To get strong expression in barley, the Chi gene was placed under the control of a powerful promoter (they used the maize Ubiquitin promoter) and a signal peptide
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