Triticeae Genomics and Genetics, 2025, Vol.16, No.2, 79-91 http://cropscipublisher.com/index.php/tgg 77 The qRT-PCR results confirmed that the transgenic plants were transcribing the introduced genes at significant levels, whereas the non-transgenic control plants showed no such transcripts (as expected). In fact, the Chi gene mRNA levels in three independent transgenic lines were dozens of times higher than in the controls, indicating that the Ubiquitin promoter was driving very high expression (constitutive “on” expression) of the chitinase gene (Chen et al., 2023). The AMP gene was also clearly transcribed in its respective transgenic lines. The qRT-PCR data showed that AMP mRNA was readily detectable in leaves and stems, but much lower in roots - likely reflecting the tissue-specific activity of the rice Actin promoter (which tends to be strong in green tissues but weaker in roots). Interestingly, the expression levels of the transgenes varied among different lines. Some lines had higher mRNA levels than others. This is commonly due to “position effects” - depending on where in the genome the transgene lands, it might be expressed more or less strongly. Also, lines with multiple insertions sometimes show gene silencing effects. In fact, the researchers noted that lines which had a single copy of the Chi gene often had higher chitinase mRNA levels than lines that had two copies. The presence of two copies could trigger a slight silencing or simply might land in less favorable genomic spots. They also checked the transgene expression at different growth stages. Notably, the antifungal gene transcripts remained high during important stages like jointing and heading. So, the transgenes weren’t just active in seedlings; they continued to be expressed throughout the plant’s development. This is good news, because it means the antifungal proteins would be present during the times when the plant is most vulnerable to certain diseases (heading is when Fusarium can infect the spikes, for instance). These consistent expression levels suggest that the antifungal genes could provide continuous protection throughout the growing season (Al-Sayaydeh et al., 2024). 4.2 Protein detection via western blot or ELISA After confirming that the transgenes produced mRNA, the next question was whether that mRNA was being translated into proteins - and how much of the antifungal proteins had accumulated in the plants. The researchers used immunological assays to detect and quantify the Chi and AMP proteins in the transgenic barley. For the Chi protein (chitinase), which has a known molecular weight of around 28 kDa and for which antibodies are readily available, they performed a Western blot analysis. They extracted soluble protein from the leaves of transgenic barley and control plants, separated the proteins by SDS-PAGE (a type of gel electrophoresis), and then transferred them onto a membrane. The membrane was probed with a polyclonal antibody that specifically recognizes plant chitinases (Mirzaee et al., 2021). On the Western blot, the transgenic plant samples showed a clear band at about 28 kDa corresponding to the chitinase, whereas the control plant samples had no such band. This told us that the chitinase protein was indeed being produced in the transgenic barley and not in the normal barley. Furthermore, by comparing the intensity of the band to known standards or controls, they estimated that the chitinase protein made up roughly 0.5%-1% of the total soluble protein in the transgenic leaves. That is a substantial level of expression (for comparison, many transgenic enzymes in plants often accumulate around 1% or less of total protein, so this was in the expected range). It also matched what has been seen in other transgenic plants expressing foreign enzymes - so nothing unusual was happening in terms of protein accumulation. Detecting the AMP protein was a bit trickier. The antimicrobial peptide is very small (around 5 kDa) and it’s hard to see such small proteins on a standard Western blot. Additionally, highly specific antibodies for the AMP were not readily available. To overcome this, the team used an indirect ELISA (enzyme-linked immunosorbent assay) to quantify the AMP protein. They basically created a sandwich ELISA: plates were coated with extracts from the barley plants, and a known concentration series of a synthetic AMP peptide was used as a standard. They then used a polyclonal antibody against the AMP and a secondary antibody with an enzyme attached (HRP, which causes a color change) to detect how much AMP was in the samples by measuring the color intensity. This ELISA revealed that the transgenic barley leaves contained the AMP peptide at levels of a few micrograms per gram of fresh weight. The AMP was also present in the seeds of the transgenic plants, though at slightly lower concentrations than in the leaves and stems. This distribution makes sense given that the Actin promoter used
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