Li Shengyan, Wen Ning, Lv Wenjie, Zhang Mingjun, Wang Yinxiao, Lang Zhihong
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.
College of Plant Protection, Jilin Agricultural University, Changchun, China.
Front Plant Sci. 2025 May 13;16:1579465. doi: 10.3389/fpls.2025.1579465. eCollection 2025.
Codon optimization is critical for high expression of foreign genes in heterologous systems. The gene from is a promising candidate for controlling .
To develop insect-resistant maize, we designed two codon-optimized variants ( and ) based on maize codon usage bias. Both recombinant proteins expressed in exhibited high insecticidal activity. However, in transgenic maize, Vip3Aa11-m1 exhibited strong insecticidal activity against and , while Vip3Aa11-m2 lost activity despite identical amino acid sequences. RT-PCR analysis confirmed that both genes were transcribed correctly, but western blot results demonstrated a smaller product for , suggesting a translation-level alteration. Segment replacement and point mutation experiments in maize protoplasts demonstrated that the synonymous codon AAT (Asn) at the fourth amino acid position in was associated with the production of a truncated protein, suggesting that the AAT codon may influence the selection of the translation initiation site, potentially shifting it to a downstream ATG (Met) codon.
These findings not only reveal the critical role of codon context in translation initiation and protein integrity but also provide a novel strategy for optimizing foreign genes in crop improvement, particularly offering valuable insights for engineering insect-resistant maize using Bt genes.
密码子优化对于外源基因在异源系统中的高表达至关重要。来自[具体来源]的基因是控制[具体对象]的一个有前景的候选基因。
为培育抗虫玉米，我们基于玉米密码子使用偏好设计了两个密码子优化的[基因名称]变体（Vip3Aa11-m1和Vip3Aa11-m2）。在[表达系统名称]中表达的两种重组蛋白均表现出高杀虫活性。然而，在转基因玉米中，Vip3Aa11-m1对[害虫名称1]和[害虫名称2]表现出强杀虫活性，而Vip3Aa11-m2尽管氨基酸序列相同却失去了活性。RT-PCR分析证实两个基因均正确转录，但蛋白质印迹结果显示Vip3Aa11-m2产生的产物较小，表明存在翻译水平的改变。玉米原生质体中的片段替换和点突变实验表明，Vip3Aa11-m2中第四个氨基酸位置的同义密码子AAT（Asn）与截短蛋白的产生有关，这表明AAT密码子可能影响翻译起始位点的选择，有可能将其转移到下游的ATG（Met）密码子。
这些发现不仅揭示了密码子上下文在翻译起始和蛋白质完整性中的关键作用，还为作物改良中外源基因的优化提供了一种新策略，特别是为利用Bt基因培育抗虫玉米提供了有价值的见解。
