Utilizing the Fungal Bicistronic System for Multi-Gene Expression to Generate Insect-Resistant and Herbicide-Tolerant Maize.

Developing simple and efficient multi-gene expression systems is crucial for multi-trait improvement or bioproduction in transgenic plants. In previous research, an -based bicistronic system from the nonpathogenic fungus efficiently expressed multiple enzyme proteins in yeast and maize, and the heterologous enzymes successfully performed their catalytic activity to reconstruct the biosynthetic pathway in the host organism. Unlike enzyme proteins, some heterologous functional proteins (such as insecticidal proteins) are dose-dependent and they need to express sufficient levels to perform their biological functions. It remains unclear whether the -based bicistronic system can achieve high expression of the functional proteins for practical applications in crops. In this study, two (Bt) insecticidal genes, and , were linked via to form a bicistron, while two glyphosate resistance genes, and , served as monocistronic selectable marker genes. Regenerated maize plants were produced through genetic transformation. RNA and immunoblot analyses revealed that the -- bicistron was transcribed as a single transcript, which was then translated into two separate proteins. Notably, the transcription and translation of were significantly positively correlated with those of . Through ELISA and leaf bioassay, we identified two transgenic maize lines, VICGG-15 and VICGG-20, that exhibited high insecticidal activity against fall armyworm (FAW; ) and Asian corn borer (ACB; ), both of which had high expression of Vip3Aa and Cry1Ab proteins. Subsequent evaluations, including silk, ear, and field bioassays, as well as glyphosate tolerance assessments, indicated that the VICGG-15 plants displayed high resistance to FAW and ACB, and could tolerate up to 3600 g acid equivalent (a.e.) glyphosate per hectare without adversely affecting phenotype or yield. Our finding established that the -based bicistronic system can achieve high expression of functional proteins in maize, and it is a potential candidate for multi-gene assembly and expression in plants.