Construction of Yeast One-Hybrid Library of Alternaria oxytropis and Screening of Transcription Factors Regulating swnK Gene Expression.

Abstract

The indolizidine alkaloid-swainsonine (SW) is the main toxic component of locoweeds and the main cause of locoweed poisoning in grazing animals. The endophytic fungi, Alternaria Section Undifilum spp., are responsible for the biosynthesis of SW in locoweeds. The swnK gene is a multifunctional complex enzyme encoding gene in fungal SW biosynthesis, and its encoding product plays a key role in the multistep catalytic synthesis of SW by fungi using pipecolic acid as a precursor. However, the transcriptional regulation mechanism of the swnK gene is still unclear. To identify the transcriptional regulators involved in the swnK gene in endophytic fungi of locoweeds, we first analyzed the upstream non-coding region of the swnK gene in the A. oxytropis UA003 strain and predicted its high transcriptional activity region combined with dual-luciferase reporter assay. Then, a yeast one-hybrid library of A. oxytropis UA003 strain was constructed, and the transcriptional regulatory factors that may bind to the high-transcriptional activity region of the upstream non-coding region of the swnK gene were screened by this system. The results showed that the high transcriptional activity region was located at -656 bp and -392 bp of the upstream regulatory region of the swnK gene. A total of nine candidate transcriptional regulator molecules, including a C2H2 type transcription factor, seven annotated proteins, and an unannotated protein, were screened out through the Y1H system, which were bound to the upstream high transcriptional activity region of the swnK gene. This study provides new insight into the transcriptional regulation of the swnK gene and lays the foundation for further exploration of the regulatory mechanisms of SW biosynthesis in fungal endophytic locoweeds.