Xue Jiaqi, Zhang Haodong, Zhao Qingmei, Cui Shengwei, Yu Kun, Sun Ruohan, Yu Yongtao
School of Animal Science and Technology, Ningxia University, Yinchuan 750021, China.
College of Biological Science and Engineering, North Minzu University, Yinchuan 750021, China.
J Fungi (Basel). 2023 Aug 3;9(8):822. doi: 10.3390/jof9080822.
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, Section 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 UA003 strain and predicted its high transcriptional activity region combined with dual-luciferase reporter assay. Then, a yeast one-hybrid library of 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.
吲哚里西啶生物碱——苦马豆素（SW）是疯草的主要有毒成分，也是放牧动物疯草中毒的主要原因。内生真菌Section spp.负责疯草中SW的生物合成。swnK基因是真菌SW生物合成中一个多功能复合酶编码基因，其编码产物在真菌以哌啶酸为前体多步催化合成SW过程中起关键作用。然而，swnK基因的转录调控机制仍不清楚。为了鉴定参与疯草内生真菌中swnK基因的转录调节因子，我们首先分析了UA003菌株中swnK基因的上游非编码区，并结合双荧光素酶报告基因检测预测其高转录活性区域。然后，构建了UA003菌株的酵母单杂交文库，并通过该系统筛选可能与swnK基因上游非编码区高转录活性区域结合的转录调节因子。结果表明，高转录活性区域位于swnK基因上游调控区的-656 bp和-392 bp处。通过酵母单杂交系统共筛选出9个候选转录调节因子分子，包括1个C2H2型转录因子、7个注释蛋白和1个未注释蛋白，它们与swnK基因上游高转录活性区域结合。本研究为swnK基因的转录调控提供了新的见解，为进一步探索内生真菌疯草中SW生物合成的调控机制奠定了基础。
