Jimenez-Sanchez Celia, Wilson Nina, McMaster Nicole, Gantulga Dash, Freedman Benjamin G, Senger Ryan, Schmale David G
School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, VA, 24061-0390, USA.
Department of Biological Systems Engineering, Virginia Tech, Blacksburg, VA, 24061, USA.
Toxicon X. 2020 Jan 23;5:100023. doi: 10.1016/j.toxcx.2020.100023. eCollection 2020 Mar.
New strategies are needed to mitigate the mycotoxin deoxynivalenol (DON) in feed and food products. Microbial DNA fragments were generated from a library of DON-tolerant microorganisms. These fragments were screened in DON-sensitive yeast strains for their ability to modify or transport DON. Fragments were cloned into a PCR8/TOPO vector, and recombined into the yeast vector, pYES-DEST52. Resulting yeast transformants were screened in the presence of 100 ppm DON. Transformants that were able to grow in the presence of DON were plated on a selective medium, and the cloned microbial DNA fragments were sequenced. BLAST queries of one microbial DNA fragment (4D) showed a high degree of similarity to an ABC transporter. A series of screening and inhibition assays were conducted with a transport inhibitor (propanol), to test the hypothesis that 4D is a mycotoxin transporter. DON concentrations did not change for yeast transformants expressing 4D. The ability of yeast transformants expressing 4D to transport DON was inhibited by the addition of propanol. Moreover, yeast transformants expressing a known efflux pump (PDR5) showed similar trends in propanol transport inhibition compared to 4D. Future work should consider mycotoxin transporters such as 4D to the development of transgenic plants to limit DON accumulation in seeds.
需要新的策略来降低饲料和食品中的霉菌毒素脱氧雪腐镰刀菌烯醇（DON）。从耐DON微生物文库中产生微生物DNA片段。在对DON敏感的酵母菌株中筛选这些片段修饰或转运DON的能力。将片段克隆到PCR8/TOPO载体中，并重组到酵母载体pYES-DEST52中。在100 ppm DON存在的情况下筛选所得的酵母转化体。能够在DON存在下生长的转化体接种在选择性培养基上，并对克隆的微生物DNA片段进行测序。对一个微生物DNA片段（4D）的BLAST查询显示与一种ABC转运蛋白有高度相似性。用转运抑制剂（丙醇）进行了一系列筛选和抑制试验，以检验4D是霉菌毒素转运蛋白这一假设。表达4D的酵母转化体的DON浓度没有变化。添加丙醇抑制了表达4D的酵母转化体转运DON的能力。此外，与4D相比，表达已知外排泵（PDR5）的酵母转化体在丙醇转运抑制方面表现出相似的趋势。未来的工作应考虑将4D等霉菌毒素转运蛋白用于开发转基因植物，以限制种子中DON的积累。
