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An efficient transformation system for Trichoderma atroviride using the pyr4 gene as a selectable marker
Brazilian Journal of Microbiology ( IF 2.2 ) Pub Date : 2020-07-06 , DOI: 10.1007/s42770-020-00329-7
Gabriela Calcáneo-Hernández 1 , Erick Rojas-Espinosa 1 , Fidel Landeros-Jaime 1 , José Antonio Cervantes-Chávez 1 , Edgardo Ulises Esquivel-Naranjo 1
Affiliation  

The development of an efficient transformation system is essential to enrich the genetic understanding of Trichoderma atroviride. To acquire an additional homologous selectable marker, uracil auxotrophic mutants were generated. First, the pyr4 gene encoding OMP decarboxylase was replaced by the hph marker gene, encoding a hygromycin phosphotransferase. Then, uracil auxotrophs were employed to determine that 5 mM uracil restores their growth and conidia production, and 1 mg ml-1 is the lethal dose of 5-fluoroorotic acid in T. atroviride. Subsequently, uracil auxotrophic strains, free of a drug-selectable marker, were selected by 5-fluoroorotic acid resistance. Two different deletions in pyr4 were mapped in four auxotrophs, encoding a protein with frameshifts at the 310 and 335 amino acids in their COOH-terminal. Six auxotrophs did not have changes in the pyr4 ORF even though a specific cassette to delete the pyr4 was used, suggesting that 5-FOA could have mutagenic activity. The Ura-1 strain was selected as a genetic background to knock out the MAPKK Pbs2, MAPK Tmk3, and the blue light receptors Blr1/Blr2, using a short version of pyr4 as a homologous marker. The ∆tmk3 and ∆pbs2 mutants selected with pyr4 or hph marker were phenotypically identical, highly sensitive to different stressors, and affected in photoconidiation. The ∆blr1 and ∆blr2 mutants were not responsive to light, and complementation of uracil biosynthesis did not interfere in the expression of blu1, grg2, phr1, and env1 genes upregulated by blue light. Overall, uracil metabolism can be used as a tool for genetic manipulation in T. atroviride.

中文翻译:

使用 pyr4 基因作为选择标记的木霉属 atroviride 高效转化系统

开发有效的转化系统对于丰富对木霉属 atroviride 的遗传理解至关重要。为了获得额外的同源选择标记,产生了尿嘧啶营养缺陷型突变体。首先,编码 OMP 脱羧酶的 pyr4 基因被 hph 标记基因取代,编码潮霉素磷酸转移酶。然后,使用尿嘧啶营养缺陷型来确定 5 mM 尿嘧啶恢复其生长和分生孢子的产生,1 mg ml-1 是 5-氟乳清酸在 T. atroviride 中的致死剂量。随后,通过 5-氟乳清酸抗性筛选出不含药物选择标记的尿嘧啶营养缺陷型菌株。pyr4 中的两个不同缺失被定位在四个营养缺陷型中,编码的蛋白质在其 COOH 末端的 310 和 335 个氨基酸处发生移码。即使使用了删除 pyr4 的特定盒式磁带,六个营养缺陷型的 pyr4 ORF 也没有变化,这表明 5-FOA 可能具有诱变活性。选择 Ura-1 菌株作为遗传背景以敲除 MAPKK Pbs2、MAPK Tmk3 和蓝光受体 Blr1/Blr2,使用短版 pyr4 作为同源标记。用 pyr4 或 hph 标记选择的 ∆tmk3 和 ∆pbs2 突变体表型相同,对不同的压力源高度敏感,并且在光分生孢子作用中受到影响。Δblr1 和 Δblr2 突变体对光没有反应,尿嘧啶生物合成的互补不干扰蓝光上调的 blu1、grg2、phr1 和 env1 基因的表达。总体而言,尿嘧啶代谢可用作 T. atroviride 基因操作的工具。
更新日期:2020-07-06
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