Cotransformation of Trichoderma harzianum with β-glucuronidase and green fluorescent protein genes provides a useful tool for monitoring fungal growth and activity in natural soils
Cotransformation of Trichoderma harzianum with β-glucuronidase and green fluorescent protein genes provides a useful tool for monitoring fungal growth and activity in natural soils
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DOI：
10.1128/aem.66.2.810-815.2000
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发表时间：
2000-02-01
影响因子：
4.4
通讯作者：
Knudsen, GR
中科院分区：
文献类型：
--
作者：
Bae, YS;Knudsen, GR
使用聚乙二醇介导的转化，将哈茨木霉与编码绿色荧光蛋白 (GFP)、β-葡萄糖醛酸酶 (GUS) 和潮霉素 B (hygB) 抗性的基因共转化，尽管在没有选择压力的情况下连续传代培养，但一种共转化体 (ThzID1-M3) 有丝分裂稳定 6 个月。 ThzID1-M3形态与野生型相似；然而，琼脂上的菌丝生长速度降低了。将ThzID1-M3制成海藻酸钙颗粒，并放置在非无菌土壤中的埋置载玻片上，并将其在核盘菌菌核中生长、形成孢子和定植的能力与野生型菌株进行比较。野生型和转化株的菌核定植水平均高于本地木霉属。在未经处理的对照中。野生型和共转化菌株之间的定植水平没有显着差异；然而，GEP 和 GUS 标记基因的存在使得引入的木霉能够与本地菌株区分开来。 GFP 活性是无损监测天然土壤中转化体菌丝生长的有用工具。在紫外落射荧光显微镜下，共转化菌丝的绿色清晰可见，而同一样品中的本土真菌几乎看不见。在土壤中培养的前 3 天内观察到发绿色荧光的分生孢子梗和分生孢子，随后形成末端和居间厚垣孢子，随后老菌丝段解体。向回收的载玻片中添加 5-溴-4-氯-3-吲哚基-β-D-葡萄糖醛酸 (X-Gluc) 底物证实了土壤中 GUS 和 GFP 的活性。我们的结果表明，与 GFP 和 GUS 共转化可以为检测和监测释放到土壤中的哈茨木霉特定菌株提供有价值的工具。
Trichoderma harzianum was cotransformed with genes encoding green fluorescent protein (GFP), beta-glucuronidase (GUS), and hygromycin B (hygB) resistance, using polyethylene glycol-mediated transformation, One cotransformant (ThzID1-M3) was mitotically stable for 6 months despite successive subculturing without selection pressure. ThzID1-M3 morphology was similar to that of the wild type; however, the mycelial growth rate on agar was reduced. ThzID1-M3 was formed into calcium alginate pellets and placed onto buried glass slides in a nonsterile soil, and its ability to grow, sporulate, and colonize sclerotia of Sclerotinia sclerotiorum was compared with that of the wild-type strain. Wild-type and transformant strains both colonized sclerotia at levels above those of indigenous Trichoderma spp. in untreated controls. There were no significant differences in colonization levels between wild-type and cotransformant strains; however, the presence of the GEP and GUS marker genes permitted differentiation of introduced Trichoderma from indigenous strains. GFP activity was a useful tool for nondestructive monitoring of the hyphal growth of the transformant in a natural soil. The green color of cotransformant hyphae was clearly visible with a UV epifluorescence microscope, while indigenous fungi in the same samples were barely visible. Green-fluorescing conidiophores and conidia were observed within the first 3 days of incubation in soil, and this was followed by the formation of terminal and intercalary chlamydospores and subsequent disintegration of older hyphal segments. Addition of 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-Gluc) substrate to recovered glass slides confirmed the activity of GUS as well as GFP in soil. Our results suggest that cotransformation with GFP and GUS can provide a valuable tool for the detection and monitoring of specific strains of T. harzianum released into the soil.