Transformation by complementation of a uracil auxotroph of the hyper lignin-degrading basidiomycete Phanerochaete sordida YK-624
Transformation by complementation of a uracil auxotroph of the hyper lignin-degrading basidiomycete Phanerochaete sordida YK-624
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DOI：
10.1007/s00253-007-1093-7
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发表时间：
2007-10-01
影响因子：
5
通讯作者：
Hirai, Hirofumi
中科院分区：
文献类型：
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作者：
Yamagishi, Kenji;Kimura, Toshiyuki;Hirai, Hirofumi
sordida Phanerochaete YK-624是一种高度降解木质素的黑子菌，比P. chrysosporium或Trametes versicolor具有更高的木质素降解选择性。为构建sordida YK-624的基因转化体系，以紫外光照射和5-氟乙酸抗性组合为选择方案，产生尿嘧啶营养缺陷突变体。利用含有P. sordida YK-624的螺旋状磷酸核糖基转移酶基因的标记质粒pPsURA5，将一株尿嘧啶营养不良菌株UV-64转化为尿嘧啶原生动物。该系统使用2 × 10(7)个原生质体产生了大约50个稳定的变子。Southern blot分析表明，转化后的pPsURA5被异位整合到所有转化子的染色体DNA中。增强的绿色荧光蛋白（EGFP）基因也被引入到UV-64中。转化后的EGFP在P. sordida甘油醛-3-磷酸脱氢酶基因启动子和终止子驱动的共转化子中表达。
Phanerochaete sordida YK-624 is a hyper lignin-degrading basidiomycete possessing greater ligninolytic selectivity than either P. chrysosporium or Trametes versicolor. To construct a gene transformation system for P. sordida YK-624, uracil auxotrophic mutants were generated using a combination of ultraviolet (UV) radiation and 5-fluoroorotate resistance as a selection scheme. An uracil auxotrophic strain (UV-64) was transformed into a uracil prototroph using the marker plasmid pPsURA5 containing the orotate phosphoribosyltransferase gene from P. sordida YK-624. This system generated approximately 50 stable transformants using 2 x 10(7) protoplasts. Southern blot analysis demonstrated that the transformed pPsURA5 was ectopically integrated into the chromosomal DNA of all transformants. The enhanced green fluorescent protein (EGFP) gene was also introduced into UV-64. The transformed EGFP was expressed in the co-transformants driven by P. sordida glyceraldehyde-3-phosphate dehydrogenase gene promoter and terminator regions.