Reiser J, Walther I S, Fraefel C, Fiechter A
Institute of Biotechnology, Swiss Federal Institute of Technology, Zürich.
Appl Environ Microbiol. 1993 Sep;59(9):2897-903. doi: 10.1128/aem.59.9.2897-2903.1993.
Two methods allowing the analysis of expression of specific lignin peroxidase (LPO) genes from white rot fungi are presented. In the first method, degenerate oligonucleotide primers derived from amino acid sequence motifs held in common among all members of the LPO gene family are used to prime the polymerase chain reaction (PCR) amplification of LPO-related nucleotide sequences from cDNA prepared by using RNA from ligninolytic cultures. The PCR products are cloned and analyzed by restriction cleavage and DNA sequencing. This method was applied to the analysis of transcripts from carbon-limited cultures of Phanerochaete chrysosporium BKM-F-1767, revealing two major classes of PCR products. One class showed DNA sequences with a high degree of similarity to the previously described CLG4 cDNA sequence (H. A. De Boer, Y. Zhang, C. Collins, and C. A. Reddy, Gene 60:93-102, 1987), whereas the other harbored DNA sequences with similarities to the L18 cDNA sequence previously described for P. chrysosporium OGC101 (T. G. Ritch, Jr., V. J. Nipper, L. Akileswaran, A. J. Smith, D. G. Pribnow, and M. H. Gold, Gene 107:119-126, 1991). The second method is based on nuclease protection assays involving isoenzyme-specific RNA probes. By using this method, the L18-related gene of P. chrysosporium BKM-F-1767 was found to be expressed under conditions of carbon and of nitrogen limitation, although the transcript levels were found to be higher in carbon-limited cultures. Furthermore, it was found that omission of veratryl alcohol addition to the culture did not affect the levels of the L18-related transcripts in carbon-limited cultures.
本文介绍了两种用于分析白腐真菌中特定木质素过氧化物酶（LPO）基因表达的方法。第一种方法是，利用从LPO基因家族所有成员共有的氨基酸序列基序衍生而来的简并寡核苷酸引物，对通过木质素分解培养物的RNA制备的cDNA中的LPO相关核苷酸序列进行聚合酶链反应（PCR）扩增。PCR产物被克隆，并通过限制性酶切和DNA测序进行分析。该方法被应用于分析黄孢原毛平革菌BKM-F-1767碳限制培养物中的转录本，揭示了两类主要的PCR产物。一类显示出与先前描述的CLG4 cDNA序列（H. A. De Boer、Y. Zhang、C. Collins和C. A. Reddy，《基因》60:93 - 102，1987）高度相似的DNA序列，而另一类含有与先前为黄孢原毛平革菌OGC101描述的L18 cDNA序列（T. G. Ritch, Jr.、V. J. Nipper、L. Akileswaran、A. J. Smith、D. G. Pribnow和M. H. Gold，《基因》107:119 - 126，1991）相似的DNA序列。第二种方法基于涉及同工酶特异性RNA探针的核酸酶保护分析。通过使用该方法，发现黄孢原毛平革菌BKM-F-1767的L18相关基因在碳限制和氮限制条件下均有表达，尽管在碳限制培养物中转录水平更高。此外，发现在碳限制培养物中不添加藜芦醇不会影响L18相关转录本的水平。
