Xu J, Nogawa M, Okada H, Morikawa Y
Department of Bioengineering, Nagaoka University of Technology, Niigata, Japan.
Appl Microbiol Biotechnol. 2000 Sep;54(3):370-5. doi: 10.1007/s002530000410.
The characteristics of regulation of the gene encoding the third xylanase (Xyn III) of a filamentous fungus, Trichoderma reesei PC-3-7, were studied by Northern blot analysis. A partial DNA sequence (185 bp) of the xyn3 gene was obtained by PCR amplification of genomic DNA of T. reesei PC-3-7 and sequenced. This xyn3 gene fragment was used as a probe for Southern and Northern blot analysis. The expression of the xyn3 gene was regulated at the transcriptional level. The xyn3 mRNA was expressed in mycelia of T. reesei PC-3-7 induced by Avicel, L-sorbose and sophorose, but not by xylose, xylooligosaccharides and birchwood xylan. Furthermore, it was observed that xyn3 was synchronously expressed with egll but not with xyn1 and xyn2 by L-sorbose, indicating that the xyn3 gene may be coordinately expressed with cellulase genes. By Southern blot analysis, the xyn3 gene was confirmed to exist as a single copy in both strains of T. reesei PC-3-7 and QM9414. However, no xyn3 mRNA appeared in the mycelia induced by any kind of inducers in T. reesei QM9414 even when total RNA was used in large excess, suggesting that the xyn3 gene in T. reesei QM9414 is in the dormant state and cannot be expressed. Therefore, T. reesei PC-3-7 may be a very useful strain for elucidating the induction mechanism of xylanase biosynthesis by cellulosic and xylanosic substrates, and also the regulatory correlation between cellulase and xylanase induction.
通过Northern印迹分析研究了丝状真菌里氏木霉PC - 3 - 7的第三种木聚糖酶(Xyn III)编码基因的调控特性。通过对里氏木霉PC - 3 - 7基因组DNA进行PCR扩增获得了xyn3基因的部分DNA序列(185 bp)并进行了测序。该xyn3基因片段用作Southern和Northern印迹分析的探针。xyn3基因的表达在转录水平受到调控。xyn3 mRNA在由微晶纤维素、L - 山梨糖和槐糖诱导的里氏木霉PC - 3 - 7菌丝体中表达,但在木糖、木寡糖和桦木木聚糖诱导下不表达。此外,观察到L - 山梨糖诱导时xyn3与egll同步表达,但与xyn1和xyn2不同步,这表明xyn3基因可能与纤维素酶基因协同表达。通过Southern印迹分析,证实xyn3基因在里氏木霉PC - 3 - 7和QM9414两个菌株中均以单拷贝形式存在。然而,即使使用大量过量的总RNA,在里氏木霉QM9414的任何诱导剂诱导的菌丝体中都未出现xyn3 mRNA,这表明里氏木霉QM9414中的xyn3基因处于休眠状态且无法表达。因此,里氏木霉PC - 3 - 7可能是用于阐明纤维素和木聚糖底物诱导木聚糖酶生物合成的诱导机制以及纤维素酶和木聚糖酶诱导之间调控相关性的非常有用的菌株。