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汪天虹, Tian-Hong WANG*, Ti LIU, Zhi-Hong WU, Shi-Li LIU, Yi LU, and Yin-Bo QU
Acta Biochimica et Biophysica Sinica 2004, 36 (10): 667-672,-0001,():
-1年11月30日
To construct strains of the filamentous fungus Trichoderma reesei with low cellobiohydrolases while high endoglucanase activity, the Pcbh1-eg3-Tcbh1 cassette was constructed and the coding sequence of the cellobiohydrolase I (CBHI) gene was replaced with the coding sequence of the eg3 gene by homologous recombination. Disruption of the cbh1 gene was confirmed by PCR, Southern dot blot and Western hybridization analysis in two transformants denoted as L13 and L29. The filter paper-hydrolyzing activity of strain L29 was 60% of the parent strain Rut C30, and the CMCase activity was increased by 33%. This relatively modest increase suggested that the eg3 cDNA under the control of the cbh1 promoter was not efficiently transcribed as the wild type cbhl gene. However our results confirmed that homologous recombination could be used to construct strains of the filamentous fungus Trichoderma reesei with novel cellulase profile. Such strains are of interest from the basic science perspective and also have potential industrial applications.
Trichoderma reesei, gene replacement, gene disruption, cbh1, eg3
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汪天虹, J. Liu, S.-Y. Sun and T.-H. Wang
Letters in Applied Microbiology 2004, 38, 277-282,-0001,():
-1年11月30日
To construct a yeast one-hybrid system and isolate transcriptional activators. Methods and Results: A 1
ACE II, promoter, trans, c, r, i, p, t, ion activator, T., reesei,, yeast one-hybrid-system,, xyn2.,
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汪天虹, Wei Huang, Tian-Hong Wang, ∗, JingWu & Chun-Qiang Liu-
Biotechnology Letters 24: 395-399, 2002.,-0001,():
-1年11月30日
A mitochondrial gene cluster, encoding proteins homologous to NADH dehydrogenase subunits II and III (ND2 and ND3) and seven tRNAs, from Trichoderma reesei QM9414 was cloned and sequenced. These genes are clustered tandemly on the mitochondrial genome of QM9414. Phylogenetic analysis showed that ND2 and ND3 were most closely related to the mitochondrialND subunits II (71% identity) and III (70% identity) from Podospora anserine. Northern dot blot analysis showed that the nd-and nd3 genes are actively transcribed in the T. reesei mitochondria.
mitochondria, NADH dehydrogenase subunits, sequence analysis, Trichoderma reesei, tRNA
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【期刊论文】Antisense inhibition of xylitol dehydrogenase gene, xdh1 from Trichoderma reesei
汪天虹, T.H. Wang, Y.H. Zhong, W. Huang, T. Liu and Y.W. You
Letters in Applied Microbiology 2005,-0001,():
-1年11月30日
To inhibit xylitol dehydrogenase (XDH) in Trichoderma reesei by antisense inhibition strategy and construct novel strains capable of accumulating xylitol. Methods and Results: The xdh1 antisense expression plasmid pGTA-xdh was constructed by inserting xdh1 DNA fragment inversely between the gpdA promoter and the trpC terminator from Aspergillus nidulans into a pUC19 plasmid backbone. Trichoderma reesei protoplasts were co-transformated with pGTA-xdh and hygromycin B resistance plasmid pAN7-1. Of 20 transformants screened from the selective medium, one transformant with the highest xylitol accumulation, designated ZY15, showed a distinct reduction (c. 52%) in XDH activity compared with the original strain Rut-C30. The results of Southern hybridization and PCR assay showed that the antisense expression cassette of xdh1 was integrated into the genome of T. reesei. The RT-PCR analysis proved that antisense RNA effectively inhibited XDH expression (c. 65%). Xylitol accumulation 2
antisense inhibition, Trichoderma reesei, xdh1,, xylitol, xylitol dehydrogenase.,
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汪天虹, Yao Hua Zhong & Xiao Li Wang & Tian Hong Wang & Qiao Jiang
Appl Microbiol Biotechnol DOI 10.100,-0001,():
-1年11月30日
Filamentous fungus Trichoderma reesei QM9414 was successfully transformed with Agrobacterium tumefaciens AGL-1 for random integration of transforming DNA (T-DNA). Co-cultivation of T. reesei conidia or protoplasts with A. tumefaciens in the presence of acetosyringone resulted in the formation of hygromycin B-resistant fungal colonies with high transformation frequency. Nine randomly selected resistant clones were proved to be stable through mitotic cell division. The integration of the hph gene into T. reesei genome was determined by PCR and dot blot analysis. Transgenic T. reesei strains were analyzed using TAIL-PCR for their T-DNA contents. The results showed that T-DNA inserts occurred evidently by fusing DNA at T-DNA borders via random recombination, which suggests that Agrobacterium-mediated transformation is a potentially powerful tool towards tagged mutagenesis and gene transfer technology for T. reesei.
Agrobacterium-mediated transformation, Trichoderma reesei, T-DNA., Insertional mutagenesis, hygromycin B resistance, TAIL-PCR
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