A novel cold-adaptive xylanolytic Penicillium strain FS010 was isolated from Yellow sea sediments. The marine fungus grew well from 4 to 20

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.

将在木聚糖上生长的瑞氏木霉（Trichodermareesei）RutC230的cDNA文库全部质粒转化已携带有毕赤氏酵母（Pichiastipitis）木糖还原酶基因的重组酿酒酵母（Saccharomycescerevisiae）菌株H475，在H475中构建了瑞氏木霉的CDNA表达亚文库。在以木糖为唯一碳源的选择性酵母合成培养基上，从该亚文库中筛选到瑞氏木霉木糖醇脱氢酶CDNA基因，该基因片段长为113kbSouthern、Northern印迹杂交分析和蛋白质凝胶电泳结果表明该基因确实来源于瑞氏木霉，所编码蛋白质分子量约为40kDa，携带有毕赤氏酵母木糖还原酶和瑞氏木霉木糖醇脱氢酶基因的重组酵母能够在以木糖为唯一碳源的培养基上生长，并能将90%以上的木糖转化为木糖醇、乙醇和其它副产品。

用20种不同的碳源（包括单一碳源和混合碳源）分别培养瑞氏木霉（Trichodermareesei）QM9414。通过一系列Northern杂交分析检测瑞氏木霉木糖还原酶（XR），木糖醇脱氢酶（XDH）以及转醛醇酶（TAL）mRNA的表达情况。实验结果证实,槐糖和木二糖是xr和xdh表达的强诱导物，阿拉伯糖和乳糖也有较强的诱导作用。葡萄糖在培养基中的存在阻遏该二基因的表达。当葡萄糖耗尽以后，培养基中不存在任何诱导物的情况下，xr和xdh以一定的基础水平进行转录。相比较,tal基因在每种碳源上都是强表达。

采用PCR技术体外扩增获得了瑞氏木霉外切葡聚糖纤维二糖水解酶（CBH）启动子和终止子序列。并以大肠杆菌质粒PUC19为骨架，在该启动子和终止子序列间加入多克隆位点，构建了瑞氏木霉强表达整合型载体PTRIL。以质粒PAN721为模板，体外扩增了带有潮霉素磷酸转移酶（hph）基因的DNA片段，将hph插入PTRIL的cbh1启动子和终止子序列之间，构建了Pcbh1-hph-Tcbh1表达盒。用此表达盒转化瑞氏木霉C30原生质体,在潮霉素平板上得到15株抗性转化子。对其中的H1转化子进行了PCR和Southern印迹分析，证实hph基因确实整合到转化子染色体DNA上，并在Pcbh1启动子控制下进行高效表达。转化子H1对潮霉素抗性达150mgPL，比出发菌株提高2倍.瑞氏木霉强表达整合型载体PTRIL的构建成功为开展瑞氏木霉分子生物学研究以及进一步的工程菌株构建工作奠定了基础。