Zhu Tingheng, Wang Weixia, Yang Xiao, Wang Kun, Cui Zhifeng
Zhejiang University of Technology, Hangzhou, China.
Plasmid. 2009 Sep;62(2):128-33. doi: 10.1016/j.plasmid.2009.06.005. Epub 2009 Jun 21.
We report the construction of two Gateway fungal expression vectors pCBGW and pGWBF. The pCBGW was generated by introducing an expression cassette, which consists of a Gateway recombinant cassette (attR1-Cmr-ccdB-attR2) under the control of fungal promoter PgpdA and a terminator TtrpC, into the multiple cloning site of fungal vector pCB1004. The pGWBF is a binary vector, which was generated from the plant expression vector pGWB2 by replacing the CaMV35S promoter with PgpdA. The pGWBF can be transformed into fungi efficiently with Agrobacterium-mediated transformation. The applicability of two newly constructed vectors was tested by generating the destination vectors pGWBF-GFP and pCBGW-GFP and examining the expression of GFP gene in Trichoderma viride and Gibberella fujikuroi, respectively. Combining with the advantage of Gateway cloning technology, pCBGW and pGWBF will be useful in fungi for large-scale investigation of gene functions by constructing the interested gene destination/expression vectors in a high-throughput way.
我们报道了两种Gateway真菌表达载体pCBGW和pGWBF的构建。pCBGW是通过将一个表达盒引入真菌载体pCB1004的多克隆位点而产生的，该表达盒由在真菌启动子PgpdA和终止子TtrpC控制下的Gateway重组盒（attR1-Cmr-ccdB-attR2）组成。pGWBF是一种二元载体，它是由植物表达载体pGWB2通过用PgpdA替换CaMV35S启动子而产生的。pGWBF可以通过农杆菌介导的转化有效地转化为真菌。通过构建目的载体pGWBF-GFP和pCBGW-GFP，并分别检测绿色荧光蛋白（GFP）基因在绿色木霉和藤仓赤霉中的表达，测试了这两种新构建载体的适用性。结合Gateway克隆技术的优势，pCBGW和pGWBF将有助于在真菌中通过高通量方式构建感兴趣的基因目的/表达载体，从而大规模研究基因功能。
