Particle bombardment and Agrobacterium-mediated transformation are the two most widely employed methods in plant genetic transformation. Vector system plays an important role in development of an efficient transgenic technology. At present, RNAi and overexpression vectors are widely used for gene function analysis. Gateway is a kind of easy and quick cloning technology, which is based on site-specific recombination characteristics of lambda phage. A number of genes can be cloned into destination vectors quickly and conveniently by using this technique. In this study, the overexpression vectors pAHC-PSK-OE and pClean-G185-OE, and the RNAi vectors pAHC-PSK-RNAi and pClean-G185-RNAi for biolistic and Agrobacterium-mediated transformation were constructed based on traditional enzyme digestion and ligation method, and gateway technology, respectively. This laid the basis for high-throughput platform for gene function analysis in monocotyledon plants.