Zhong Shaobin, Leng Yueqiang, Bolton Melvin D
Department of Plant Pathology, North Dakota State University, Fargo, ND, USA.
Methods Mol Biol. 2012;835:623-33. doi: 10.1007/978-1-61779-501-5_40.
RNA-mediated gene silencing is one of the major tools for functional genomics in fungi and can be achieved by transformation with constructs that express hairpin (hp) RNA with sequences homologous to the target gene(s). To make an hpRNA expression construct, a portion of the target gene can be amplified by PCR and cloned into a vector as an inverted repeat. The generic gene-silencing vectors such as the pSilent1 and pSGate1 have been developed and are available for RNA-mediated gene-silencing studies. In this protocol, we describe construction of hpRNA-expressing constructs using both pSilent1 and pSGate1. With pSilent1, the PCR products of the target gene are inserted into the vector by conventional cloning (i.e., restriction enzyme digestion and ligation). For pSGate1, the PCR products of the target gene are inserted into the vector through the Gateway-directed recombination system. In this chapter, we describe the construction of RNAi vectors for RNA-mediated gene silencing using both pSilent1 and pSGate1.
RNA介导的基因沉默是真菌功能基因组学的主要工具之一，可通过用表达与靶基因序列同源的发夹（hp）RNA的构建体进行转化来实现。为构建hpRNA表达构建体，可通过PCR扩增靶基因的一部分，并作为反向重复序列克隆到载体中。已经开发出了诸如pSilent1和pSGate1等通用的基因沉默载体，可用于RNA介导的基因沉默研究。在本方案中，我们描述了使用pSilent1和pSGate1构建hpRNA表达构建体的方法。对于pSilent1，通过常规克隆（即限制性内切酶消化和连接）将靶基因的PCR产物插入载体。对于pSGate1，通过Gateway定向重组系统将靶基因的PCR产物插入载体。在本章中，我们描述了使用pSilent1和pSGate1构建用于RNA介导的基因沉默的RNAi载体的方法。
