Building blocks method, efficient gene silencing of multiple genes

在后基因组时代，功能基因组学的一个主要障碍是基因家族成员之间的功能冗余。为了克服这一困难，我们开发了一种名为构建块方法的RNAi策略，即从一个目的基因中选择一个40-60bp的序列，然后将来自多个基因家族成员的序列组合在一起，通过合成DNA来触发人工RNAi。本研究采用构建块方法对木聚糖酶和纤维素酶两个基因家族进行了敲除，以阐明它们在稻瘟病菌致病中的作用。在得到的KD突变体中，所有目的基因的转录水平都显著降低。KD突变体感病叶片的病斑数量、侵染率和侵染细胞的程度均降低。因此，我们得出结论，木聚糖酶和纤维素酶在稻曲霉在感染植株中的垂直穿透和水平扩展中起着重要的作用。

In the post-genomics era, a major obstacle to functional genomics is functional redundancy among gene family members. To overcome this difficulty, we have developed an RNAi strategy designated building blocks method, in which a 40-60 bp sequence is chosen from a target gene, and such sequences from multiple gene family members were combined to make an artificial RNAi trigger by synthetic DNA. In this study, the building blocks method was applied to knock-down (KD) two gene families, xylanases and cellulases, to elucidate their roles in the pathogenicity of the blast fungus, Magnaporthe oryzae. In the resulting KD mutants, the transcript levels of all the target genes were significantly decreased. The number of lesions, rate of penetration, and extent of infected cells were all reduced in KD mutant-infected leaves. Thus, we concluded that xylanases and cellulases play significant roles in vertical penetration and horizontal expansion of M. oryzae in infected plants.