title: Construction of a genomewide RNAi mutant library in rice.
abstract: Long hairpin RNA (hpRNA) transgenes are a powerful tool for gene function studies in plants, but a genomewide RNAi mutant library using hpRNA transgenes has not been reported for plants. Here, we report the construction of a hpRNA library for the genomewide identification of gene function in rice using an improved rolling circle amplification-mediated hpRNA (RMHR) method. Transformation of rice with the library resulted in thousands of transgenic lines containing hpRNAs targeting genes of various function. The target mRNA was down-regulated in the hpRNA lines, and this was correlated with the accumulation of siRNAs corresponding to the double-stranded arms of the hpRNA. Multiple members of a gene family were simultaneously silenced by hpRNAs derived from a single member, but the degree of such cross-silencing depended on the level of sequence homology between the members as well as the abundance of matching siRNAs. The silencing of key genes tended to cause a severe phenotype, but these transgenic lines usually survived in the field long enough for phenotypic and molecular analyses to be conducted. Deep sequencing analysis of small RNAs showed that the hpRNA-derived siRNAs were characteristic of Argonaute-binding small RNAs. Our results indicate that RNAi mutant library is a high-efficient approach for genomewide gene identification in plants.
authors.affiliation: Biotechnology Research Institute, The National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Jie
authors.initials: J
authors.lastname: Zheng
authors.forename: Yanzhong
authors.initials: Y
authors.lastname: Luo
authors.forename: Tao
authors.initials: T
authors.lastname: Xu
authors.forename: Qiuxue
authors.initials: Q
authors.lastname: Zhang
authors.forename: Lan
authors.initials: L
authors.lastname: Zhang
authors.forename: Miaoyun
authors.initials: M
authors.lastname: Xu
authors.forename: Jianmin
authors.initials: J
authors.lastname: Wan
authors.forename: Ming-Bo
authors.initials: MB
authors.lastname: Wang
authors.forename: Chunyi
authors.initials: C
authors.lastname: Zhang
authors.forename: Yunliu
authors.initials: Y
authors.lastname: Fan
mesh_terms: D004252:DNA Mutational Analysis; D055786:Gene Knockout Techniques; D015698:Genomic Library; D005810:Multigene Family; D012275:Oryza / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics; D034622:RNA Interference
keywords: RNAi; Rice; gene silencing; hairpin RNA; library; siRNA