Rnai-based Construction Of A Mutant Library In Arabidopsis

RNA interference (RNAi) is a conserved mechanism of gene expression and regulation in most organisms, and plays an important role in developmental regulation and genome maintainence. It is a complex process involving multiple steps and factors, and greatly relies on double-stranded RNA derived siRNAs which are targeted to the complementary mRNA and eventually cause mRNA degredation. Due to its high efficiency in gene expression inhibition, RNAi has been developed into a powerful tool for genetic analysis. A genome-wide analysis of gene function has already been performed using RNAi in nematode, Drosophila and mammalian cells; however, there are still no such reports in plants at whole-genome level.In this study, 3 Arabidopsis cDNA libraries were converted into an RNAi library based on rolling circle amplification-mediated hairpin RNA system (RMHR). The RNAi library was composed of 6.85×105 clones, out of which 12,326 clones were randomly chosen for sequencing and found to represent 2,593 unique genes accounting for 21% of the seqeenced clones and 10% of the total predicted Araibidopsis genes. The results indicate that a nearly whole genome-covered RNAi library was constructed.Over 5,000 Arabidopsis transgenic RNAi lines were obtained via Agrobacterium tumefaciens-mediated transformation of the RNAi library above mentioned, and part of them displayed various phenotype. Expession analysis of the sequenced mutant lines revealed that the target transcripts were significantly decreased, indicating that hpRNA functioned in transgenics as expected. More interestingly and importantly, some of the transgenic lines with the hpRNA targets belonging to multi-copy genes or gene families displayed striking phenotypes, whereas interruption of the single gene hardly produced any visible phenotype by tranditional method such as T-DNA insertion. As compared with other approaches, the RNAi technology can bring about great advantages in functional analysis of multi-copy genes or gene family.The advantages of RMHR system were fully reflected when an Arabidopsis cDNA library was converted to an RNAi library in our study. The system would facilitate the construction of hpRNA library irrespective of cDNA lengths or sequences, and overcome some disadvantages of the methods used to construct shRNA library in animal cells. Finally, it can also tackle the problem of the low silencing efficiency of transitive RNAi library depending on RDRP activity in plants. Therefore, RMHR system is viewed as an advanced and high throughput system with great potential in functional genomics research in animals, plants and other eukaryotic microorganisms.