Rolling Circle Amplification-Mediated Long Hairpin RNA Library Construction in Plants

Gene silencing has been used widely in gene function studies and crop plant modification. Long hairpin RNA (lhRNA) results in high efficiency of gene silencing; however, constructing multiple lhRNA vectors using traditional approaches is both time consuming and costly. Also, most of the existing approaches are based on sequence-specific cloning of individual sequences and are therefore not suitable for preparing hpRNA libraries from a pool of mixed target sequences. The rolling circle amplification (RCA)-mediated hairpin RNA (RMHR) construction system is suitable for generating hairpin libraries from any gene of interest or pool of genes. Using the RMHR system, a long-hairpin RNA (lhRNA) library is generated from an Arabidopsis cDNA population containing known and unknown genes. Our results indicate that the RMHR technique permits the rapid, efficient, and low-cost preparation of genome-wide lhRNA expression libraries.