RNA interference (RNAi) has been used to develop efficient strategies to silence targeted genes in a wide range of species (1,2,3). Hairpin RNA (hpRNA) constructs were commonly introduced into genomes to express hpRNAs that could induce degradation of target RNAs through RNAi machines (4,5). In plants, intron-containing hairpin RNA (ihpRNA) constructs with a spliceable intron as spacer sequence had the highest efficiency with 80% to approximately 100% transformants showing silencing of target genes (6,7,8). Currently, the ihpRNA technology has become one of the most powerful tools for gene discovery and gene engineering in plants (9,10,11,12).

To facilitate generation of ihpRNA constructs, several generic vectors with a functional intron were reported (7,8). However, these methods generally required amplification of target sequences with long primers and/or several rounds of restrictions and ligations. Therefore, a simple and efficient method for rapid generation of ihpRNA constructs was urgently in demand. To this end, we developed a novel PCR-mediated method designated directed amplification of ihpRNA (DA-ihpRNA), to amplify an ihpRNA construct in one tube directly from genomic DNA. The resultant ihpRNA construct could be cloned into any expression vector as conveniently as a sense or antisense gene.

The DA-ihpRNA method is depicted in Figure 1A. The final ihpRNA construct was designed to contain inverted repeats of an exon (Figure 1 A, exon2) with its flanking upstream intron as spacer sequence. Two primers, the flanking primer and the bridge primer, were used, and the concentration of the bridge primer was lowered to perform an asymmetric PCR. In the early cycles, a fragment consisting of the intron and exon2 was exponentially amplified. With the bridge primer exhausted in the late cycles, the main amplification would be the linear amplification of the antisense strand of the intron and exon2, which was primed by the flanking primer. The bridge primer contained a 5′ heel reverse complementary to the 5′ end of exon2, and the 3′ ends of the excessive antisense strands would anneal to the internal complementary sequences in the same or another single strands and initiate the synthesis of the reverse complementary sequence of exon2 (Figure 1 A). The resultant single strands would form the antisense strand of the final ihpRNA construct and could be used as a template to synthesize the double-stranded ihpRNA construct in the next PCR cycle.

Figure 1. Direct amplification of intron-containing hairpin RNA (DA-ihpRNA) construct (Click to enlarge)

As a test, the DA-ihpRNA method was first used to amplify an ihpRNA construct of a small GTPase protein gene of cotton (GhRacA; GenBank® accession no. DQ667981) (13). The target ihpRNA construct consisted of two inverted repeats of the GhRacA exon 8 intervened by the flanking upstream intron (intron 7). The PCR of 25 µL contained 1 µL boiled Escherichia coli culture harboring the cloned genomic GhRacA gene or around 100 ng cotton genomic DNA, 1× Ex Taq™ buffer (TaKaRa, Dalian, China), 200 µM each dNTPs, 2 mM MgCl₂, 400 nM flanking primer (Figure 1D, 5′-CGACTTGATCCTGATTGTCT-3′, annealing to the 3′ end of GhRacA exon 8), 1.5 U Ex Taq DNA polymerase (TaKaRa), and various concentrations (400, 200, 40, or 20 nM) of the bridge primer (Figure 1D, 5′-AAGTTCCTCCCGCAGAAGCCAAGTCGAGGATGTC-3′, the sequences annealed to the 3′ end of exon 7 are in bold, while those that are reverse complementary to the 5′ end of exon 8 are in italic). The reaction started at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 56°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 5 min. The interest fragment was gel-purified, cloned into a vector pUCm-T (Sangon, Shanghai, China) by AT cloning according to manufacturer′s instructions, and sequenced by Invitrogen (Shanghai, China) in an ABI PRISM® 3700 DNA sequence analyzer (Applied Biosystems, Foster City, CA, USA).

As shown in Figure 1, B and C, similar amplification products were obtained with the cloned and uncloned genomic DNA as template. When the concentration of the bridge primer was high, the fragment containing a single copy of the intron and the target exon was the only amplification product. However, when the bridge primer concentration was lowered to 40 and 20 nM (one-tenth and one-twentieth of the concentration of the flanking primer, respectively), a fragment of about 700 bp was amplified. Sequencing analysis confirmed that this fragment was 689 bp in length and contained two self-complementary arms and a spacer as expected (Figure 1D), suggesting that the ihpRNA construct of the GhRacA gene was successfully amplified from both cloned and uncloned cotton genomic DNA. Additionally, we had harnessed the DA-ihpRNA method to amplify a total of 14 ihpRNA constructs, with the inverted arms of 102–352 bp and the spacer of 85–693 bp, from cotton, rice, and Arabidopsis (data not shown). With appropriate primers, all these ihpRNA constructs (100%) had been successfully amplified and cloned, indicating that the DA-ihpRNA method was quite reliable.

High efficiency of DA-ihpRNA method required the template DNA containing an intron-containing spacer of appropriate length (100 to approximately 500 bp), which presumably facilitated annealing of the 3′ end of excess single strands to internal complementary sequence. Furthermore, to decrease the PCR suppression effect of the inverted repeats in the last step (14), the sequence of inverted arm should excess 100 bp. However, compared with the previous methods to generate ihpRNA constructs (7,8), the DA-ihpRNA method bypassed the construction of generic vectors, required less or shorter primers, and reduced cloning steps. Most important of all, by using this method, ihpRNA constructs could be amplified directly from uncloned genomic DNAs as long as the sequence was available. With several genomes sequenced and more and more sequence information available in the web, the DA-ihpRNA method would be increasingly valuable to generate ihpRNA constructs for the functional analyses of various intron-containing genes.

In addition to the ihpRNA constructs, other hpRNA constructs without intron in the spacer were also useful in some systems, showing high gene silencing efficiency (2,3). In these cases, any DNA of appropriate length could be used as a template to amplify hpRNA construct using the DA-ihpRNA method, which might broaden the application of the DA-ihpRNA method to generate constructs for RNAi. In short, with suitable template available, the DA-ihpRNA method was simple and efficient for rapid generation of hpRNA constructs for gene functional analyses via RNAi.