Abstract
Hairpin RNA-based RNA interference (hpRNAi) has become a powerful tool for exploring gene function in reverse genetics. Although, several methods are available for making constructs that express hpRNAi, multiple time-consuming cloning steps are usually involved. Here, we introduce an efficient and flexible hpRNAi vector construction method via the isothermal in vitro recombination system (IR-hpRNAi). For an IR-hpRNAi reaction, two PCR products of a target gene sequence are generated, which containS complementary ends (~20 bp) to each other and to the ends of linearized vector, are fused in a way of head-to-head or tail-to-tail into the vector. This IR-hpRNAi method offers two options to construct the RNAi vectors. Using this method, we created a IR-hpRNAi construct for the Arabidopsis PDS3 gene,and verified the silencing effect via Agrobacterium-mediated transformation. The IR-hpRNAi system rules out the requirement of engineering restriction enzyme cutting sites in target DNA fragments, and is ligation-independent. Thus, this method has advantages over the other hpRNAi construction methods.
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Abbreviations
- ER:
-
17β-Estradiol
- hpRNAi:
-
Hairpin RNA-based RNA interference
- IR:
-
Isothermal in vitro recombination system
- IR-hpRNAi:
-
HpRNAi vector via the isothermal in vitro recombination system
- PDS3:
-
Phytoene desaturase 3
- RT-PCR:
-
Reverse transcription PCR
- WT:
-
Wild type
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Acknowledgments
We are grateful to N.H. Chua (Rockefeller University, NY, USA) for inducible T-DNA binary vectors pER8 and to X.J. Wang (South China Normal University, Guangzhou, China) for the seed of Arabidopsis Columbia ecotype. This work was supported by grants from Ministry of Science and Technology of China (2012AA10A303 and 2011CB100204) and National Natural Science Foundation of China (30871331).
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Jiang, Y., Xie, M., Zhu, Q. et al. One-step cloning of intron-containing hairpin RNA constructs for RNA interference via isothermal in vitro recombination system. Planta 238, 325–330 (2013). https://doi.org/10.1007/s00425-013-1896-y
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DOI: https://doi.org/10.1007/s00425-013-1896-y