Article | . 2017 Vol. 35, Issue. 3
New Hairpin RNAi Vector with Brassica rapa ssp. pekinensis Intron for Gene Silencing in Plants



Department of Horticultural Biotechnology, Kyung Hee University1
Biosafety Division, National Academy of Agricultural Science, Rural Development Administration2




2017.. 323:332


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Homology-specific transcriptional and post-transcriptional silencing, an intrinsic mechanism of gene regulation in most eukaryotes, can be induced by anti-sense, co-suppression, or hairpinbased double-stranded RNA. Hairpin-based RNA interference (RNAi) has been applied to analyze gene function and genetically modify crops. However, RNAi vector construction usually requires high-cost cloning steps and large amounts of time, or involves methods that are protected by intellectual property rights. We describe a more effective method for generating intron-spliced RNAi constructs. To produce intron-spliced hairpin RNA, an RNAi cassette was ligated with the first intron and splicing sequences of the Brassica rapa ssp. pekinensis histone deacetylase 1 gene. This method requires a single ligation of the PCR-amplified target gene to SpeI-NcoI and SacI-BglII enzyme sites to create a gene-specific silencing construct. We named the resulting binary vector system pKHi and verified its functionality by constructing a vector to silence DIHYDROFLAVONOL 4-REDUCTASE (DFR ), transforming it into tobacco plants, and confirming DFR gene-silencing via PCR, RT-qPCR, and analysis of the accumulation of small interfering RNAs. Reduction of anthocyanin biosynthesis was also confirmed by analyzing flower color of the transgenic tobacco plants. This study demonstrates that small interfering RNAs generated through the pKHi vector system can efficiently silence target genes and could be used in developing genetically modified crops



1. 3,000 rice genomes project (2014) The 3,000 rice genomes project. Gigascience 3:7. doi:10.1186/2047-217X-3-7  

2. Abdurakhmonov IY, Ayubov MS, Ubaydullaeva KA, Buriev ZT, Shermatov SE, Ruziboev HS, Shapulatov UM, Saha S, Ulloa M, et al (2016) RNA interference for functional genomics and improvement of cotton (Gossypium sp.). Front Plant Sci 7:202. doi:10.3389/ fpls.2016.00202  

3. Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana . Nature 408:796-815. doi:10.1038/35048692  

4. Aufsatz W, Mette MF, van der Winden J, Matzke AJ, Matzke M (2002) RNA-directed DNA methylation in Arabidopsis. Proc Natl Acad Sci USA 99:16499-16506. doi:10.1073/pnas.162371499  

5. Childers CC, Brecht JK (1996) Colored sticky traps for monitoring Frankliniella bispinosa (Morgan)(Thysanoptera: Thripidae) during flowering cycles in citrus. J Econ Entomol 89:1240-1249. doi:10.1093/jee/89.5.1240  

6. Clemente TE, Cahoon EB (2009) Soybean oil: genetic approaches for modification of functionality and total content. Plant Physiol 151:1030–1040. doi:10.1104/pp.109.146282  

7. Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11-15  

8. Gan X, Stegle O, Behr J, Steffen JG, Drewe P, Hildebrand KL, Lyngsoe R, Schultheiss SJ, Osborne EJ, et al (2011) Multiple reference genomes and transcriptomes for Arabidopsis thaliana. Nature 477:419-423. doi:10.1038/nature10414  

9. Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989-994. doi:10.1007/BF00014672  

10. Haun W, Coffman A, Clasen BM, Demorest ZL, Lowy A, Ray E, Retterath A, Stoddard T, Juillerat A, et al (2014) Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family. Plant Biotechnol J 12:934-940. doi:10.1111/pbi.12201  

11. He Q, Shen Y, Wang M, Huang M, Yang R, Zhu S, Wang L, Xu Y, Wu R (2011) Natural variation in petal color in Lycoris longituba revealed by anthocyanin components. PLoS ONE 6:e22098. doi:10.1371/journal.pone.0022098  

12. Heilersig HJ, Loonen A, Bergervoet M, Wolters AM, Visser RG (2006) Post-transcriptional gene silencing of GBSSI in potato: effects of size and sequence of the inverted repeats. Plant Mol Biol 60:647-662. doi:10.1007/s11103-005-5280-6  

13. Helliwell C, Waterhouse P (2003) Constructs and methods for high-throughput gene silencing in plants. Methods 30: 289-295. doi:10.1016/S1046-2023(03)00036-7  

14. Helliwell CA, Wesley SV, Wielopolska AJ, Waterhouse PM (2002) High-throughput vectors for efficient gene silencing in plants. Funct Plant Biol 29:1217-1225. doi:10.1071/FP02033  

15. Hirai S, Kodama H (2008) RNAi vectors for manipulation of gene expression in higher plants. Open Plant Sci J 2:21-30.doi:10.2174/1874294700802010021  

16. Holton TA, Cornish EC (1995) Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell 7:1071-1083. doi:10.1105/tpc.7.7.1071  

17. Jyotishwaran G, Kotresha D, Selvaraj T, Srideshikan SM, Rajvanshi PK, Jayabaskaran C (2007) A modified freeze-thaw method for efficient transformation of Agrobacterium tumefaciens. Curr Sci 93:770-772  

18. Kumar R, Vashisth D, Misra A, Akhtar MQ, Jalil SU, Shanker K, Gupta MM, Rout PK, Gupta AK, Shasany AK (2016) RNAi downregulation of cinnamate-4-hydroxylase increases artemisinin biosynthesis in Artemisia annua . Sci Rep 6:26458. doi:10.1038/ srep26458  

19. Kim SB, Yu JG, Lee GH, Park YD (2012) Characterization of Brassica rapa S-adenosyl-L-methionine synthetase gene including its roles in biosynthesis pathway. Hortic Environ Biotechnol 53:57–65. doi:10.1007/s13580-012-0084-5  

20. Lee GH, Kang YJ, Yi SK, Lim SB, Park YD (2010) Development of a highly effective T-DNA inserted mutant screening method in a Chinese cabbage (Brassica rapa L. spp. pekinensis) reverse genetics system. Plant Biotechnol Rpt 4:201-211. doi:10.1007/s11816-010-0137-0  

21. Lee WS, You JA, Chung H, Lee YH, Baek NI, Yoo JS, Park YD (2008) Molecular cloning and biochemical analysis of Dihydroflavonol 4-Reductase (DFR) from Brassica rapa ssp. pekinesis (Chinese cabbage) using a heterologous system. J Plant Biol 51:42-47. doi:10.1007/bf03030739  

22. Liu Q, Singh SP, Green AG (2002) High-stearic and High-oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing. Plant Physiol 129:1732-1743. doi:10.1104/pp.001933  

23. Mo R, Zhang N, Yang S, Zhang Q, Luo Z (2016) Development of a transient ihpRNA-induced gene silencing system for functional analysis in Persimmon (Diospyros kaki Thunb.) Korean J Hortic Sci Technol 34:314–323. doi:10.12972/kjhst.20160032  

24. Niu SS, Xu CJ, Zhang WS, Zhang B, Li X, Lin-Wang K, Ferguson IB, Allan AC, Chen KS (2010) Coordinated regulation of anthocyanin biosynthesis in Chinese bayberry (Myrica rubra ) fruit by a R2R3 MYB transcription factor. Planta 231:887-899. doi:10.1007/ s00425-009-1095-z  

25. Pall GS, Hamilton AJ (2008) Improved northern blot method for enhanced detection of small RNA. Nat Protoc 3:1077-1084.doi:10.1038/nprot.2008.67  

26. Park JH, Kim HS, Lee GH, Yu JG, Park YD (2016) Stable inheritance of an integrated transgene and its expression in phenylethylisothiocyanate-enriched transgenic Chinese cabbage. Korean J Hortic Sci Technol 34:112–121. doi:10.12972/kjhst.20160013  

27. Park JS, Yu JG, Park YD (2017) Characterization of a drought tolerance-related gene of Chinese cabbage in a transgenic tobacco plant.Hortic Environ Biotechnol 58:48-55. doi:10.1007/s13580-017-0157-6  

28. Schmitzer V, Veberic R, Osterc G, Stampar F (2010) Color and phenolic content changes during flower development in groundcover rose. J Am Soc Hortic Sci 135:195-202  

29. Siu FK, Lee LT, Chow BK (2008) Southwestern blotting in investigating transcriptional regulation. Nat Protoc 3:51-58. doi:10.1038/nprot.2007.492  

30. Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM (2000) Total silencing by intron-spliced hairpin RNAs. Nature 407:319-320. doi:10.1038/35030305  

31. Song X, Cao X (2016) Small RNA extraction and detection in rice (Oryza sativa ). Curr Protoc Plant Biol 1:79-87. doi:10.1002/cppb.20005  

32. Tan J, Wang M, Tu L, Nie Y, Lin Y, Zhang X (2013) The flavonoid pathway regulates the petal colors of cotton flower. PLoS One 8:e72364. doi: 10.1371/journal.pone.0072364  

33. Wang MB, Waterhouse PM (2000) High-efficiency silencing of a beta-glucuronidase gene in rice is correlated with repetitive transgene structure but is independent of DNA methylation. Plant Mol Biol 43:67-82. doi:10.1023/A:1006490331303  

34. Wang X, Wang H, Wang J, Sun R, Wu J, Liu S, Bai Y, Mun JH, Bancroft I, et al (2011) The genome of the mesopolyploid crop species Brassica rapa. Nat Genet 43:1035-1039. doi:10.1038/ng.919  

35. Wassenegger M (2000) RNA-directed DNA methylation. Plant Mol Biol 43:203-220. doi:10.1007/978-94-011-4183-3_6  

36. Wesley SV, Helliwell CA, Smith NA, Wang MB, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, et al (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 27:581-590. doi:10.1046/j.1365-313X.2001.01105.x  

37. Xia Z, Zhao Z, Chen L, Li M, Zhou T, Deng C, Zhou Q, Fan Z (2016) Synergistic infection of two viruses MCMV and SCMV increases the accumulations of both MCMV and MCMV-derived siRNAs in maize. Sci Rep 6:20520. doi:10.1038/srep20520  

38. Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13:134. doi:10.1186/1471-2105-13-134  

39. Ye R, Chen Z, Lian B, Rowley MJ, Xia N, Chai J, Li Y, He XJ, Wierzbicki AT, Qi Y (2016) A dicer-independent route for biogenesis of siRNAs that direct DNA methylation in Arabidopsis. Mol Cell 61:222-235. doi:10.1016/j.molcel.2015.11.015