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Prediction of VIGS efficiency by the Sfold program and its reliability analysis in Gossypium hirsutum

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  • Life & Medical Sciences
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Science Bulletin

Abstract

Genetic transformation in some plant species, including cotton (Gossypium hirsutum), is hampered by laborious and time-consuming processes and often unachievable. Virus-induced gene silencing (VIGS) by double-stranded RNAs can serve as a reverse-genetics tool to determine gene function. However, knockdown levels vary greatly when using a tobacco rattle virus-based vector that carries different cDNA fragments of a gene. How to choose the optional target fragment for high interference efficiency is very challenging. Addressing this challenge requires increasing the efficacy of small interference RNA (siRNA) in target fragment. Here, we describe a method to assess VIGS efficiency by comparing the following parameters of siRNA in target sequence: the disruption energy of the target (ΔGdisruption), the differential stability of siRNA duplex ends (DSSE), and the internal stability at positions 9–14 of the siRNA antisense strand (AIS), which are calculated by Sfold program (http://sfold.wadsworth.org). We find that the siRNAs with low ΔGdisruption, high DSSE and high AIS have high activity and easily result in high VIGS efficiency by experimentally testing the actual knockdown levels of the four target genes, GhPDS, GhCLA1, GhAOS1, and GhCXE1 via choosing different target sequences for each gene. Therefore, the Sfold program can be used to analyze target sequences when carrying out VIGS design to increase gene-silencing effects in plants.

摘要

包括棉花在内的许多植物的遗传转化都是费时费力的,经常无法获得转基因植株.病毒 诱导的基因沉默(VIGS)现在已经成为研究基因功能的有效工具.但是,同一基因的不同 的片段可能会产生不同的干涉效率.如何选择合适的靶标片段提高干涉效率是目前面临的主 要挑战.为了解决这个问题,我们需要分析靶标片段的siRNA的干涉效率.本文我们利用 Sfold软件分析siRNA的ΔGdisruption, DSSE和 AIS 来比较靶标片段的干涉效率.通过VIGS 实验验证,发现具有低的ΔGdisruption,高的DSSE和高的AIS的siRNA可以提高VIGS的效 率.因此,Sfold软件可以提前预测基因的靶标片段干涉效率的高低来设计VIGS实验.

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References

  1. Robertson D (2004) VIGS vectors for gene silencing: many targets, many tools. Annu Rev Plant Biol 55:495–519

    Article  Google Scholar 

  2. Lindbo JA, Silva-Rosales L, Proebsting WM et al (1993) Induction of a highly specific antiviral state in transgenic plants: implications for regulation of gene expression and virus resistance. Plant Cell 5:1749–1759

    Article  Google Scholar 

  3. Dougherty WG, Lindbo JA, Smith HA et al (1994) RNA-mediated virus resistance in transgenic plants: exploitation of a cellular pathway possibly involved in RNA degradation. Mol Plant Microbe Interact 7:544–552

    Article  Google Scholar 

  4. Ratcliff F, Martin-Hernandez AM, Baulcombe DC (2001) Technical advance: tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J 25:237–245

    Article  Google Scholar 

  5. MacFarlane SA, Popovich AH (2000) Efficient expression of foreign proteins in roots from tobravirus vectors. Virology 267:29–35

    Article  Google Scholar 

  6. Mallory AC, Parks G, Endres MW et al (2002) The amplicon-plus system for high-level expression of transgenes in plants. Nat Biotechnol 20:622–625

    Article  Google Scholar 

  7. Baulcombe D (1999) Viruses and gene silencing in plants. Arch Virol Suppl 15:189–201

    Google Scholar 

  8. Abbink TE, Peart JR, Mos TN et al (2002) Silencing of a gene encoding a protein component of the oxygen-evolving complex of photosystem II enhances virus replication in plants. Virology 295:307–319

    Article  Google Scholar 

  9. Teycheney PY, Tepfer M (2001) Virus-specific spatial differences in the interference with silencing of the chs-A gene in non-transgenic petunia. J Gen Virol 82:1239–1243

    Article  Google Scholar 

  10. Gao X, Britt RC Jr, Shan L et al (2011) Agrobacterium-mediated virus-induced gene silencing assay in cotton. J Vis Exp 54:2938

    Google Scholar 

  11. Luo KQ, Chang DC (2004) The gene-silencing efficiency of siRNA is strongly dependent on the local structure of mRNA at the targeted region. Biochem Biophys Res Commun 318:303–310

    Article  Google Scholar 

  12. Lacomme C, Hrubikova K, Hein I (2003) Enhancement of virus-induced gene silencing through viral-based production of inverted-repeats. Plant J 34:543–553

    Article  Google Scholar 

  13. Far RKK, Sczakiel G (2003) The activity of siRNA in mammalian cells is related to structural target accessibility: a comparison with antisense oligonucleotides. Nucleic Acids Res 31:4417–4424

    Article  Google Scholar 

  14. Lee NS, Dohjima T, Bauer G et al (2002) Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol 20:500–505

    Article  Google Scholar 

  15. Bohula EA, Salisbury AJ, Sohail M et al (2003) The efficacy of small interfering RNAs targeted to the type 1 insulin-like growth factor receptor (IGF1R) is influenced by secondary structure in the IGF1R transcript. J Biol Chem 278:15991–15997

    Article  Google Scholar 

  16. Overhoff M, Alken M, Far RKK et al (2005) Local RNA target structure influences siRNA efficacy: a systematic global analysis. J Mol Biol 348:871–881

    Article  Google Scholar 

  17. Westerhout EM, Ooms M, Vink M et al (2005) HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome. Nucleic Acids Res 33:796–804

    Article  Google Scholar 

  18. Lu R, Martin-Hernandez AM, Peart JR et al (2003) Virus-induced gene silencing in plants. Methods 30:296–303

    Article  Google Scholar 

  19. Baulcombe D (2004) RNA silencing in plants. Nature 431:356–363

    Article  Google Scholar 

  20. Ding Y, Chan CY, Lawrence CE (2004) Sfold web server for statistical folding and rational design of nucleic acids. Nucleic Acids Res 32:W135–W141

    Article  Google Scholar 

  21. Li FG, Fan GY, Lu CR et al (2015) Genome sequence of cultivated upland cotton (Gossypium hirsutum TM-1) provides insights into genome evolution. Nat Biotechnol 33:524–530

    Article  Google Scholar 

  22. Liu YL, Schiff M, Dinesh-Kumar SP (2002) Virus-induced gene silencing in tomato. Plant J 31:777–786

    Article  Google Scholar 

  23. Pang JH, Zhu Y, Li Q et al (2013) Development of agrobacterium-mediated virus-induced gene silencing and performance evaluation of four marker genes in Gossypium barbadense. PLoS ONE 8:e73211

    Article  Google Scholar 

  24. Wu YR, Llewellyn DJ, Dennis ES (2002) A quick and easy method for isolating good-quality RNA from cotton (Gossypium hirsutum L.) tissues. Plant Mol Biol Rep 20:213–218

    Article  Google Scholar 

  25. Ge XY, Zhang CJ, Wang QH et al (2015) iTRAQ protein profile differential analysis between somatic globular and cotyledonary embryos reveals stress, hormone, and respiration involved in increasing plantlet regeneration of Gossypium hirsutum L. J Proteome Res 14:268–278

    Article  Google Scholar 

  26. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408

    Article  Google Scholar 

  27. Schwarz DS, Hutvagner G, Du T et al (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell 115:199–208

    Article  Google Scholar 

  28. Khvorova A, Reynolds A, Jayasena SD (2003) Functional siRNAs and miRNAs exhibit strand bias. Cell 115:209–216

    Article  Google Scholar 

  29. Mandel MA, Feldmann KA, Herrera-Estrella L et al (1996) CLA1, a novel gene required for chloroplast development, is highly conserved in evolution. Plant J 9:649–658

    Article  Google Scholar 

  30. Bartley GE, Viitanen PV, Pecker I et al (1991) Molecular cloning and expression in photosynthetic bacteria of a soybean cDNA coding for phytoene desaturase, an enzyme of the carotenoid biosynthesis pathway. Proc Natl Acad Sci USA 88:6532–6536

    Article  Google Scholar 

  31. Ozawa R, Arimura G, Takabayashi J et al (2000) Involvement of jasmonate- and salicylate-related signaling pathways for the production of specific herbivore-induced volatiles in plants. Plant Cell Physiol 41:391–398

    Article  Google Scholar 

  32. Reymond P, Weber H, Damond M et al (2000) Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis. Plant Cell 12:707–720

    Article  Google Scholar 

  33. Lleperuma NR, Marshall SD, Squire CJ et al (2007) High-resolution crystal structure of plant carboxylesterase AeCXE1, from Actinidia eriantha, and its complex with a high-affinity inhibitor paraoxon. Biochemistry 46:1851–1859

    Article  Google Scholar 

  34. Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286:950–952

    Article  Google Scholar 

  35. Estevez JM, Cantero A, Romero C et al (2000) Analysis of the expression of CLA1, a gene that encodes the 1-deoxyxylulose 5-phosphate synthase of the 2-C-methyl-D-erythritol-4-phosphate pathway in Arabidopsis. Plant Physiol 124:95–104

    Article  Google Scholar 

  36. Chan CY, Carmack CS, Long DD et al (2009) A structural interpretation of the effect of GC-content on efficiency of RNA interference. BMC Bioinformatics 10(Suppl 1):S33

    Article  Google Scholar 

  37. Shao Y, Chan CY, Maliyekkel A et al (2007) Effect of target secondary structure on RNAi efficiency. RNA 13:1631–1640

    Article  Google Scholar 

  38. Schubert S, Grunweller A, Erdmann VA et al (2005) Local RNA target structure influences siRNA efficacy: systematic analysis of intentionally designed binding regions. J Mol Biol 348:883–893

    Article  Google Scholar 

  39. Reynolds A, Leake D, Boese Q et al (2004) Rational siRNA design for RNA interference. Nat Biotechnol 22:326–330

    Article  Google Scholar 

Download references

Acknowledgments

This work was supported by Major Program of Joint Funds (Sinkiang) of the National Natural Science Foundation of China (No. U1303282).

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    Authors and Affiliations

    1. State Key Laboratory of Cotton Biology, Institute of Cotton Research, Chinese Academy of Agricultural Sciences, Anyang, 455000, China

      Xiaoyang Ge, Jie Wu, Chaojun Zhang, Qianhua Wang, Zuoren Yang, Zhaoen Yang, Zhenzhen Xu, Ye Wang, Lili Lu, Xueyan Zhang & Fuguang Li

    2. Department of Plant Genetics and Breeding, Key Laboratory of Crop Heterosis and Utilization of Ministry of Education, Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing, 100193, China

      Xiaoyang Ge & Jinping Hua

    3. College of Science, China Agricultural University, Beijing, 100193, China

      Yuxia Hou

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    1. Xiaoyang Ge
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    Xiaoyang Ge, Jie Wu and Chaojun Zhang contributed equally to this work.

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    Ge, X., Wu, J., Zhang, C. et al. Prediction of VIGS efficiency by the Sfold program and its reliability analysis in Gossypium hirsutum . Sci. Bull. 61, 543–551 (2016). https://doi.org/10.1007/s11434-016-1032-z

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    • DOI: https://doi.org/10.1007/s11434-016-1032-z

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