3 Biotech

, 8:198 | Cite as

An improved method for rapid analysis of promoters using modified sonication-assisted transient assay

  • Chetan Chauhan
  • Gauri Joshi
  • Darshna Chaudhary
  • Sandip Das
Original Article

Abstract

We present here a modified, sonication-assisted transient transformation assay for rapid analysis of cis-regulatory elements. We tested promoter elements from MIR159B locus of Brassica juncea by generating stable transgenic lines and compared the transcriptional activity of GUS reporter with that of the transient assay method. To obtain reliable and repeatable results, and to omit false-positive data, we optimized several parameters including sonication duration and cycle and concentration of Agrobacterium tumefaciens measured as optical density (O.D.) at 600 nm. To the best of our knowledge, this is the first report of promoter characterization of MIR159B from Brassica juncea, and comparative analysis of stable and transient lines. Our analysis shows that the protocol described herein allows understanding promoter activity/transcriptional control in tissues other than leaf or protoplast which have remained the mainstay for transient analysis thus far. We tested reporter gene GUS under the control of constitutive promoter, CaMV 35S, and MIR159b from Brassica juncea. We optimized the duration of sonication (5-, 10- and 15-min cycle), bacterial density (measured as O.D at 600 nm = 0.6/0.8/1.0) and Agro-infection time (5, 10, 15 min), and co-cultivation (12-, and 24-h). Sonication cycle of 10-min, followed by Agro-infection and co-cultivation with Agrobacterium tumefaciens with O.D. 600 nm = 0.8 and for 12 h was found to be optimum. We could successfully express reporter genes in deep-seated tissues such as floral organs and pollen grains where it was previously not possible to perform transient assay. Constitutive GUS activity was observed when reporter was placed under control of the constitutive promoter of CaMV 35S. Reporter GUS when placed under transcriptional control of MIR159b promoter from Brassica juncea showed reporter activity in floral tissues, in mature pollen grains. Comparative analysis of reporter activity from stable transgenic lines at T2 generation with that of transient assay system reveals identical to near-identical reporter activity. Transient assay could be successfully performed in tissues collected not only from Arabidopsis thaliana, but also from Brassica juncea and Brassica nigra to demonstrate its wide applicability. Our modified method thus has the potential of quick and rapid analysis of promoter activity and allows us to record the developmental dynamics and spatio-temporal expression pattern driven by specific promoters. Suitable modification and controls should also allow analysis of hormonal regulation and identification of trans-factors via DNA–protein interactions. Furthermore, this method can also be extended to study promoters under various environmental conditions that otherwise do not allow growth and complete life cycle of healthy plants and can be modified to test reporter activity in other non-model plants or plants with long life cycle.

Keywords

Sonication Promoter Transient assay Brassica MIR159 

Notes

Acknowledgements

The research was supported by a Department of Biotechnology, Govt. Of India (DBT) Grant (Grant number BT/PR14532/AGR/36/673/2010) to SD, and a DBT project Junior Research Fellowship (JRF) to CC. GJ is supported by JRF/SRF from the University Grants Commission (UGC). SD would also like to acknowledge the financial assistance received from Delhi University under the R&D grant support.

Author contributions

CC, DC and SD were involved in planning of the study. CC and GJ performed the experiments and collected data. CC, DC and SD analysed the data and wrote the manuscript. All authors have read and approved the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no competing interest that may be construed as conflict of interest.

Supplementary material

13205_2018_1219_MOESM1_ESM.tif (7.1 mb)
Supplementary material 1 (TIFF 7225 kb) Supplementary Figure 1: Comparative analysis of full-length 1.0 kb (Bj-p_159B-A06-1kb) and a 5’-nested deletion of 0.6kb (Bj-p_159B-A06-0.6) promoter driving GUS through transient and stable transgenic lines in flower and siliques of Arabidopsis thaliana reveals no reporter activity
13205_2018_1219_MOESM2_ESM.tif (4.8 mb)
Supplementary material 2 (TIFF 4872 kb) Supplementary Figure 2: Sonication-assisted transient assay can be used in Brassica species: Sonication-assisted transient expression analysis of CaMV35S promoter::GUS reporter was performed in unopened floral buds and open flowers of Brassica nigra which are not commonly used for transient assay using 10 minutes of sonication-rest-sonication cycle, 12 hours of co-cultivation with A. tumefaciens at O.D = 0.8. Scale bar represents 200 µm

References

  1. Abel S, Theologis A (1994) Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression. Plant J 5(3):421–427CrossRefGoogle Scholar
  2. Allen RS, Li J, Alonso-Peral MM, White RG, Gubler F, Millar AA (2010) MicroR159 regulation of most conserved targets in Arabidopsis has negligible phenotypic effects. Silence 1:18CrossRefGoogle Scholar
  3. Alvarez-Buylla ER, Benítez-Keinrad M, Corvera-Poiré A, Cador ÁC, de Foltera S, de Buenb AG, Garay-Arroyo A, García-Ponce, Jaimes-Miranda F, Pérez-Ruiz RV, Piñeyro-Nelson A, Sánchez-Corraleset YE (2010) Flower development. Arabidopsis Book 8:e0127.  https://doi.org/10.1199/tab.0127 CrossRefGoogle Scholar
  4. Caboche M (1990) Liposome-mediated transfer of nucleic acids in plant protoplasts. Physiol Plant 79:173–176CrossRefGoogle Scholar
  5. Cheng F, Liu S, Wu J, Fang L, Sun S, Liu B, Li P, Hua W, Wang X (2011) BRAD, the genetics and genomics database for Brassica plants. BMC Plant Biol 11:136CrossRefGoogle Scholar
  6. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16:735–743CrossRefGoogle Scholar
  7. De Smet I, Lau S, Ehrismann JS, Axiotis I, Kolb M, Kientz M, Weijers D, Jürgens G (2013) Transcriptional repression of BODENLOS by HD-ZIP transcription factor HB5 in Arabidopsis thaliana. J Exp Bot 64:3009–3019CrossRefGoogle Scholar
  8. Gilbert S (2002) Genomic regulatory systems: development and evolution, by Eric H. Arabidopsis. Am J Med Genet 108:341–342CrossRefGoogle Scholar
  9. Harmer SL (2009) The circadian system in higher plants. Annu Rev Plant Biol 60:357–377CrossRefGoogle Scholar
  10. Hauptmann RM, Ozias-Akins P, Vasil V, Tabaeizadeh Z, Rogers SG, Horsch RB, Vasil IK, Fraley RT (1987) Transient expression of electroporated DNA in monocotyledonous and dicotyledonous species. Plant Cell Rep 6:265–270CrossRefGoogle Scholar
  11. Jain A, Das S (2016) Synteny and comparative analysis of miRNA retention, conservation, and structure across Brassicaceae reveals lineage- and sub-genome-specific changes. Funct Integr Genomics 16:253–268CrossRefGoogle Scholar
  12. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907Google Scholar
  13. Joersbo M, Brunstedt J (1992) Sonication: a new method for gene transfer to plants. Physiol Plant 85:230–234CrossRefGoogle Scholar
  14. Kagale S, Robinson SJ, Nixon J et al (2014) Polyploid evolution of the Brassicaceae during the Cenozoic era. Plant Cell 26:2777–2791CrossRefGoogle Scholar
  15. Kuijt SJ, Greco R, Agalou A, Shao J, CJ‘t Hoen C, Övernäs E, Osnato M, Curiale S, Meynard D, van Gulik R, de Faria Maraschin S (2014) Interaction between the GROWTH-REGULATING FACTOR and KNOTTED1-LIKE HOMEOBOX families of transcription factors [W]. Plant Physiol 164(4):1952–1966CrossRefGoogle Scholar
  16. Kumari G, Kusumanjali K, Srivastava PS, Das S (2013) Isolation and expression analysis of MIR165a and REVOLUTA from Brassica species. Acta Physiol Plant 35:399–410CrossRefGoogle Scholar
  17. Kusumanjali K, Kumari G, Srivastava PS, Das S (2012) Sequence conservation and divergence in MIR164C1 and its target, CUC1, in Brassica species. Plant Biotechnol Rep 6:149–163CrossRefGoogle Scholar
  18. Lee MW, Yang Y (2006) Transient expression assay by agroinfiltration of leaves. Arabidopsis protocols. Humana Press, New York, pp 225–229CrossRefGoogle Scholar
  19. Leuzinger K, Dent M, Hurtado J, Stahnke J, Lai H, Zhou X, Chen Q (2013) Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins. J Vis Exp 2013(77):50521Google Scholar
  20. Levy M, Rachmilevitch S, Abel S (2005) Transient agrobacterium-mediated gene expression in the Arabidopsis hydroponics root system for subcellular localization studies. Plant Mol Biol Rep 23(2):179–184CrossRefGoogle Scholar
  21. Li J-F, Park E, von Arnim AG, Nebenführ A (2009) The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species. Plant Meth 5:6CrossRefGoogle Scholar
  22. Morikawa H, Iida A, Yamada Y (1989) Transient expression of foreign genes in plant cells and tissues obtained by a simple biolistic device (particle-gun). Appl Microbiol Biotechnol 31:320–322CrossRefGoogle Scholar
  23. Ouwerkerk PBF, Meijer AH (2001) Yeast One-Hybrid Screening for DNA-Protein Interactions. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) Current protocols in molecular biology. Wiley, HobokenGoogle Scholar
  24. Plackett ARG, Thomas SG, Wilson ZA, Hedden P (2011) Gibberellin control of stamen development: a fertile field. Trends Plant Sci 16:568–578CrossRefGoogle Scholar
  25. Ren B (2000) Genome-wide location and function of DNA binding proteins. Science 290:2306–2309CrossRefGoogle Scholar
  26. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  27. Ueki S, Lacroix B, Krichevsky A, Lazarowitz SG, Citovsky V (2008) Functional transient genetic transformation of Arabidopsis leaves by biolistic bombardment. Nat Protoc 4(1):71CrossRefGoogle Scholar
  28. Wang Y, Sun F, Cao H, Peng H, Ni Z, Sun Q, Yao Y (2012) TaMIR159 directed wheat TaGAMYB cleavage and its involvement in anther development and heat response. PLoS one 7:e48445CrossRefGoogle Scholar
  29. Wu H-Y, Liu K-H, Wang Y-C, Wu J-F, Chiu W-L, Chen C-Y, Wu S-H, Sheen J, Lai E-M (2014) AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings. Plant Methods 10:19CrossRefGoogle Scholar
  30. Yang Y, Li R, Qi M (2000) In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves. Plant J 22:543–551CrossRefGoogle Scholar
  31. Yoo S-D, Cho Y-H, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of BotanyUniversity of DelhiDelhiIndia
  2. 2.Centre for BiotechnologyMaharshi Dayanand UniversityRohtakIndia

Personalised recommendations