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Comparative analysis of synthetic DNA promoters for high-level gene expression in plants

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Abstract

Main conclusion

We have designed two near- constitutive and stress-inducible promoters (CmYLCV9.11 and CmYLCV4); those are highly efficient in both dicot and monocot plants and have prospective to substitute the CaMV 35S promoter.

We performed structural and functional studies of the full-length transcript promoter from Cestrum yellow leaf curling virus (CmYLCV) employing promoter/leader deletion and activating cis-sequence analysis. We designed a 465-bp long CmYLCV9.11 promoter fragment (−329 to +137 from transcription start site) that showed enhanced promoter activity and was highly responsive to both biotic and abiotic stresses. The CmYLCV9.11 promoter was about 28-fold stronger than the CaMV35S promoter in transient and stable transgenic assays using β-glucuronidase (GUS) reporter gene. The CmYLCV9.11 promoter also demonstrated stronger activity than the previously reported CmYLCV promoter fragments, CmpC (−341 to +5) and CmpS (−349 to +59) in transient systems like maize protoplasts and onion epidermal cells as well as transgenic systems. A good correlation between CmYLCV9.11 promoter-driven GUS-accumulation/enzymatic activities with corresponding uidA-mRNA level in transgenic tobacco plants was shown. Histochemical (X-Gluc) staining of transgenic seedlings, root and floral parts expressing the GUS under the control of CmYLCV9.11, CaMV35S, CmpC and CmpS promoters also support the above findings. The CmYLCV9.11 promoter is a constitutive promoter and the expression level in tissues of transgenic tobacco plants was in the following order: root > leaf > stem. The tobacco transcription factor TGA1a was found to bind strongly to the CmYLCV9.11 promoter region, as shown by Gel-shift assay and South-Western blot analysis. In addition, the CmYLCV9.11 promoter was regulated by a number of abiotic and biotic stresses as studied in transgenic Arabidopsis and tobacco plants. The newly derived CmYLCV9.11 promoter is an efficient tool for biotechnological applications.

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Acknowledgments

We are very much grateful to Kentucky Tobacco Research and Development Center (KTRDC) for facilities and support. This work was supported by the KY state KTRDC Grant to IBM. Part of the work was done at the Institute of life Sciences (ND and SS); supported by ILS and Council for Scientific and Industrial Research, Govt. of India. The authors would like to thank Ms. Bonnie Kinney, KTRDC, for her excellent care of the experimental tobacco plants and Mr. Abhimanyu Das, ILS for his technical support in DNA–protein interaction studies.

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  1. KTRDC, College of Agriculture, University of Kentucky, Lexington, KY, 40546, USA

    Dipak Kumar Sahoo & Indu B. Maiti

  2. Department of Gene Function and Regulation, Institute of Life Sciences, Department of Biotechnology, Government of India, Chandrasekharpur, Bhubaneswar, Odisha, India

    Shayan Sarkar & Nrisingha Dey

  3. Department of Radiation Oncology, Feinberg School of Medicine, Northwestern University, Ward-13-002, 303 East Chicago Ave., Chicago, IL, 60611, USA

    Sumita Raha

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  1. Dipak Kumar Sahoo
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Correspondence to Dipak Kumar Sahoo, Indu B. Maiti or Nrisingha Dey.

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425_2014_2135_MOESM1_ESM.tif

Fig. S1 The DNA sequence of the full-length transcript promoter of the cestrum yellow leaf curling virus (CmYLCV). A 865-bp fragment (-729 to + 137) with respect to the transcription start site (TSS), with genomic coordinates 5,700 to 6,565 (GenBank Accession No. NC004324) is presented from left to right in the 5′ to 3′ direction. Arrows in the sequence indicate the deletion fragments CmpC, CmpS, Cm4 and Cm9.11, as shown. The TATA box (TATAAAT) and the transcription start site (TSS, +1, nucleotide A) are indicated. (TIFF 2467 kb)

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Sahoo, D.K., Sarkar, S., Raha, S. et al. Comparative analysis of synthetic DNA promoters for high-level gene expression in plants. Planta 240, 855–875 (2014). https://doi.org/10.1007/s00425-014-2135-x

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