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Isolation and characterization of a 3-hydroxy-3-methylglutaryl coenzyme A reductase 2 promoter from Salvia miltiorrhiza

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Abstract

The aim of the present study was to isolate and characterize the 5′ regulatory region of Salvia miltiorrhiza 3-hydroxy-3-methylglutaryl coenzyme A reductase 2 gene. The entire fragment is 2696 bp long and consists of the promoter, 5′UTR and 85 nucleotide 5′ fragments of the CDS region. The results of in silico bioinformatic tests indicate that the promoter region contains repetitions of many potential cis-active elements serving as the recognition sites for transcription factors. Data obtained from the in silico tests are verified by co-expression studies based on A. thaliana microarray data to make them more probably to occur. The bioinformatic analysis indicated no tandem repeats or CpNpG islands in the promoter. However, potential target sites for miRNA 156 and miRNA 159 were found in the 5′ UTR segment. In addition, two possible polymorphic sites, A2719G and A2744C, were found in the CDS region. Finally, the activity of isolated fragment was evaluated experimentally by quantitative RT–PCR. The promoter activity of the isolated 2696 bp HMGR2 gene fragment was confirmed by RT–PCR studies of in vitro cultured, transformed S. miltiorrhiza plants. Analysis of the RT–PCR results revealed temporal changes in the promoter activity occurring in response to treatment by five abiotic factors: auxin, gibberellin, salicylic acid, abscisic acid and 100 mM NaCl.

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Abbreviations

ABA:

Abscisic acid

CDS:

Coding DNA sequence

IAA:

Indole-3-aceticacid

GA:

Gibberellic acid

HMGR:

3-hydroxy-3-methylglutaryl coenzyme A reductase

IAA:

Indole-3-aceticacid

MeJ:

Methyl jasmonate

PP2A:

Ser/Thr protein phosphatase 2A

5′UTR:

5′ untranslated region

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Acknowledgements

Authors are extremely grateful to Dean Elżbieta Mikiciuk-Olasik, the former Dean of the Faculty of Pharmacy, Medical University of Łódź, and Dean Daria Orszulak-Michalak for providing financial support. Authors gratefully acknowledge the technical assistance of Wacław Prószyński.

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Authors and Affiliations

  1. Department of Pharmaceutical Biotechnology, Medical University of Łódź, Muszyńskiego 1, 90-151, Lodz, Poland

    Piotr Szymczyk

  2. Department of Biology and Pharmaceutical Botany, Medical University of Łódź, Muszyńskiego 1, 90-151, Lodz, Poland

    Renata Grąbkowska & Ewa Skała

  3. Department of Pharmaceutical Biochemistry and Molecular Diagnostics, Medical University of Łódź, Muszyńskiego 1, 90-151, Lodz, Poland

    Marta Żebrowska, Ewa Balcerczak & Agnieszka Jeleń

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  1. Piotr Szymczyk
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  2. Renata Grąbkowska
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  3. Ewa Skała
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Correspondence to Piotr Szymczyk.

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Szymczyk, P., Grąbkowska, R., Skała, E. et al. Isolation and characterization of a 3-hydroxy-3-methylglutaryl coenzyme A reductase 2 promoter from Salvia miltiorrhiza . J. Plant Biochem. Biotechnol. 27, 223–236 (2018). https://doi.org/10.1007/s13562-017-0434-6

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