Isolation and characterization of a 3-hydroxy-3-methylglutaryl coenzyme A reductase 2 promoter from Salvia miltiorrhiza
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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.
KeywordsmiRNA Polymorphic site Promoter RT–PCR Trans-factor
Coding DNA sequence
3-hydroxy-3-methylglutaryl coenzyme A reductase
Ser/Thr protein phosphatase 2A
5′ untranslated region
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.
Compliance with ethical standards
Conflict of interest
All authors declare that they have no conflict of interest.
- Cao XY, Li Ch-G, Mao Q, Zheng ZJ, Jiang JH (2011) Molecular cloning and expression analysis of a leaf-specific expressing 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase gene from Michelia chapensis Dandy. J Med Plant Res 5:3868–3875Google Scholar
- Khan S, Qureshi MI, Kamaluddin Alam T, Abdin MZ (2007) Protocol for isolation of genomic DNA from dry and fresh roots of medicinal plants suitable for RAPD and restriction digestion. Afr J Biotechnol 6:175–178Google Scholar
- Liao Y, Xu F, Huang X, Zhang W, Cheng H, Li L, Cheng S, Shen Y (2015) Promoter analysis and transcriptional profiling of Ginkgo biloba 3-hydroxy-3-methylglutaryl coenzyme A reductase (GbHMGR) gene in abiotic stress responses. Not Bot Horti Agrobo 43:25–34Google Scholar
- Teh KY, Abdullah JO (2016) An overview of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) in plants. Pertanika J Sch Res Rev 2:100–107Google Scholar
- Wang K (ed) (2006) Agrobacterium protocols. In: Methods in molecular biology, vol 343. Humana Press Inc, TotowaGoogle Scholar