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Characterization of NtREL1, a novel root-specific gene from tobacco, and upstream promoter activity analysis in homologous and heterologous hosts

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Abstract

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A novel root-specific gene and its upstream promoter were cloned and characterized for potential application in root-specific expression of transgenes.

Abstract

The root is an important plant organ subjected to many biotic and abiotic stresses, such as infection by Ralstonia solanacearum. To isolate tobacco root-specific promoters for genetic applications, microarray screening was performed to identify genes highly and specifically expressed in the root. One root-specific gene encoding an extensin-like protein (NtREL1) was isolated, and its expression pattern was further characterized by both microarray analysis and reverse transcription-polymerase chain reaction. NtREL1 was highly expressed only in roots but not in any other organ. NtREL1 expression was affected by hormone treatment (salicylic acid, abscisic acid, and ethephon) as well as low temperature, drought, and R. solanacearum infection. A full-length 849 bp cDNA containing a 657-nucleotide open reading frame was cloned by Rapid Amplification of cDNA Ends. Subsequently, a fragment of 1,574 bp upstream of NtREL1 was isolated by flanking PCR and named pNtREL1. This promoter fragment contains TATA, GATA, and CAAT-boxes, the basic elements of a promoter, and six root-specific expression elements, namely OSE1, OSE2, ROOTMOTIFTAPOX1, SURECOREATSULTR11, P1BS, and WUSATAg. A construct containing the bacterial uidA reporter gene (β-glucuronidase, GUS) driven by the pNtREL1 promoter was transformed into tobacco plants. GUS staining was only detected in the root, but not in leaves and stems. Additionally, transgenic tobacco plants containing peanut resveratrol synthase gene (AhRS) driven by the pNtREL1 promoter produced resveratrol only in the root. Thus, the pNtREL1 promoter can be used to direct root-specific expression of target genes to protect the root from stress or for biological studies.

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Abbreviations

SA:

Salicylic acid

ABA:

Abscisic acid

ETH:

Ethephon

RT-PCR:

Reverse transcript polymerase chain reaction

CTAB:

Cetyltrimethyl ammonium bromide

BLAST:

Basic local alignment search tool

ORF:

Open reading frame

RACE:

Rapid amplification of cDNA ends

UTR:

Untranslated regions

UV:

Ultra-violet

CaMV:

Cauli flower mosaic virus

GUS:

β-Glucuronidase

RS:

Resveratrol synthase

HPLC:

High-performance liquid chromatography

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Acknowledgments

We thank Dr. Ronglong Pan (National Tsing Hua University, Taiwan) for a critical reading and revision of the article. This work was funded by Fujian Provincial Tobacco Corporation (Grant No.: 2008-191, Grant No.: 2012-042 and Grant No.: 2015-176).

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Authors and Affiliations

  1. Fujian Provincial Key Laboratory of Crop Molecular and Cell Biology, Fuzhou, 350002, Fujian, China

    Chong Zhang, Shufang Pan, Hua Chen, Tiecheng Cai, Chunhong Zhuang, Ye Deng, Yuhui Zhuang, Yuanhuan Zeng & Weijian Zhuang

  2. College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, 350002, Fujian, China

    Chong Zhang, Hua Chen, Tiecheng Cai, Ye Deng & Weijian Zhuang

  3. Institute of Tobacco Agricultural Science and Technology, Fujian Bureau of Tobacco, Fuzhou, 350000, Fujian, China

    Shunhui Chen

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  1. Chong Zhang
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  2. Shufang Pan
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  3. Hua Chen
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Correspondence to Shunhui Chen or Weijian Zhuang.

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Communicated by Z.-Y. Wang.

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Zhang, C., Pan, S., Chen, H. et al. Characterization of NtREL1, a novel root-specific gene from tobacco, and upstream promoter activity analysis in homologous and heterologous hosts. Plant Cell Rep 35, 757–769 (2016). https://doi.org/10.1007/s00299-015-1918-2

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