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Two novel positive cis-regulatory elements involved in green tissue-specific promoter activity in rice (Oryza sativa L ssp.)

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Abstract

In plant genetic engineering, using tissue-specific promoters to control the expression of target gene is an effective way to avoid potential negative effects of using constitutive promoter, such as metabolic burden and so on. However, until now, there are few tissue-specific promoters with strong and reliable expression that could be used in crop biotechnology application. In this study, based on microarray and RT-PCR data, we identified a rice green tissue-specific expression gene DX1 (LOC_Os12g33120). The expression pattern of DX1 gene promoter was examined by using the β-glucuronidase (GUS) reporter gene and analyzed in transgenic rice plants in different tissues. Histochemical assays and quantitative analyses of GUS activity confirmed that P DX1 :GUS was highly expressed in green tissues. To identify the regulatory elements controlling the expression of the DX1 gene, a series of 5′ and 3′ deletions of DX1 promoter were fused to GUS gene and stably introduced into rice plants. In addition, gel mobility shift assays and site-directed mutagenesis studies were used, allowing for the identification of two novel tissue-specific cis-acting elements (GSE1 and GSE2) within P DX1 . GSE1 acted as a positive regulator in all green tissues (leaf, sheath, stem and panicle). Compared with GSE1, GSE2 acted as a positive regulator only in sheath and stem tissue, and had a weaker effect on gene expression. In addition, P DX1 :GUS was not expressed in anther and seed, this characteristic reduced the potential ecological risk and potential food safety issues. Taken together, our results strongly suggest that the identified promoter, P DX1 , and its cis regulatory elements, GSE1 and GSE2, are potentially useful in the field of rice transgenic breeding.

Key message

We have isolated and characterized the rice green tissue-specific promoter P DX1 , and identified two novel positive cis-acting elements in P DX1 .

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Abbreviations

CaMV35S:

Cauliflower Mosaic Virus 35S

GUS:

β-Glucuronidase

4-Mu:

4-Methylumbelliferone

TSS:

Transcription start site

EMSA:

Electrophoretic mobility shift assay

GSE1:

Green tissue-specific cis-acting element 1

GSE2:

Green tissue-specific cis-acting element 2

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Acknowledgments

This research was funded by the National Program of Transgenic Variety Development of China (2011ZX11001-001, the development of insect-resistant transgenic rice).

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Authors and Affiliations

  1. National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research, Huazhong Agricultural University, Wuhan, 430070, People’s Republic of China

    Rongjian Ye, Fei Zhou & Yongjun Lin

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  1. Rongjian Ye
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  2. Fei Zhou
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  3. Yongjun Lin
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Corresponding author

Correspondence to Yongjun Lin.

Additional information

Communicated by M. Jordan.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Figure 1 online. Schematic diagram of the T-DNA region of binary vectors used in this study.

Supplementary Figure 2 online. The expression pattern of the DX1 gene in 30 different tissues of Minghui 63 (O. sativa L. ssp. indica).

Supplementary Table 1 online. The known and identified tissue-specific cis-acting elements (GSE1 and GSE2) and the basal regulatory elements in the P DX1 promoter.

Supplementary material 1 (DOC 785 kb)

Supplementary material 2 (DOC 735 kb)

Supplementary material 3 (DOC 79 kb)

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Ye, R., Zhou, F. & Lin, Y. Two novel positive cis-regulatory elements involved in green tissue-specific promoter activity in rice (Oryza sativa L ssp.). Plant Cell Rep 31, 1159–1172 (2012). https://doi.org/10.1007/s00299-012-1238-8

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