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Isolation of the maize Zpu1 gene promoter and its functional analysis in transgenic tobacco plants

  • Genetic Transformation and Hybridization
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Abstract

By screening a genomic library of maize, a 2.2 kb 5′ flanking fragment of Zpu1 gene, encoding the pullulanase-type starch debranching enzyme, was isolated. Promoter fragments of various lengths, including the full 5′ flanking sequence (−2267 to −1) (Z1), a 3′ deletion (−2267 to −513) (Z5) and three 5′ deletions extending to −1943 (Z2), −1143 (Z3) and −516 (Z4) upstream of the translational initiation codon (ATG), were fused to the GUS reporter gene and introduced into tobacco. When these constructs were tested in transgenic tobacco plants, seed-preferred GUS activity was observed in pZ1-transgenic lines. In pZ2-transgenic lines, the GUS activity was not only restricted to seeds, but was also detected in calyxes, petals, stamens and mature leaves. At the same time, negligible GUS activity was detected in roots, stems, young leaves, stigmas and ovaries from the transgenic tobacco plants, which had integrated the full isolated sequence of Zpu1 promoter or its deletions. Deletion analysis indicated that the promoter contained a putative positive cis-regulatory element and the proximal region (−516 to −1) was essential for directing the expression of gus reporter gene. Analysis of GUS activity during the fruit development and seed germination suggested that Zpu1 promoter is active both in starch anabolism and in starch catabolism, which is consistent with the function of the endogenous gene in maize. GUS activity in leaves under light and darkness confirmed that Zpu1 promoter functions in the starch degradation of photosynthetic tissues in the dark phase of the diurnal cycle.

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Abbreviations

CaMV:

Cauliflower mosaic virus

DAF:

Days after flowering

DBE:

Debranching enzyme

4-MU:

4-Methylumbelliferone

GUS:

β-Glucuronidase

MUG:

4-Methylumbelliferyl-β-d-glucuronide

TSS:

Transcriptional start site

X-gluc:

5-Bromo-4-chloro-3-indolyl-β-d-glucuronide

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Acknowledgments

The authors thank Dr. Ulrike Goebel (Max Planck Institute for Chemical Ecology, Germany) for providing the Perl script. This work was supported by the National Special Program for Research and Industrialization of Transgenic Plants.

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Authors and Affiliations

  1. Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100081, China

    Guoying Wang

  2. State Key Laboratory of Agrobiotechnology and Department of Seed Sciences and Technology, China Agricultural University, Beijing, 100094, China

    Xiaoping Chen, Zhangying Wang, Riliang Gu, Junjie Fu, Jianhua Wang, Ying Zhang, Maoyan Wang, Jinpeng Zhang, Jinping Jia & Guoying Wang

  3. Institute of Crop Sciences, Guandong Academy of Agricultural Sciences, Guanzhou, 510640, China

    Xiaoping Chen & Zhangying Wang

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  1. Xiaoping Chen
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  2. Zhangying Wang
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Correspondence to Guoying Wang.

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Communicated by H. Ebinuma.

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Chen, X., Wang, Z., Gu, R. et al. Isolation of the maize Zpu1 gene promoter and its functional analysis in transgenic tobacco plants. Plant Cell Rep 26, 1555–1565 (2007). https://doi.org/10.1007/s00299-007-0372-1

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  • DOI: https://doi.org/10.1007/s00299-007-0372-1

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